CMT2C research papers explained

At a glanceEach paper has a relevance score and an evidence score out of 5, a short takeaway, and a full analysis you can open: what the study did, what it found (with short quotes from the paper), the problems with the research, and plain explanations. The analyses were written with the help of an AI reader, and every quote was checked against the paper itself, so please treat each one as a careful first reading. How to read the scores.

Papers

1. #1Full paperMulticenter patient registry (cross-sectional, with limited longitudinal follow-up) plus patient questionnaire

   TRPV4 neuromuscular disease registry highlights bulbar, skeletal and proximal limb manifestations

   Kosmanopoulos GP, Donohue JK, Hoke M et al. · 2025 · Brain: a journal of neurology

   Relevance 5/5Evidence 4/53 moderate · 5 minor problems

   This is the largest description so far of people with TRPV4 neuromuscular disease, including CMT2C. It suggests that leg weakness (including near the hips), voice and breathing problems, and spine and foot problems are common. How fast the condition changes is still uncertain, because most people were seen only once.

   Full analysis: findings, problems and plain explanations

   The study

   Type

   Multicenter patient registry (cross-sectional, with limited longitudinal follow-up) plus patient questionnaire

   Who or what

   95 people with TRPV4 variants, from 74 families, were enrolled at 12 Inherited Neuropathy Consortium centers in the USA, UK and Italy between 2009 and 2023; the 68 with pathogenic or likely pathogenic variants (or lab evidence of pathogenicity) were analyzed. The median age at first visit was 39 (interquartile range 18 to 60). 40 of the 68 (34 adults, 6 children) completed a TRPV4 questionnaire. 13 had repeat CMT examination scores over 2 to 9 years. The TRPV4 group was compared with 1680 people with CMT1A from the same natural history study.

   What they did

   Trained evaluators scored each person with standard CMT scales (CMTNS, CMTES, CMTSS, a video version, and CMTPedS for some children). The team also wrote a TRPV4-specific questionnaire, given once, about voice, breathing, skeleton, bladder, skin and other symptoms. They compared TRPV4 patients with CMT1A patients and looked at how scores related to age, gene variant and age of onset.

   What they found

   • Symptoms most often started very early. In the questionnaire group, 54% said symptoms began in the first 2 years of life, with a second, smaller peak at 34 to 48 years. Diagnosis often came much later.

     “Patient-reported age of symptom onset showed a bimodal distribution, with 54% having symptom onset within the first 2 years of life”Results, 'TRPV4-related neuromuscular disease is motor predominant with bimodal age of onset'

   • Compared with CMT1A, people with TRPV4 disease had more leg weakness and walking difficulty, and more weakness near the hips and shoulders (proximal weakness). On examination 40% had proximal leg weakness, against 4% in CMT1A.

     “These patient-reported symptoms were consistent with clinical exams showing that 40% of TRPV4 patients had proximal leg weakness versus 4% of CMT1A patients”Results, 'TRPV4 clinical features are distinct from CMT1A'

   • Most people (68%) reported no sensory symptoms, but examination often found reduced feeling: reduced pinprick sense in 47% and reduced vibration sense at a similar rate to CMT1A (84% versus 83%).

     “However, clinical examination showed diminished pinprick sensation in 47% of TRPV4 patients and diminished vibration sensation at similar rates to CMT1A patients”Results, 'TRPV4 clinical features are distinct from CMT1A'

   • Voice, breathing and skeletal problems were common: vocal fold weakness 55%, shortness of breath 55%, ventilatory support 11%, scoliosis 64% and arthrogryposis 33% (questionnaire figures).

     “Many patients were affected by vocal fold weakness (55%) and shortness of breath (55%), and 11% required ventilatory support. Skeletal abnormalities were common, including scoliosis (64%), arthrogryposis (33%) and foot deformities.”Abstract; detailed in Results, 'Patient quality of life is driven by motor and bulbar symptoms'

   • In the 13 people with repeat examinations over 2 to 9 years, the CMT examination score rose by an average of +0.81 points per year, but change varied widely between people. Across the whole group, age was only weakly linked to severity (R² = 0.066).

     “Analysing the change in CMTES from baseline in this cohort showed an average change of +0.81 in CMTES per year”Results, 'Charcot-Marie-Tooth disease exam scores demonstrate minimal disease progression'

   • Weakness of the shoulders and upper arms did not go with less sensory involvement, so the authors could not use it to separate CMT2C from scapuloperoneal SMA (SPSMA). People whose symptoms began at or before age 2 reported less sensory change and less worsening than people whose symptoms began later.

     “Thus, we were not able to validate proximal arm weakness as a clinical sign to distinguish between CMT2C and SPSMA.”Discussion, 'Clinical heterogeneity and subtype analysis'

   What the authors concluded

   TRPV4 neuromuscular disease mainly affects movement rather than feeling, often includes weakness near the hips and shoulders, and commonly involves the vocal folds, breathing and the skeleton. CMT2C and SPSMA look like one overlapping spectrum, while infantile-onset disease may be a distinct form that changes less over time. Standard CMT scores may not capture change well, so future drug trials should use measures of proximal weakness, walking, vocal fold function and breathing.

   Problems with the research

   Every study has limits. Listing them shows how much weight each finding can bear.

   • ModerateLikely over-representation of severe cases

     People with severe disease, and parents of severely affected children, may have been more motivated to join. This could make the condition look more severe and early onset more common than it really is.

     “there are likely to be ascertainment biases in our cohort due to increased motivation to participate in patients with severe disease and in parents of severely affected children”Discussion, 'Limitations and future directions'

   • ModerateMostly one time point

     Most conclusions about progression come from comparing people of different ages at a single visit, not from following the same people. Only 13 had repeat scores, and their results varied a lot, so the rate of change is uncertain.

     “the patient data in our analysis is largely cross-sectional and thus provides a limited assessment of disease progression”Discussion, 'Limitations and future directions'

   • ModerateMany figures are self-reported

     Vocal fold, breathing, skeletal, bladder and onset figures come from a questionnaire filled in once by 40 of the 68 people. Terms such as stridor, arthrogryposis and skeletal dysplasia, and recalled age of onset, may be reported inaccurately. The authors themselves urge caution.

     “Thus, the validity of patient responses to such questionnaire items should be viewed with some degree of caution.”Discussion, 'Limitations and future directions'

   • MinorPercentages differ between data sources

     Table 2 (registry, about 66 people) gives scoliosis in 26 (39%) and wheelchair use required in 21 (32%), while the questionnaire gives scoliosis 64% and the CMT1A comparison gives wheelchair use 19%. Text and table also differ slightly for foot surgery (48% versus 49%). These come from different subgroups and questions, so no single percentage should be read as exact.

     “and a wheelchair (1% CMT1A versus 19% TRPV4)”Results, 'TRPV4 clinical features are distinct from CMT1A'; compare Table 2

   • MinorSmall internal inconsistency on children's follow-up

     The Results say longitudinal CMTPedS scores were available for two patients, but the Limitations say three of 13. This does not change the main findings but shows how few children had repeat scores.

     “we had longitudinal CMTPedS scores for only 3 of 13 patients”Discussion, 'Limitations and future directions' (compare Results: 'longitudinal scores were available for two patients')

   • MinorOnly people with symptoms were included

     The registry only included people with symptoms, and some qualified on a first-degree relative's genetic result rather than their own. So it cannot say how often people carrying a TRPV4 variant have no symptoms (reduced penetrance).

     “Only patients with personal genetic testing results or results from a first degree relative and symptoms consistent with TRPV4-related neuromuscular disease were considered for analysis.”Materials and methods, 'Study design and patient enrolment'

   • MinorMany comparisons, no stated correction

     The infantile versus later-onset comparisons tested many symptoms one by one in small groups defined by recalled age of onset. The paper does not mention adjusting for multiple comparisons, so some 'significant' differences could arise by chance. The authors say the difference between the onset groups needs to be confirmed in prospective studies.

     “Two-tailed P- values of <0.05 were considered statistically significant.”Materials and methods, 'Statistical analysis'

   • MinorIndustry links declared

     Several authors take part in a TRPV4 natural history study supported by a company (Actio Biosciences), one receives company support for lab work, and others consult for drug companies. These are openly declared; the paper itself does not test a drug.

     “is supported by Actio Biosciences”Competing interests

   Three ways to explain it

   Scientific

   In a multicenter registry of 68 patients with pathogenic TRPV4 variants, disease was motor-predominant with frequent non-length-dependent proximal weakness, vocal fold weakness (55%) and skeletal involvement, and subclinical sensory loss was common on examination. Onset was bimodal, with 54% of questionnaire respondents reporting onset within the first 2 years. Proximal arm weakness did not discriminate CMT2C from SPSMA. Infantile-onset patients reported less sensory involvement and showed little correlation between age and CMTES. Because the data are largely cross-sectional, partly self-reported and probably subject to ascertainment bias, estimates of progression remain provisional.

   Easier

   Researchers collected information on 68 people with disease-causing changes in the TRPV4 gene, including people with CMT2C. Weakness, especially in the legs and sometimes in muscles near the hips and shoulders, was more common than numbness, although doctors often found reduced feeling when they tested for it. Just over half reported weakness of the vocal folds (the voice box muscles), and just as many reported shortness of breath. Spine and foot problems were common. Most people were seen only once and were volunteers, so how quickly the condition changes is still not clear.

   Simple

   Scientists checked 68 people who have a change in the TRPV4 gene. Many had weak legs, a weak voice, trouble breathing, a curved back, or feet with an unusual shape. Most people were only seen once, so nobody knows yet exactly how fast it changes.

   Why these ratings

   Relevance 5/5: The largest study so far of people with TRPV4 neuromuscular disease, including CMT2C, describing symptoms and change over time in ways patients can relate to.

   Evidence 4/5: A large multicenter cohort by the standards of a rare disease, using standard examination scores, but with data mostly from one visit, partly self-reported, and probably skewed toward more severe cases.

   Funding and conflicts of interest

   Funded by the US National Institutes of Health (NINDS K08 NS102509, NINDS R35 NS122306), the Muscular Dystrophy Association (629305) and the Inherited Neuropathies Consortium (U54 NS065712, part of the NCATS Rare Diseases Clinical Research Network). Several authors take part in a TRPV4 natural history study (NCT05600764) supported by Actio Biosciences; one author receives Actio Biosciences support for laboratory TRPV4 work; two authors list consultancy or advisory work for various drug companies.

   PubMed 38917025 · DOI

2. #2Full paperGenetic linkage study of two families, with cell, frog egg (oocyte) and protein structure experiments

   Mutations in TRPV4 cause Charcot-Marie-Tooth disease type 2C

   Landouré G, Zdebik AA, Martinez TL et al. · 2010 · Nature genetics

   Relevance 5/5Evidence 4/53 moderate · 2 minor problems

   This is one of the papers that first linked CMT2C to TRPV4. It shows that limb, breathing and voice weakness, hearing loss and bladder urgency can occur in the same families, with very different severity even between a mother and daughter. The idea that an overactive channel harms nerves comes from cell experiments, not from studies in patients.

   Full analysis: findings, problems and plain explanations

   The study

   Type

   Genetic linkage study of two families, with cell, frog egg (oocyte) and protein structure experiments

   Who or what

   Two large families with CMT2C (one reported before), with affected people in 3 generations; their clinical details are listed in a table. The variants were checked in 209 ethnically matched controls. Lab work used cultured dorsal root ganglion (DRG) cells, HEK293 and HeLa cells and Xenopus frog eggs made to produce normal or mutant TRPV4, human spinal cord and cartilage samples, mice lacking TRPV4 and normal mice, and a crystal structure of the chicken TRPV4 ankyrin repeat domain.

   What they did

   The team examined members of two CMT2C families and used genetic markers to narrow the disease region on chromosome 12. They sequenced all 38 predicted protein-coding genes in that region and found TRPV4 changes. They then put normal and mutant TRPV4 into cultured cells and frog eggs to see how the channel behaved and whether it harmed the cells.

   What they found

   • Both families showed linkage to chromosome 12q24 (LOD scores 3.1 and 6.9). Sequencing found two different changes at the same TRPV4 position, R269C in one family and R269H in the other, and neither was found in 209 controls.

     “These sequence variants were not present in 209 ethnically matched controls. The identified mutations predict substitutions at the same amino acid (R269C, R269H) in the TRPV4 protein.”Main text, genetic results

   • Severity varied greatly within families: a mother had mild weakness starting late in life, while her daughter had severe weakness of all four limbs and breathing failure.

     “Marked variability of disease severity is demonstrated by mild, late onset weakness in subject F1.III.2, but severe quadriparesis and respiratory failure in her daughter, subject F1.IV.4.”Figure 1 legend

   • Affected people had weakness at the far ends of the limbs and in the diaphragm and voice box muscles, with fewer sensory complaints. Hearing loss was documented in ten people, and nine reported bladder urgency and incontinence.

     “Bilateral sensorineural hearing loss was documented in ten subjects, and nine subjects complained of bladder urgency and incontinence.”Main text, clinical description

   • Two other families with a CMT2C-like illness had no TRPV4 change, suggesting more than one gene can cause this picture.

     “Sequencing of TRPV4 in two other families with a CMT2C-like phenotype failed to identify mutations, and in one linkage to chromosome 12q24.11 was excluded, suggesting that CMT2C may be genetically heterogeneous”Main text, genetic results

   • In frog eggs, the mutant channels passed more current than normal TRPV4 at rest and when stimulated by heat or a chemical activator. The amount of channel at the cell surface was about the same, so the extra activity was not due to more channels.

     “Oocytes expressing R269C and R269H TRPV4 showed a 2.5 fold increase in channel currents compared to WT under resting conditions (18°C)”Main text, electrophysiology; Figure 3

   • Mutant TRPV4 killed more cultured cells than normal TRPV4: four-fold more DRG cell death at 48 hours, and in HEK293 cells two-fold at 24 hours and about six-fold at 48 hours. Channel blockers (ruthenium red, RN1734) or a pore-blocking change (M680K) prevented this.

     “The CMT2C mutations caused a four-fold increase in cell death in DRG neurons compared to wild type (WT) TRPV4 at 48 hours”Main text, toxicity experiments; Figure 2

   What the authors concluded

   Changes in TRPV4 cause CMT2C, showing that a gene already linked to skeletal dysplasias can also cause degeneration of peripheral nerves. The mutant channels are overactive and let in too much calcium, which harmed nerve-type cells in culture even though nerve cells make little TRPV4. Because R269 sits on a different face of the ankyrin repeat region from known skeletal dysplasia changes, the authors suggest the different diseases may come from effects on different partner proteins. They also suggest hearing loss and bladder problems may partly reflect loss of normal TRPV4 function.

   Problems with the research

   Every study has limits. Listing them shows how much weight each finding can bear.

   • ModerateUnaffected carriers judged only on limited home examination

     Two relatives carried the disease-linked stretch of DNA but were judged unaffected after only a brief examination at home, with no nerve tests described. We cannot tell whether they had mild signs, would develop symptoms later, or were truly unaffected. The pedigrees also mark some people as 'unknown disease status'. The paper makes no penetrance claim, so this is a gap in information rather than a flawed conclusion.

     “Two individuals in family 1 carried the disease allele but were not found to be affected during limited examination in their homes.”Main text, linkage results

   • ModerateLab models are far from human nerves

     The channel experiments used cells and frog eggs made to produce large amounts of TRPV4. The frog egg work used a mouse version of TRPV4, and the paper cited for the DRG cells describes an immortalized DRG cell line. Cell death in a dish does not show that the same process happens in patients' nerves.

     “For expression in Xenopus oocytes, TRPV4 cDNA constructs were obtained by PCR of mouse kidney cDNA and inserted into pTLB.”Materials and Methods, expression in mammalian cells and Xenopus oocytes

   • ModerateMechanism stated more firmly than the data allow

     The discussion says the team 'have shown' that the mutations are toxic through calcium overload. This was shown in cultured cells, not in patients or animals carrying the mutation. The idea that hearing and bladder problems reflect loss of function rests on mice lacking TRPV4, not on the patients.

     “Here we have shown that despite low expression levels of TRPV4 in neurons, the TRPV4 CMT2C mutations are highly toxic due to increased channel activity causing calcium overload.”Main text, discussion

   • MinorClinical measures not fully described

     Weakness and sensory loss are graded as mild, moderate or severe without a named scale. The method used to document hearing loss is not given, some items were not done, and worsening in the cold is based on patient report. This limits how precisely the clinical picture can be compared with other studies.

     “Most affected patients reported worsening of hand weakness in the cold.”Main text, clinical description

   • MinorSmall reporting slip in a figure legend

     The Figure 2 legend refers to panel 'f', but the legend only describes panels a to e. This does not change the findings.

     “Data in d, f are averaged from three independent experiments; error bars, s.e.m.”Figure 2 legend

   How “unaffected” relatives were checked

   Pedigrees mark people as affected, unaffected or of 'unknown disease status', but the paper does not say how unaffected relatives were assessed. Two relatives in family 1 carried the disease allele yet 'were not found to be affected during limited examination in their homes'; no nerve conduction tests are described for them.

   Three ways to explain it

   Scientific

   Linkage analysis in two autosomal dominant CMT2C kindreds (LOD 3.1 and 6.9) mapped the disease to a 2.6 Mb interval on chromosome 12q24, where sequencing identified heterozygous TRPV4 missense variants at the same conserved ankyrin repeat domain residue (R269C, R269H), absent from 209 controls. In heterologous expression systems the mutants showed gain of function, with raised basal and stimulated currents without increased surface expression, and caused TRPV4-dependent cell death that channel inhibitors prevented. Phenotypic expression was highly variable within families, and two carriers appeared unaffected on limited home examination.

   Easier

   The researchers studied two big families with CMT2C and tracked the illness to a small stretch of chromosome 12. There they found a spelling change in the TRPV4 gene, at the same spot in both families. When they put the changed gene into cells grown in the lab, the channel it makes let in more calcium than normal and many cells died. How badly people were affected varied a lot, even between a mother and her daughter.

   Simple

   TRPV4 is like a door that lets calcium into cells. In two families with CMT2C, the gene for this door had a small mistake that left the door open too much, at least in cells grown in a dish. People in the same family could be affected in very different ways.

   Why these ratings

   Relevance 5/5: One of the founding papers showing that TRPV4 changes cause CMT2C, with clinical details on affected family members.

   Evidence 4/5: Strong family linkage, with changes at the same amino acid in both families and none in controls, backed by consistent lab experiments, though the lab work uses cells and frog eggs made to overproduce the channel rather than human nerves.

   Funding and conflicts of interest

   Supported by intramural funds from NINDS at NIH, the Johns Hopkins Department of Neurology, the David and Elaine Potter Charitable Foundation, NIH grant R01GM081340 and a McKnight Scholar Award. The authors state they have no competing financial interests.

   PubMed 20037586 · DOI

3. #3Full paperGene discovery study in two families, with tissue pathology and cell experiments (letter-length research article)

   Scapuloperoneal spinal muscular atrophy and CMT2C are allelic disorders caused by alterations in TRPV4

   Deng HX, Klein CJ, Yan J et al. · 2010 · Nature genetics

   Relevance 5/5Evidence 4/52 moderate · 5 minor problems

   This paper is part of the original evidence that changes in TRPV4 cause CMT2C, and that the same gene causes the related condition SPSMA. Its early lab results suggested the faulty channel lets in too much calcium, though this was shown only in kidney-derived cells.

   Full analysis: findings, problems and plain explanations

   The study

   Type

   Gene discovery study in two families, with tissue pathology and cell experiments (letter-length research article)

   Who or what

   Two previously described families: a large New England family of French-Canadian origin with scapuloperoneal spinal muscular atrophy (SPSMA) and an American family of English and Scottish descent with CMT2C. Over 600 control DNA samples for the SPSMA change and over 700 for the CMT2C change. Muscle, brain and spinal cord tissue from one person with SPSMA. Human embryonic kidney (HEK293) cells made to produce normal or mutant TRPV4. The number of people tested in each family is not given in the text.

   What they did

   The authors sequenced the genes in the chromosome 12 region already linked to both conditions, using DNA from the SPSMA family, and then tested TRPV4 in the CMT2C family. They checked whether each change was present in affected relatives and absent from controls. They then put normal and mutant TRPV4 into HEK293 cells and measured where the channel sat, how much calcium entered and how much current flowed.

   What they found

   • After the team sequenced every known gene in the shared region, the only change that tracked with disease in the SPSMA family was a TRPV4 change, R316C.

     “We then sequenced a total of 62 genes, including all the 56 known and predicted genes in the minimum 4-Mb CMT2C region, using DNA samples from individuals with SPSMA.”Main text, genetic analysis

   • R316C co-segregated with SPSMA in the family and was absent from over 600 control samples.

     “The mutation causing this substitution was not present in the SNP database or in over 600 control samples.”Main text

   • The original CMT2C family carried a different TRPV4 change, R269H, which co-segregated with disease and was absent from over 700 controls. This showed SPSMA and CMT2C are allelic (caused by changes in the same gene).

     “The G806A mutation was not present in the SNP database, and we did not detect it in over 700 control samples.”Main text

   • In HEK293 cells, both mutant channels reached the cell surface normally but gave higher resting calcium and larger responses to several stimuli than normal TRPV4.

     “the presence of the TRPV4 mutant protein resulted in substantially higher basal calcium levels than wtTRPV4 protein, suggesting an increased constitutive activity for the mutants.”Main text, calcium imaging; Figures 4 and 5

   • Electrical recordings in a small number of cells showed that those with the CMT2C-linked R269H channel had a TRPV4 conductance about six times that of cells with normal TRPV4.

     “The conductance of R269H-expressing cells was ~6 times larger (0.35 ± 0.5 in wild type and 2.1 ± 0.6 in the mutant, 9 and 6 cells, respectively, P < 0.01).”Figure 6 legend

   • In the one person with SPSMA studied after death, muscle showed damage typical of nerve disease, while motor neurons in the spinal cord were present in normal numbers, pointing to a problem in the peripheral motor nerves.

     “brain and spinal cord sections revealed normal numbers of motor neurons in the motor cortex and spinal cord.”Main text, first paragraph after abstract

   What the authors concluded

   SPSMA and CMT2C are caused by different changes in the same gene, TRPV4. The disease-linked changes increase the channel's calcium activity in cells, suggesting a gain of function, which the authors say points to a disease mechanism and possible future treatments. They propose grouping the related conditions under the name TRPV4-associated peripheral neuropathy and bony dysplasia (TRPV4-PNAB).

   Problems with the research

   Every study has limits. Listing them shows how much weight each finding can bear.

   • ModerateFamily members and how they were assessed are not described

     The paper relies on earlier publications for the clinical description of both families. It reports only that affected members with available DNA carried the change. It does not say how many people were tested, how unaffected relatives were assessed, or whether any carriers were unaffected, so penetrance cannot be judged from this paper.

     “All the affected members whose DNA samples were available for sequencing analysis had the R316C substitution.”Figure 2 legend

   • ModerateLab tests in kidney-derived cells overproducing the channel

     Calcium and current measurements were made in HEK293 cells given extra TRPV4, not in nerve cells or patient cells. This shows the mutant channel can be more active in this setting, but how far it reflects what happens in human nerves is uncertain.

     “we analyzed the calcium channel activity of transiently transfected HEK293 cells”Main text, calcium imaging

   • MinorSome supporting data not shown

     Two supporting results, the cell-surface protein measurement and the block of extra calcium entry by ruthenium red, are stated but not shown, so readers cannot check them.

     “The increased calcium channel activity could be inhibited by the TRPV antagonist ruthenium red (data not shown).”Main text, calcium imaging

   • MinorCurrent recordings limited and selective

     Electrical recordings were done only for R269H and in a small number of cells (9 and 6). About half of the cells given TRPV4 showed no typical TRPV4 current, so which cells were analyzed could affect the result.

     “about half of the wild-type (22 out of 47) and mutant (15 out of 27) TRPV4-transfected HEK293 cells did not show rectifying TRPV4 currents.”Main text, electrophysiology

   • MinorPathology from a single person

     The tissue findings that support a peripheral nerve origin come from one person with SPSMA, so they may not represent everyone with the condition, and no CMT2C tissue was studied.

     “Pathological studies of biopsy and autopsy samples from an individual with SPSMA revealed”Main text, first paragraph after abstract

   • MinorSmall inconsistencies

     The text lists stickleback among species where the affected sites are conserved, while the Figure 2 legend lists dog and cattle instead. Ruthenium red is given as 10 μM in one Methods passage and 20 μM elsewhere. These do not affect the main genetic finding.

     “Both Arg316 and Arg269 of TRPV4 are conserved among human, rat, mouse, chicken, stickleback and zebrafish”Main text; Figure 2 legend; Online Methods

   • MinorMechanism and treatment ideas are speculative

     The abstract says the findings imply a disease mechanism and possible therapy options, and the Discussion offers four possible mechanisms. These are reasonable hypotheses but were not tested in this paper.

     “implying a pathogenic mechanism and possible options for therapy for these disorders.”Abstract

   How “unaffected” relatives were checked

   Not described in this paper. Affected status rests on earlier clinical and neurophysiological reports of the two families, and the text states only that affected members with available DNA carried the change. It does not say how unaffected relatives were assessed or whether any of them carried the change. Quote: 'All the affected members whose DNA samples were available for sequencing analysis had the R316C substitution.' (Figure 2 legend).

   Three ways to explain it

   Scientific

   Sequencing of the shared 12q24 linkage interval identified heterozygous TRPV4 missense mutations in the ankyrin repeat-containing region, R316C in a large SPSMA family and R269H in a large CMT2C family. Each co-segregated with disease in the affected members tested and was absent from over 600 and over 700 controls, respectively, establishing SPSMA and CMT2C as allelic. In transiently transfected HEK293 cells both mutants trafficked to the plasma membrane and showed higher basal and stimulated calcium than wild type, and R269H gave a larger ruthenium red-sensitive conductance, suggesting gain of function. The family phenotyping and penetrance are not reported here.

   Easier

   The researchers studied two large families, one with SPSMA and one with CMT2C, and found that both conditions come from changes in the same gene, TRPV4. Each family had its own change, found in every affected relative whose DNA was tested but not in hundreds of unrelated control samples. When they put the changed gene into kidney cells grown in the lab, the channel let in more calcium than normal. The paper does not describe how healthy-looking relatives were checked.

   Simple

   Two families with different nerve and muscle illnesses turned out to have small mistakes in the same gene, TRPV4. TRPV4 makes a door that lets calcium into cells, and in lab cells the faulty door let in more calcium than usual, like a door that does not close properly.

   Why these ratings

   Relevance 5/5: This is one of the original reports identifying TRPV4 as the gene behind CMT2C, using the original CMT2C family.

   Evidence 4/5: The genetic evidence is strong (whole-region sequencing, co-segregation in two large families, hundreds of controls), but family details are not given here and the lab work is limited to kidney-derived cells overproducing the channel.

   Funding and conflicts of interest

   Supported by the National Institute of Neurological Disorders and Stroke (NS050641), the Les Turner ALS Foundation, the Vena E. Schaff ALS Research Fund, the Harold Post Research Professorship, the Herbert and Florence C. Wenske Foundation, the David C. Asselin MD Memorial Fund, the Help America Foundation, the Les Turner ALS Foundation/Herbert C. Wenske Foundation Professorship and the Epilepsy Foundation. Competing interests: not stated in this version.

   PubMed 20037587 · DOI

4. #4Full paperFamily genetic study (linkage and sequencing) with cell experiments

   Alterations in the ankyrin domain of TRPV4 cause congenital distal SMA, scapuloperoneal SMA and HMSN2C

   Auer-Grumbach M, Olschewski A, Papić L et al. · 2010 · Nature genetics

   Relevance 5/5Evidence 4/53 moderate · 4 minor problems

   This is one of the first papers to show that changes in TRPV4 cause CMT2C and related conditions, and that relatives with the same change can have different diagnoses. Its cell experiments pointed to reduced channel function, but these came from a non-nerve cell line and the authors could not rule out other explanations.

   Full analysis: findings, problems and plain explanations

   The study

   Type

   Family genetic study (linkage and sequencing) with cell experiments

   Who or what

   Five families (FAM_1, FAM_2, A5, FAM_3 and FAM_4) with congenital distal SMA, scapuloperoneal SMA or HMSN2C (CMT2C), including a family from the Netherlands (FAM_2) and one from New Zealand (A5). FAM_1 had ten living affected individuals. Three TRPV4 variants were found (R269H, R315W, R316C). Healthy European controls were screened (162 for exon 5 and 304 for exon 6). Normal and mutant human TRPV4 were expressed in HeLa cells, and muscle and sural nerve biopsies from one person with HMSN2C were stained.

   What they did

   They used a genome-wide SNP scan in one large family to narrow the disease region to chromosome 12q23-24, then sequenced genes in that region and found a TRPV4 change. They screened further families and healthy controls for TRPV4 changes. They then put normal and mutant human TRPV4 into HeLa cells and measured where the protein went and how much calcium and electrical current the channels let through.

   What they found

   • One large family (FAM_1) included people with all three diagnoses, from mild to severe, showing that one inherited cause can look different in different relatives.

     “We identified a large family (FAM_1; see anonymized pedigree, Supplementary Fig. 1) with ten living individuals showing a mild to severe congenital distal SMA, SPSMA or HMSN2C phenotype.”Main text, page 2

   • Three TRPV4 missense variants (R269H, R315W, R316C), all in the ankyrin repeat domain, were found in five families. They were present in every affected person tested and absent from hundreds of healthy European controls.

     “The mutations causing these three substitutions were present in all affected individuals but were not found in 162 (looking at exon 5) and 304 (looking at exon 6) healthy European control individuals.”Main text, page 2

   • In HeLa cells, the mutant TRPV4 proteins clumped inside the cell instead of reaching the cell surface, and surface levels were markedly reduced (completely absent for R316C).

     “mutant TRPV4 proteins formed cytoplasmic aggregates without plasma membrane involvement”Main text, pages 3 to 4; Figure 2

   • Cells carrying only mutant TRPV4 responded less to swelling: calcium peaks were about 30% lower for R269H and R316C and about 75% lower for R315W, and currents were 90% smaller.

     “The R269H and R316C mutants showed a significantly decreased (~30%) calcium peak compared to wild-type (Fig. 4c), whereas the calcium peak of the R315W mutant was decreased by ~75%.”Main text, page 4; Figures 4 and 5

   • When normal and mutant TRPV4 were expressed together, which is closer to the situation in a person carrying one changed copy, the cells' responses were not significantly different from normal.

     “When HeLa cells coexpressed wild-type and any of the mutant TRPV4 proteins, we observed no significant change in basal calcium level (data not shown) or response to hypo-osmotic challenge”Main text, page 4; Figure 4d

   • The authors proposed that too little working channel (haploinsufficiency) was the most likely mechanism, while saying they could not rule out the mutant protein interfering with the normal one.

     “Haploinsufficiency can be considered the most likely underlying pathomechanism, although we cannot fully exclude adverse interaction of wild-type and mutant TRPV4 subunits.”Main text, page 5

   What the authors concluded

   Changes in the ankyrin domain of TRPV4 cause congenital distal SMA, scapuloperoneal SMA and HMSN2C, which the authors describe as a new channelopathy. They propose that the changes disrupt channel maturation so that fewer working channels reach the cell surface, with too little working channel (haploinsufficiency) as the most likely mechanism, and suggest the findings may help diagnosis and treatment development.

   Problems with the research

   Every study has limits. Listing them shows how much weight each finding can bear.

   • ModerateMechanism tested only in a non-nerve cell line with overexpression

     HeLa cells are a human cancer cell line, not nerve or muscle cells, and the TRPV4 protein was made in large amounts from added DNA. How the channel behaves in these cells may differ from how it behaves in human nerves, so the conclusion that the variants reduce channel activity is uncertain.

     “To evaluate the consequences of the TRPV4 mutations, we expressed differentially tagged (polyhistidine (His)- or Flag-tagged) mutant and wild-type human TRPV4 in HeLa cells.”Main text, page 3

   • ModerateThe experiment closest to patients showed no defect

     People with these conditions carry one normal and one changed copy of TRPV4. When cells were given both, they behaved like normal cells, so the cell results do not by themselves explain how one changed copy causes disease. The authors' preferred mechanism (haploinsufficiency) is therefore a proposal rather than a demonstrated finding.

     “Coexpression (+) of the mutant channels with the wild-type protein did not result in a significant difference in the Fura-2 ratio change evoked by the hypo-osmotic solution.”Figure 4 legend; main text, page 4

   • ModerateCarrier status of unaffected relatives not reported

     The paper reports that the variants were found in all affected people, but the main text does not say whether apparently unaffected relatives were tested or whether any of them carried a variant. So the paper cannot tell us anything about reduced penetrance in these families.

     “present in all affected individuals”Main text, page 2

   • MinorTwo families not re-examined

     Affected people in two of the five families were not examined again for this study, so their diagnoses rely on earlier reports.

     “The affected individuals from families FAM_2 and A5 were not re-examined for this study.”Online Methods, Subjects

   • MinorLinkage evidence partly not shown

     The first scan pointed to three possible regions with LOD scores above 2, which is suggestive rather than conclusive, and these data were not shown. The later sequencing in several families strengthens the result, so this mainly limits how far readers can check the first step.

     “observed linkage to three chromosomal regions with log10 of odds (lod) scores >2 for several SNP markers, including the chromosome 12q23-24 region (data not shown)”Main text, page 2

   • MinorSmall reporting inconsistencies and small cell numbers

     The methods say at least five cells were recorded per construct, but the figure legends give n of at least 4. The methods say values are shown as standard error, while the Figure 4 legend says standard deviation. The electrical recordings rest on small numbers of cells.

     “Whole membrane currents were measured for at least five randomly selected Ds-Red-positive cells per TRPV4 expression construct”Online Methods, Electrophysiology; Figure 5 legend

   • MinorClinical details mostly outside the main text

     The main text says little about each person's symptoms, nerve test results, age of onset or severity. These are in supplementary tables that were not part of the copy analyzed here, so the clinical picture in each family could not be checked.

     “Although many of the affected individuals in our study showed mild to severe scoliosis (Supplementary Table 3)”Main text, page 3

   How “unaffected” relatives were checked

   The methods state that unaffected relatives and spouses were examined, including nerve testing, by experienced neurologists, clinicians and geneticists: 'Affected and unaffected family members and spouses underwent a detailed clinical, neurological and neurophysiological examination following standard methods'. However, affected members of two families (FAM_2 and A5) were not re-examined for this study, and the main text does not say whether any unaffected relatives carried the TRPV4 variants.

   Three ways to explain it

   Scientific

   Linkage in a large pedigree and sequencing across five families identified heterozygous TRPV4 missense variants (R269H, R315W, R316C) at conserved arginines in the ankyrin repeat domain, present in all affected individuals with congenital distal SMA, SPSMA or HMSN2C and absent from European controls. In HeLa cells the mutant channels showed cytoplasmic aggregation, reduced surface expression and reduced hypo-osmotic and 4αPDD-evoked responses, but co-expression with wild type gave a wild-type response. The authors favored haploinsufficiency while not excluding a dominant-negative interaction.

   Easier

   The researchers found that changes in the TRPV4 gene caused three related inherited nerve conditions, including CMT2C, in five families, and that the changes were missing in hundreds of healthy people. One large family had all three conditions, showing that the same gene change can look different in different relatives. In lab-grown cells (not nerve cells), the changed TRPV4 protein got stuck inside the cell and let in less calcium. But when normal and changed protein were mixed, the cells behaved normally, so how the change causes disease was left uncertain.

   Simple

   Scientists found that a change in the TRPV4 gene causes CMT2C and two similar illnesses, and that people in one family can be affected in different ways. TRPV4 makes a door that lets calcium into cells. In their lab cells, the changed doors got stuck inside instead of reaching the cell's surface. But the scientists were not sure this is what happens in real nerves.

   Why these ratings

   Relevance 5/5: One of the papers that first identified TRPV4 as the gene behind HMSN2C (CMT2C) and its related conditions.

   Evidence 4/5: The genetic link is supported by five families, with the variants present in all affected members and absent from hundreds of controls, but the proposed mechanism rests on overexpression in one non-nerve cell line.

   Funding and conflicts of interest

   Supported by the Austrian Science Fund (FWF, P19455-B05), the Oesterreichische Nationalbank (ÖNB, project 13010) and the Interdisciplinary Centre for Clinical Research BIOMAT within the Faculty of Medicine, RWTH Aachen; one author was a Heisenberg fellow of the Deutsche Forschungsgemeinschaft (DFG). No competing interests statement appears in this author manuscript.

   PubMed 20037588 · DOI

5. #5Full paper (scan)Genetic linkage study in one large family

   The gene for HMSN2C maps to 12q23-24: a region of neuromuscular disorders

   Klein CJ, Cunningham JM, Atkinson EJ et al. · 2003 · Neurology

   Relevance 5/5Evidence 4/52 moderate · 4 minor problems

   This early study of one large family narrowed down where on chromosome 12 the CMT2C gene lies, but did not identify the gene itself. It also shows how differently the condition affected relatives in one family, from a baby with severe disease to adults whose only sign was a hoarse voice.

   Full analysis: findings, problems and plain explanations

   The study

   Type

   Genetic linkage study in one large family

   Who or what

   One large American family of English and Scottish descent with HMSN2C (CMT2C). Clinical evaluations, cell lines and DNA were available on 73 individuals; the genome-wide scan used 41 (17 affected, 17 unaffected, 7 unaffected and unrelated spouses), and the pedigree legend says genetic analysis was performed on 44. 18 people were judged clinically affected. 382 DNA markers were used in the initial scan.

   What they did

   The team revisited the family several times over about 10 years and decided who was affected using a neuropathy impairment score (NIS) and a combined score of nerve conduction tests, plus, in some people, voice and larynx assessment and diaphragm nerve stimulation. They then searched the whole genome for DNA markers shared by affected relatives, narrowed the region with extra markers and haplotype analysis, and checked the ataxin-2 gene in that region for a triplet repeat expansion.

   What they found

   • The condition in this family was linked to the long arm of chromosome 12 (12q24), with a maximum two-point LOD score of 4.73 and a multipoint LOD score of 5.17 at 119.0 cM.

     “The initial genome-wide scan identified a region at 12q24, and fine mapping provided a maximal lod score of 4.73”Abstract, page 1 (journal p. 1151); Table, page 3 (p. 1153)

   • Haplotype analysis narrowed the region to about 5.0 cM (12q23.3-24.21). All patients identified as affected shared a conserved haplotype.

     “Haplotype analysis narrowed the region to approximately 5.0 cM between D12S1646,D12S1330 and D12S105,D12S1339 (12q23.3-24.21).”Abstract, page 1 (p. 1151); Results, page 4 (p. 1154)

   • The ataxin-2 (SCA2) gene lies in this region, but no triplet repeat expansion or point mutation in the repeat was found; the repeat length in the first four people tested was 21, and no other family member had an expansion. The disease gene itself was not identified.

     “Ataxin-2, the gene responsible for spinocerebellar ataxia type 2 (SCA2), localizes to this region, but no triplet repeat expansion or point mutations within the repeat were found.”Abstract, page 1 (p. 1151); Results, page 4 (p. 1154)

   • Voice and breathing muscle involvement was very common: 16 of the 18 affected people had some vocal cord, diaphragm or intercostal (rib muscle) weakness, often subtle, and in several a hoarse voice was the only sign.

     “Of the 18 clinically felt to be affected, all but 2 had varying degrees of vocal cord, diaphragm, or intercostal weakness.”Results, page 2 (p. 1152)

   • Severity varied greatly within the family. Impairment scores ranged from 8 to 164 points; the youngest generation's proband presented at 1 month while her grandmother presented at 80, and her mother (NIS 50) was mildly affected.

     “The NIS of affected individuals varied from 8 (IV-50) to 164 (IV-58) points, which are both unequivocally abnormal.”Subjects and methods, page 2 (p. 1152); Figure 1 legend; Results, page 4 (p. 1154)

   • One relative classified as unaffected carried the same disease-linked haplotype as affected relatives.

     “Also of note is one individual classified as unaffected who carried the affected haplotype combination.”Results, page 4 (p. 1154)

   What the authors concluded

   The gene for HMSN2C (CMT2C) lies at 12q23-24, in a region also linked to scapuloperoneal spinal muscular atrophy, congenital distal spinal muscular atrophy and SCA2. The authors suggest these conditions might share a genetic cause, and say further studies are needed to find the specific gene change.

   Problems with the research

   Every study has limits. Listing them shows how much weight each finding can bear.

   • MinorLocation only, gene not identified

     The study narrows the search to a chromosome region but does not identify the gene or the mutation. The authors are clear about this, so the conclusion does not overreach, but the paper cannot say which gene causes CMT2C.

     “Whereas we have identified the approximate location of the gene for HMSN2C (CMT2C), further studies are needed to demonstrate the specific gene alteration”Discussion, page 6 (p. 1156)

   • ModerateSome 'affected' labels not based on examination

     People without signs of disease were counted as affected if they had both an affected parent and an affected child, and the pedigree also marks some ancestors as 'affected by history'. This is a recognized approach in linkage studies, but it means not every affected label rests on an examination, and family members shown this way should not be counted as examined patients.

     “Also designated as affected were persons (without overt manifestations) in a lineage having both an affected parent and child.”Subjects and methods, page 2 (p. 1152); Figure 2 legend key, page 3 (p. 1153)

   • ModerateUnaffected carrier not described

     One person classified as unaffected carried the disease haplotype, which could mean reduced penetrance, mild undetected disease, or an age too young for signs to appear. The paper does not give this person's age, NIS score or nerve test results, and does not discuss what it means, so no conclusion about penetrance can be drawn.

     “IV-53 is clinically unaffected.”Figure 4 legend, page 4 (p. 1154)

   • MinorCandidate gene only partly checked

     Only the triplet repeat region of ataxin-2 was examined, so other changes in that gene were not excluded. The authors say so.

     “Further work will be required to exclude alternative mutations in the SCA2 gene.”Discussion, page 5 (p. 1155)

   • MinorAnticipation inferred from three people

     Earlier onset in each generation (grandmother at 80, mother at 37, daughter at 1 month) was based on three people. The authors note that variable expression may mimic anticipation, so this pattern is not evidence that CMT2C gets worse in each generation.

     “These three individuals were chosen because of possible anticipation seen in each successive generation”Results, page 4 (p. 1154)

   • MinorMarker names inconsistent between text and figures

     The Figure 3 legend lists D12S29, where the text and the figure itself use D12S79, and the Figure 4 legend gives the flanking markers as D12S105,D12S1583, where the abstract gives D12S105,D12S1339. These look like typing errors and do not change the main result.

     “Multipoint lod scores utilizing five markers at 12q24 (D12S78, D12S1583, D12S1646, D12S29, D12S86).”Figure 3 legend, page 4 (p. 1154)

   How “unaffected” relatives were checked

   Relatives were examined. A person was called 'putatively unaffected' only if older than 23, without neuropathic symptoms, with an NIS under 2 and with summed nerve conduction scores below the 50th percentile of normal: 'Persons designated as putatively unaffected 1) were older than 23 years, were without neuropathic symptoms, and had an NIS of <2 points; and 2) had summated nerve conduction scores at <50th percentile of normal.' (Subjects and methods, p. 1152). However, some people were labeled affected from history or family structure, and one person classified as unaffected (IV-53) carried the disease haplotype, but no further details are given.

   Three ways to explain it

   Scientific

   Genome-wide linkage analysis in a single large autosomal dominant HMSN2C kindred gave a maximal two-point LOD score of 4.73 and a multipoint LOD score of 5.17 at 12q24, and recombinant haplotypes narrowed the locus to about 5.0 cM at 12q23.3-24.21. No CAG expansion or point mutation was found in the ataxin-2 repeat. Expressivity was highly variable (NIS 8 to 164), and one clinically unaffected relative carried the disease haplotype, which raises but does not resolve the question of reduced penetrance.

   Easier

   The researchers studied one very large family with CMT2C to find which part of the DNA travels with the condition. Affected relatives shared the same stretch of chromosome 12, which narrowed the search to a small region, but the team did not find the gene itself. Severity varied hugely within the family, and many affected people had weakness of the voice box or breathing muscles. One relative who seemed unaffected carried the same stretch of DNA, but the paper gives no detail about that person.

   Simple

   Scientists studied the DNA of one big family with CMT2C, like detectives looking for the street where a missing gene lives. They found the right street on chromosome 12, but not the exact house. Some family members were very weak and others only had a croaky voice.

   Why these ratings

   Relevance 5/5: Directly about a family with CMT2C, documenting voice and breathing involvement, wide variability and a possible unaffected carrier, although its main result is a chromosome region rather than a gene.

   Evidence 4/5: Careful clinical and nerve-test classification and LOD scores above 3 at several markers give solid evidence for the location, but it is one family and the gene was not found.

   Funding and conflicts of interest

   Supported in part by the National Institute of Neurological Disorders and Stroke (NS36797), the Muscular Dystrophy Association, and the Mayo Gene Discovery Shared Resource (Mayo Comprehensive Cancer Center Grant) from the National Cancer Institute (CA15083). Competing interests not stated.

   PubMed 12682323 · DOI

6. #6Full paperExpert clinical review (GeneReviews chapter, created May 2014, last updated December 2025)

   Autosomal Dominant TRPV4-Related Disorders

   McCray BA, Schindler A, Hoover-Fong JE et al. · 2014 (updated December 2025) · University of Washington, Seattle (WA)

   Relevance 5/5Evidence 3/52 moderate · 4 minor problems

   This chapter is a standard clinical summary of TRPV4-related neuromuscular disease, including CMT2C. It describes a wide range of possible features. It also says that the age of onset and severity in any one person cannot be predicted, and that treatments aimed at the cause have so far only been tested in animals.

   Full analysis: findings, problems and plain explanations

   The study

   Type

   Expert clinical review (GeneReviews chapter, created May 2014, last updated December 2025)

   Who or what

   A summary of published research on autosomal dominant TRPV4-related disorders: three neuromuscular forms (CMT2C, scapuloperoneal SMA and congenital distal SMA) and five skeletal dysplasias. No new patients were studied. It draws on cited studies, including a registry cohort of 40 people and screening studies of 422 and 443 people with CMT2 or hereditary motor neuropathy.

   What they did

   Four clinicians and researchers summarized the published literature on how TRPV4-related disorders look, how they are diagnosed and inherited, and how they are managed. Because no clinical practice guidelines exist, the management and surveillance sections are based on the authors' own experience. The chapter was last fully updated on 4 December 2025.

   What they found

   • CMT2C, scapuloperoneal SMA and congenital distal SMA were first described as three separate conditions, but the review says they are probably one overlapping spectrum. Different forms can appear in members of the same family.

     “these phenotypes likely represent an overlapping spectrum of disease and can often not be clearly distinguished”Clinical Description, Neuromuscular Disorders

   • Age of onset varies hugely. Symptoms usually begin between early childhood and age 25, but onset ranges from birth to after the eighth decade of life.

     “disease onset can range from birth, with breathing difficulties and delayed walking, to after the eighth decade”Clinical Description, Neuromuscular Disorders

   • The condition mainly affects movement, and weakness can be patchy (non-length dependent) and uneven between sides, often affecting the shoulders (scapular winging) as well as the feet and hands. Vocal fold weakness is described as a hallmark. Breathing weakness, sleep apnea, hearing loss, scoliosis, joint contractures, bladder problems and skin changes can also occur. Tethered spinal cord was reported in 7.5% of a registry cohort of 40 people.

     “Laryngeal dysfunction is a hallmark of TRPV4-related neuromuscular disease and can occur in each subtype”Clinical Description, Neuromuscular Disorders; Suggestive Findings

   • Penetrance is reduced and expressivity is highly variable, so a person's type of disease, age of onset and severity cannot be predicted from the genetic result.

     “Specific phenotype, age of onset, and disease severity cannot be accurately predicted because of reduced penetrance and highly variable expressivity.”Summary, Genetic counseling

   • The weight of evidence from cells, flies and mice suggests the disease variants make the channel overactive (gain of function). In knock-in mice (mice engineered to carry a disease variant), a drug that blocks TRPV4 rescued them from the disease. TRPV4-blocking drugs are being studied as a possible treatment, but no disease-modifying treatment is available to people yet.

     “Trpv4 knock-in mice develop a severe, rapidly progressive fatal neuromuscular phenotype that can be rescued by pharmacologic inhibition of TRPV4 ion channel activity”Molecular Pathogenesis; Therapies Under Investigation

   • TRPV4 variants are found in roughly 1% to 3.5% of people with CMT2 or hereditary motor neuropathy. The figure rises to between 9% and 16% in people with CMT2 plus unusual features such as vocal fold weakness or diaphragm weakness.

     “estimates ranging from ~1% to 3.5% of Charcot-Marie-Tooth neuropathy type 2 (CMT2) / hereditary motor neuropathy (HMN)”Prevalence

   What the authors concluded

   TRPV4-related neuromuscular disease is a motor-predominant nerve condition with a wide range of severity and onset. It often involves the voice box, breathing, hearing and skeleton, and is best managed by a team focused on symptoms. Disease variants most likely cause gain of channel function. TRPV4-blocking drugs are under study after success in mice, but there is no disease-modifying treatment yet.

   Problems with the research

   Every study has limits. Listing them shows how much weight each finding can bear.

   • MinorReview, not new data

     The chapter summarizes other studies. How much each statement can be trusted depends on the study behind it, and the chapter does not grade the strength of that evidence.

   • ModerateManagement advice based on personal experience

     The suggested checks and treatments come from the authors' clinical experience, not from trials or formal guidelines. They are sensible expert opinion, but they have not been tested to show they improve outcomes.

     “In the absence of published guidelines, the following recommendations are based on the authors' personal experience managing individuals with this disorder.”Management

   • ModerateReduced penetrance stated without figures or method

     The chapter says penetrance is reduced but gives no estimate of how often carriers stay unaffected and does not say how apparently unaffected carriers in the underlying studies were examined. Elsewhere it notes that some people are so mildly affected that their condition goes unnoticed, so some 'unaffected' carriers may in fact be mildly affected.

     “Penetrance is reduced with the neuromuscular disease-associated pathogenic variants.”Penetrance

   • MinorArithmetic inconsistency in prevalence figure

     13 out of 422 is well above 1%, so the label '<1%' does not match the numbers given. The overall range quoted in the same paragraph (about 1% to 3.5%) is not affected, but the error suggests this figure should be checked against the original study.

     “Fawcett et al [2012] determined that 13 (<1%) of 422 individuals with a CMT2 (axonal CMT) phenotype were heterozygous for a TRPV4 pathogenic variant”Prevalence

   • MinorAuthors review their own work; no competing interests statement

     The two lead authors are leading researchers whose own laboratories produced much of the mechanism and drug work cited, and a company (Actio Biosciences) is listed among supporters of research in this field. This does not make the review wrong, but the chapter has no separate competing interests statement, so readers cannot see whether any author has a financial link to treatments under study.

     “Her laboratory discovered TRPV4 pathogenic variants that cause neuromuscular disease, developed cell and animal models of disease, and demonstrated that TRPV4 small-molecule antagonists can ameliorate disease features in these models.”Chapter Notes, Author Notes

   • MinorTreatment evidence so far comes from mice

     The promising drug results described come from genetically altered mice, not from people. The review itself is careful to say that a key part of the mouse mechanism has not been shown in patients.

     “These findings suggest that disruption of neural vascular barriers could contribute to disease pathogenesis, although such a mechanism has not been demonstrated in affected individuals.”Molecular Pathogenesis, Mechanism of disease causation

   How “unaffected” relatives were checked

   This is a review, not a family study, so it did not assess anyone itself. It states that penetrance is reduced but does not describe how apparently unaffected carriers in the underlying studies were assessed. It warns that an apparently unaffected parent may turn out to be affected on examination: 'Evaluation of parents may determine that one is affected but has escaped previous diagnosis because of a milder phenotype (possibly associated with parental mosaicism) or reduced penetrance in the parent with the pathogenic variant.'

   Three ways to explain it

   Scientific

   TRPV4-related neuromuscular disease (CMT2C, SPSMA and CDSMA) is presented as an autosomal dominant, motor-predominant, often non-length-dependent neuropathy or neuronopathy spectrum with frequent laryngeal, respiratory, auditory and skeletal involvement. Penetrance is reduced and expressivity highly variable within and between families. Most neuromuscular variants are missense changes at conserved arginines on the ankyrin repeat domain, and the preponderance of evidence supports gain of channel function. TRPV4 antagonists rescue knock-in mouse models, but efficacy in patients has not been shown.

   Easier

   CMT2C and two related nerve conditions are caused by changes in the same gene, TRPV4, and are best thought of as one condition with many forms. It mainly weakens muscles, often including the shoulders, and commonly affects the voice box, breathing, hearing and spine. People with the same gene change, even in one family, can be affected very differently, and some carriers show few or no signs. The gene change seems to make a calcium channel too active; blocking it helped in mice, but this has not yet been shown in people.

   Simple

   TRPV4 is like a door that lets calcium into cells. In this condition the door seems to be stuck too far open, which can weaken muscles, the voice and breathing. People in the same family can be affected very differently, and medicines that close the door have so far only been tested in mice.

   Why these ratings

   Relevance 5/5: A clinical overview written specifically about TRPV4 neuromuscular disease, with CMT2C as a named core phenotype.

   Evidence 3/5: A careful, well-referenced expert review, but it presents no new data, its management advice rests on the authors' experience, and penetrance is described without figures.

   Funding and conflicts of interest

   No formal funding or competing interests statement appears in the chapter. The Acknowledgments say research on TRPV4-related neuromuscular diseases has been supported by the Inherited Neuropathies Consortium, NINDS, the Muscular Dystrophy Association, Actio Biosciences, and affected individuals and families. The Author Notes say Dr. Sumner's laboratory showed that TRPV4 small-molecule antagonists ameliorate disease features in models, and that both lead authors are actively involved in clinical research on this condition.

   PubMed 24830047 · GeneReviews chapter; series began 1993.

7. #7Full paperCase report with literature review and cell experiments

   TRPV4 mutations causing mixed neuropathy and skeletal phenotypes result in severe gain of function

   Taga A, Peyton MA, Goretzki B et al. · 2022 · Annals of clinical and translational neurology

   Relevance 5/5Evidence 3/54 moderate · 3 minor problems

   Some people with TRPV4 neuropathy also have skeletal differences, and in the published cases reviewed here these mixed cases tended to start early and to get worse over time. In cells, a TRPV4 blocker worked less well against some changes than others, which may matter for future drug trials; this has not been tested in people.

   Full analysis: findings, problems and plain explanations

   The study

   Type

   Case report with literature review and cell experiments

   Who or what

   One girl aged 2 years 7 months (31 months) with a new R616G TRPV4 change; a review of 36 previously reported people with both nerve and skeletal features, from 19 different TRPV4 changes (nine families plus several people with new, non-inherited changes); and lab tests of normal TRPV4 and seven mutant forms (R269C, D333G, S542Y, R616Q, R616G, L619P, W785C) in MN-1 cells (a mouse motor neuron and neuroblastoma hybrid cell line), HEK293T cells and MDCK dog kidney cells.

   What they did

   They described the girl's symptoms, tests and genetic result. They searched English-language PubMed reports up to February 2021 for people with TRPV4 changes who had both nerve and skeletal features and summarized them in a table. They then made cells produce selected TRPV4 forms, measured calcium levels, cell death and how well the TRPV4-blocking compound GSK2193874 (GSK219) switched the channel off, and used an existing 3D structure to suggest why two changes are so strong.

   What they found

   • The girl had a severe sensory and motor neuropathy with paralysis of both vocal cords, sleep apnea and a tethered spinal cord, and eventually needed a tracheostomy. Her change was new (not found in either parent) and was seen in only 25% of sequencing reads (suggesting it may be present in only some of her cells). She had no definite skeletal dysplasia, although a different change at the same position (R616Q) is reported to cause skeletal dysplasia.

     “We report a novel R616G mutation causing an asymmetric sensorimotor neuropathy with complete bilateral vocal cord paralysis and no definitive evidence of skeletal dysplasia.”Discussion 4.1; Results 3.1

   • The literature review found 36 previously reported people with both nerve and skeletal features, from 19 different TRPV4 changes. CMT2C or HMSN2 accounted for 9 (25%), congenital distal SMA for 14 (39%) and scapuloperoneal SMA for 4 (11%).

     “we identified 36 individual cases of mixed neuropathy and skeletal phenotypes arising from 19 different mutations.”Results 3.2

   • In these mixed cases, symptoms began earlier on average (2.7 ± 3.9 years) than in the isolated neuropathy cases the authors reviewed (10.9 ± 16.0 years). Weakness often affected the upper legs as well as the lower legs (proximal lower limb weakness in 21 of 29 assessed, 72%).

     “Age of onset varied from congenital to young adulthood, with an average age of 2.7 ± 3.9 years.”Results 3.2

   • Where follow-up information was published, the condition worsened over time in 21 of 23 people (91%), including some with congenital distal SMA, which is often thought of as fairly static.

     “there was evidence of disease progression in 21 out of 23 patients (91%) for which longitudinal information was reported.”Results 3.2; Discussion 4.2

   • In cells, R616G and L619P raised resting calcium the most, caused the most cell death (26.3% and 28.5% compared with 7.2% for normal TRPV4) and needed far more blocker to switch off (IC50 about 216 nM and 198 nM compared with 8 nM for normal TRPV4).

     “the most dramatic effects seen in the R616G and L619P mutants, in which IC50 values were 216 nM and 198 nM, respectively”Results 3.4; Table 4

   • There was no clear rule linking a particular change to a mixed nerve and skeletal picture, and in some families only some members with the same change had skeletal involvement.

     “we did not observe definitive genotype-phenotype correlation between specific TRPV4 mutations and combined neuropathy and skeletal dysplasia phenotypes”Results 3.2; Discussion 4.2

   What the authors concluded

   Mixed nerve and skeletal TRPV4 disease is more common than first thought, starts early and usually progresses. Changes behind severe or mixed forms tend to keep the channel open even at rest, cause more cell damage and respond less to a TRPV4 blocker, which the authors link to structural disruption of the S5 part of the channel. They suggest that blocker doses be checked for each specific change before clinical trials, that children may be the best group in which to show benefit in trials, and that people with TRPV4 changes be checked for skeletal involvement.

   Problems with the research

   Every study has limits. Listing them shows how much weight each finding can bear.

   • ModerateLiterature review method

     Only English-language PubMed reports were searched, cases were selected using the authors' judgment about possible skeletal dysplasia, and the number of papers screened is not given. Published cases tend to be the unusual or severe ones, so the figures on age of onset and progression may not represent everyone with these changes.

     “A PubMed review of English language literature using the search terms”Methods 2.2

   • ModerateAge-of-onset comparison not formally tested

     The isolated neuropathy comparison group is not defined in the Methods, no statistical test is reported, and the spread of ages is very wide. The earlier onset in mixed cases is a pattern in published reports, not a measured result in a defined group.

     “By comparison, the average age of onset for patients with isolated neuropathy in the case reports reviewed was 10.9 ± 16.0 years.”Results 3.2

   • ModerateInterpretation goes further than the results

     The abstract says the findings 'demonstrate' that baseline calcium 'correlates' with mixed phenotypes. Only seven changes were tested, no correlation was calculated, the Results say no definitive pattern was found, and R616G (no definite skeletal dysplasia) and R616Q (skeletal dysplasia with mild calcium rise) do not fit a simple rule.

     “did not reveal definitive patterns of channel function that uniformly distinguished neuropathy, skeletal dysplasia, and mixed phenotype mutants.”Results 3.3; Abstract, Interpretation

   • ModerateCell models are far from people

     The channels were overproduced with a fluorescent tag in a mouse hybrid cell line, HEK293T cells and dog kidney cells. MN-1 cells have no TRPV4 of their own, so they carried only mutant channels, whereas patients carry one normal and one changed copy. Blocker doses that work in a dish may not match what is needed or safe in people.

     “these cells do not express endogenous TRPV4”Results 3.3

   • MinorStructural explanation is a prediction

     The idea that R616G and L619P break a helix in the channel comes from looking at the position of these amino acids on a published structure, not from measuring the structure of the mutant channels.

     “the R616G and L619P mutations are predicted to disrupt the S5 helix structure”Results 3.5

   • MinorYoung case with possible mosaicism

     The girl was 31 months old, so skeletal features could still appear. Mosaicism was suggested by the 25% read fraction but the authors say they have no direct evidence of it. Her case therefore cannot show that R616G never affects bone.

     “Finally, we cannot rule out that additional and more significant skeletal abnormalities will appear in our patient during further follow-up.”Discussion 4.1

   • MinorSmall errors in the text

     L619P is written as 'L169P' in one place and 'L619Q' in a figure legend, and D333G's blocker response (IC50 20.7 nM) is described as similar to normal TRPV4 (8.0 nM). These look like slips; Table 4 gives the values.

     “the dramatic toxicity of the nearby L169P mutation”Results 3.5; Figure 4 legend; Table 4

   How “unaffected” relatives were checked

   For the new case, both parents tested negative for the change on targeted sequencing and there was 'no family history of neurological disorders'. Her 2-month-old brother was described as 'healthy without signs of neuromuscular disease'; the paper does not say whether he was examined by a neurologist or tested genetically. In the literature review, affected status was taken from the published reports rather than reassessed, and one reviewed family (R186Q) was reported to show incomplete penetrance.

   Three ways to explain it

   Scientific

   A de novo, possibly mosaic TRPV4 R616G variant caused a congenital sensorimotor axonal neuropathy with bilateral vocal cord paralysis and no definite skeletal dysplasia, whereas R616Q at the same residue is reported in skeletal dysplasia. Review of 36 published mixed neuropathy and skeletal cases (19 variants) showed early mean onset and frequent progression but no definitive genotype-phenotype correlation. On heterologous expression, R616G and L619P produced marked constitutive calcium elevation, cytotoxicity and right-shifted GSK219 inhibition (IC50 216 and 198 nM versus 8 nM), which structural modeling attributed to S5 helix disruption. This suggests that the magnitude of gain of function may relate to severe or mixed phenotypes and to antagonist sensitivity.

   Easier

   The researchers describe a toddler with a new change in the TRPV4 gene who had severe nerve damage and paralyzed vocal cords, but no clear bone disease, even though a different change at the same spot is known to cause bone disease. They also gathered 36 published cases of people with both nerve and bone problems and found that these problems usually started in early childhood and often got worse. In lab-grown cells, the strongest changes let calcium in all the time, killed more cells and needed much more of a blocking compound to switch the channel off. This suggests that future drug trials may need to check the dose for each specific gene change.

   Simple

   TRPV4 is like a door that lets calcium into cells. With some gene changes the door is stuck wide open all the time, and in lab cells those doors did more harm and were harder to close with a door-blocking chemical. Some children with these changes have problems with both their nerves and their bones.

   Why these ratings

   Relevance 5/5: It is about people with TRPV4 neuropathy, including CMT2C, who also have skeletal features, and it shows in cells that a TRPV4 blocker may work differently depending on the specific change.

   Evidence 3/5: A single case, a review of published reports and careful cell experiments; the cell work is far from patients and the review is not a formal systematic review.

   Funding and conflicts of interest

   Funded by the American Academy of Neurology (Neuroscience Research Training Fellowship), Deutsche Forschungsgemeinschaft (grant 390713860), Max Planck Graduate Center, Muscular Dystrophy Association (grant 629305) and the US National Institute of Neurological Disorders and Stroke (K08 NS102509 and R35 NS122306). The authors declare no conflicts of interest.

   PubMed 35170874 · DOI

8. #8Full paper (scan)Single-center cohort screening study with two case descriptions and one family pedigree

   Incidence and Clinical Features of TRPV4-Linked Axonal Neuropathies in a USA Cohort of Charcot-Marie-Tooth Disease Type 2

   Deng S, Feely SME, Shi Y et al. · 2020 · Neuromolecular medicine

   Relevance 5/5Evidence 3/51 major · 4 moderate · 4 minor problems

   In one USA clinic group, TRPV4 explained CMT2 in 2 of 62 people whose cause was otherwise unknown. Within one family, the condition ranged from mild to severe, with voice and breathing problems in some. The claim that some relatives carry the variant without disease is not backed by examining or testing them.

   Full analysis: findings, problems and plain explanations

   The study

   Type

   Single-center cohort screening study with two case descriptions and one family pedigree

   Who or what

   62 unrelated people with CMT2 from a USA cohort (Wayne State University, Detroit) who had no mutations in MFN2, GARS, NEFL or GDAP1, plus 192 control samples. Two people with disease-causing TRPV4 variants are described in detail: a 61-year-old man with R316C and a family history of the condition, and a 30-year-old man with R269C whose family history was unknown. Relatives of the 61-year-old man are described from family history only.

   What they did

   The researchers read all 15 coding exons of the TRPV4 gene by Sanger sequencing in 62 people with CMT2 whose genetic cause was unknown. They checked a third, uncertain variant in 192 controls and in a public database. They described the symptoms, muscle strength and nerve test results of the two people with disease-causing variants, and drew a family tree for one of them.

   What they found

   • 2 of the 62 people with unexplained CMT2 had a disease-causing TRPV4 variant (R316C and R269C), which the authors give as 3.2%.

     “accounting for 3.2% (2/62) in this cohort”Discussion, p. 70

   • A third variant, I571M, was also found in one of 192 controls. Database figures suggest about 15% of Latino people carry it, so the authors judged it harmless or of very low penetrance.

     “suggests that the TRPV4-I571M is either a benign variant or has a very low penetrance”Results, p. 69

   • The man with R316C had symptoms from childhood, distal leg weakness, loss of vibration and temperature feeling in the feet, a herniated disc, and high-frequency hearing loss. His nerve conduction speeds were normal, with mildly reduced amplitudes in some nerves.

     “In addition, he had hearing loss at high frequencies.”Results, p. 70

   • In the R316C family, severity varied widely. Onset ranged from age 10 to the 20s, vocal cord paralysis was reported in three relatives, and one niece had breathing problems from vocal cord and phrenic nerve paralysis plus severe scoliosis. The authors also describe three relatives as non-penetrant.

     “the disease onset ranged from 10 to 20s, but non-penetrance of the disease was also observed in three members (II-2, IV-5, and IV-11)”Discussion, p. 71

   • The man with R269C had balance problems, frequent tripping and difficulty swallowing liquids since childhood, distal weakness in arms and legs, fine tremor and vocal cord paralysis. Hearing seemed normal on a bedside test, and nerve tests suggested an axonal neuropathy.

     “who complained of increasing difficulty with balancing, tripping, and liquid swallowing since childhood”Results, p. 70

   What the authors concluded

   The authors conclude that TRPV4-related CMT2C makes up a sizable fraction of their USA CMT2 cohort (2 of 62 unexplained cases, or 2.1% of the original cohort of 96). They say it varies widely between people, and that vocal cord and phrenic nerve paralysis, scapular weakness and wasting, skeletal dysplasia and hearing loss are signs that should point doctors toward TRPV4.

   Problems with the research

   Every study has limits. Listing them shows how much weight each finding can bear.

   • MajorNon-penetrance claimed without examining or testing the relatives

     The paper says three relatives (II-2, IV-5 and IV-11) showed non-penetrance, meaning they carry the variant but have no disease. None of them had a DNA test, and the paper does not say any of them was examined by a neurologist or had nerve conduction studies. Their carrier status is inferred from the family tree (IV-5 and IV-11 each have an affected daughter), and their unaffected status is not documented. II-2 is described only as not appearing to have problems. This is weak evidence for reduced penetrance; mild disease could have been missed.

     “His maternal grandmother (II-2) did not appear to have motor and sensory problems”Results, p. 70

   • ModerateVariant not confirmed in relatives; relatives described second-hand

     Only the two patients were genotyped, so the paper does not show that the affected relatives carry R316C (no segregation testing). The symptoms of the mother, brother, nieces and great-grandfather are reported as descriptions or past diagnoses, not as examinations by the study team, so details such as onset age and vocal cord paralysis in relatives are second-hand.

     “DNA samples from other family members were unavailable for testing.”Results, p. 70

   • ModerateVery small numbers behind the conclusions

     The frequency estimate and the description of the clinical picture rest on two people and one family. A figure based on 2 out of 62 is very uncertain, and calling it a sizable fraction is an interpretation rather than a measured result.

     “This suggests that TRPV4-linked CMT2C accounts for a sizable fraction of our CMT2 cohort.”Discussion, p. 70

   • ModeratePromised comparison with non-TRPV4 patients not shown

     The abstract says TRPV4 patients were compared with patients without TRPV4 variants, but the paper presents no clinical data on the patients without TRPV4 variants. So the claim that certain signs point to TRPV4 is not tested against that comparison group.

     “Clinical features of TRPV4-linked patients were compared with those lacking TRPV4 mutations.”Abstract, p. 68

   • ModerateSome suggestive signs come from earlier studies, not this cohort

     The conclusion lists scapular weakness and wasting and skeletal dysplasia as suggestive signs. Scapular weakness is not specifically described in anyone in this paper, and no skeletal dysplasia was shown on imaging (one niece had severe scoliosis). These parts of the conclusion lean on previous studies.

     “Consistent with the previous findings (Auer-Grumbach et al. 2010; Deng et al. 2010; Landoure et al. 2010; Zimon et al. 2010; Echaniz-Laguna et al. 2014; Klein et al. 2011), our data suggest”Discussion, p. 71

   • MinorVariant name inconsistent

     The Discussion calls the second variant R269H, while the abstract, Results, figure and tables call it R269C. This looks like a typing error but could confuse readers.

     “We identified two disease-causing TRPV4 mutations (R316C and R269H)”Discussion, p. 70

   • MinorSporadic case not checked

     The R269C patient is called an apparently sporadic case, but his family history was unknown and his parents were not tested, so it is not known whether the variant was new or inherited.

     “His family history was unknown.”Results, p. 70

   • MinorHearing assessed only roughly

     Normal hearing in the R269C patient was judged by whether he could hear finger rubbing, a simple bedside check, and the paper does not say how the proband's high-frequency hearing loss was measured. Mild hearing loss could be missed or under-described.

     “He could hear finger rubbing bilaterally, suggesting normal hearing.”Results, p. 70

   • MinorOne clinic cohort, called 'incidence'

     The cohort comes from one USA center and was previously studied. The paper calls its figure an incidence, but it is the share of patients in this clinic group, not a rate in the wider USA population.

     “A CMT2 cohort of 96 unrelated patients (Wayne State University, Detroit, MI) was previously studied by Saporta et al. (2011).”Materials and Methods, p. 69

   How “unaffected” relatives were checked

   No relative was examined or genetically tested, as far as the paper reports. Non-penetrance in II-2, IV-5 and IV-11 is inferred from the family tree. II-2 is described from history only ('did not appear to have motor and sensory problems'), and the paper gives no description at all of how IV-5 and IV-11 were assessed. The paper states: 'DNA samples from other family members were unavailable for testing.'

   Three ways to explain it

   Scientific

   Sanger sequencing of all 15 coding exons of TRPV4 in 62 genetically unexplained CMT2 probands identified two previously reported pathogenic variants, R316C and R269C, a yield of 3.2% (2/62), or 2.1% (2/96) of the full cohort; a third variant, I571M, was judged benign or of very low penetrance. Phenotypes were markedly variable and included vocal cord and phrenic nerve paralysis, scoliosis and high-frequency hearing loss, with electrophysiology consistent with an axonal neuropathy. Non-penetrance in three relatives is inferred from the pedigree without genotyping or documented examination, so no penetrance estimate can be drawn from this family.

   Easier

   The team checked the TRPV4 gene in 62 people with the nerve-fiber type of CMT (type 2) whose cause was unknown. Two of them had a TRPV4 change already known to cause disease. These two men, and relatives in one family, differed a lot: some had paralyzed vocal cords that weakened the voice or breathing, one had trouble hearing high-pitched sounds, and severity varied greatly. The paper says some relatives carry the change without symptoms, but those relatives were not tested and, as far as the paper says, not examined.

   Simple

   Scientists looked at one gene, TRPV4, in 62 people with a nerve illness called CMT and found a change in it in 2 of them. TRPV4 makes a tiny door that lets calcium into cells, and earlier studies suggest these changes leave the door open too much. Even in one family, the illness was very different from person to person. We cannot be sure that some relatives carry the change without being ill, because nobody checked them.

   Why these ratings

   Relevance 5/5: Describes people with TRPV4-related CMT2C, including voice, breathing and hearing problems and variation within a family, and estimates how often TRPV4 explains CMT2 in a USA clinic cohort.

   Evidence 3/5: A reasonable genetic screen of a defined cohort, but only two people with TRPV4 variants were found, relatives were neither tested nor examined, and the cohort comes from one center.

   Funding and conflicts of interest

   Supported by NIH (NS078287, NS099623 to H.-X.D., and U54NS065712 to M.E.S.); M.E.S. is also supported by the MDA and CMTA. Conflict of interest: Dr. Michael E. Shy is a consultant for Alnylam, Inflectis, and Acceleron; the other authors declare no competing financial interests.

   PubMed 31468327 · DOI

9. #9Full paperGenetic screening study with two family reports and cell experiments

   TRPV4 mutations and cytotoxic hypercalcemia in axonal Charcot-Marie-Tooth neuropathies

   Klein CJ, Shi Y, Fecto F et al. · 2011 · Neurology

   Relevance 5/5Evidence 3/53 moderate · 4 minor problems

   In this study, TRPV4 changes were found in only 2 of 17 families with a CMT2C picture. In one family, the same change caused vocal cord weakness in one person but not in two others. The cell results support the idea that the faulty channel lets in too much calcium, but the effect of blocking it was shown only in cultured cells here.

   Full analysis: findings, problems and plain explanations

   The study

   Type

   Genetic screening study with two family reports and cell experiments

   Who or what

   53 unrelated probands: 17 families with a CMT2C picture (vocal cord and diaphragm involvement) and 36 people or families with CMT2 without these features, plus 800 control samples. Two families with TRPV4 changes are described in detail: an R232C family (a 43-year-old man, his mother and his sister) and the family of a 30-year-old man with a new R316H change not found in his parents. Lab work used two standard cell lines (HEK293 and HeLa) made to produce normal TRPV4 or the R232C, R316H or previously known R269H forms.

   What they did

   They read the coding part of the TRPV4 gene in all 53 probands and checked any changes in relatives and in 800 controls. They described the two families with changes, using examination, nerve conduction tests, breathing tests and direct views of the vocal cords. They then put normal or mutant TRPV4 into cultured cell lines to see where the protein went, how much calcium entered the cells and whether the cells died, with and without the channel blocker ruthenium red.

   What they found

   • TRPV4 changes were found in only 2 of the 17 families with a CMT2C picture, and in none of the 36 people or families with CMT2 without vocal cord or diaphragm involvement.

     “only 2 out of 17 screened CMT2C families had identified mutations in TRPV4 and no TRPV4 mutation was found in 36 screened undifferentiated CMT2 families”Discussion

   • A 30-year-old man with breathlessness, a weak voice and vocal cord paresis had a new change, R316H, that was not present in either parent (parentage was confirmed genetically); both parents were normal on examination.

     “R316H has not been reported before and is a de novo mutation.”Results

   • In the R232C family only the 43-year-old son had vocal cord paralysis. His mother and sister had no symptoms, but examination found distal weakness and nerve tests showed an axonal neuropathy.

     “R232C is from a CMT2C family, but only the proband has vocal cord involvement whereas other affected do not.”Discussion; Methods, CMT2C kindred 1

   • Both mutant channels reached the cell surface normally in both cell lines, so the changes did not seem to stop the channel from being made or delivered.

     “Both mutant TRPV4 proteins had normal subcellular localization in HEK293 and HeLa cells.”Abstract; Results

   • Cells with R232C or R316H had higher resting calcium than cells with normal TRPV4. After stimulation with a TRPV4 activator, R232C (and the known R269H) also gave a larger response, but R316H did not differ significantly.

     “TRPV4R232C and TRPV4R316H showed increased basal intracellular calcium concentration vs the wtTRPV4 (p < 0.0005)”Results; Figure 4 legend

   • In HeLa cells the mutant channels caused more cell death than normal TRPV4, and ruthenium red prevented this. In HEK293 cells the rise in cell death was not statistically significant.

     “All observed cell death could be suppressed by TRPV antagonist ruthenium red”Results; Figure 5

   What the authors concluded

   Changes in the ankyrin repeat part of TRPV4, including a new change that arose for the first time in one patient, cause axonal CMT2. People with the same change can have CMT2 with or without vocal cord weakness. The mutant channels let too much calcium into cells (gain of function) rather than too little, and blocking the channel prevented cell death in the dish. The authors see calcium channel blockade as an area worth investigating for treatment.

   Problems with the research

   Every study has limits. Listing them shows how much weight each finding can bear.

   • ModerateCell experiments far from people

     The channels were overproduced in two standard laboratory cell lines. No nerve cells, animals or patients were tested, and ruthenium red was only used in the dish. The results show what the mutant channels can do in these cells, not what happens in human nerves.

     “HEK293 or HeLa were transfected, incubated either with or without ruthenium red (20 μM) for 48 hours”Methods, Cell viability assay

   • ModerateCalcium and cell death measured in different cell lines

     Calcium was measured only in HEK293 cells, where cell death did not rise significantly. Cell death was significant only in HeLa cells. The Discussion still says all three mutations raise calcium 'with decreased cell viability', which joins results from two different systems.

     “We observed a trend toward increased cell death in mutant TRPV4-expressing HEK293 without reaching statistical significance”Results

   • ModerateVariable vocal cord involvement rests on one small family

     The claim that the same change can cause CMT2 with or without vocal cord weakness comes from three affected people in one family. The text says vocal cord and diaphragm involvement was absent in the mother and sister but does not say exactly which tests were used for them.

     “His mother and sister were asymptomatic, but distal muscle weakness was elicited and vocal cord and diaphragm involvement was absent.”Methods, CMT2C kindred 1

   • MinorTreatment idea is speculative

     The idea that calcium channel blockers deserve study draws on Parkinson's disease research and on ruthenium red in cultured cells. No medicine was tested in people with CMT2C. The authors frame it as an area for investigation, which is appropriate, but it is not evidence of a treatment.

     “Calcium channel inhibitors provide a rational new approach for investigation of TRPV4-mediated axonal neuropathies.”Discussion

   • MinorTitle wording may confuse

     Readers may take 'hypercalcemia' to mean high calcium in the blood. In this paper it means raised calcium inside cultured cells. Blood calcium in patients was not studied.

     “Intracellular hypercalcemia is a common pathway of diverse pathogenesis.”Discussion

   • MinorStatistics treat individual cells as samples

     The calcium results pool more than 3,000 cells per condition, and the number of independent repeat experiments for the calcium tests is not stated. Very small p-values can arise this way even if the experiment was run only a few times.

     “(n >3,000 cells for each condition)”Figure 4 legend

   • MinorSmall reporting inconsistencies

     The text gives p < 0.0005 for the calcium differences while the Figure 4 legend gives p < 0.005. The Results also describe all 53 probands as having vocal cord paralysis, although the 36 undifferentiated cases are described elsewhere as lacking vocal cord or diaphragm involvement.

     “a total of 53 unrelated CMT2 probands with vocal cord paralysis including subjects with diaphragmatic weakness (n = 17) and undifferentiated CMT2 (n = 36)”Results; Figure 4 legend; Introduction

   How “unaffected” relatives were checked

   Relatives were examined in person. The Methods say 'Family history or clinical involvements were confirmed by direct examination in kindred evaluation.' In the R232C family the mother and sister had no symptoms, but examination found distal weakness and nerve conduction tests showed axonal neuropathy, so they were counted as affected. In the R316H family the parents were 'unaffected by neuropathy as determined by normal clinical examinations'; the text does not say whether they also had nerve conduction tests, and it does not say how the man's 2 siblings and his children were judged 'normal'. The authors note that in their earlier R269H family, repeated examinations including specialized nerve conduction tests were needed to find very mildly affected people.

   Three ways to explain it

   Scientific

   Sequencing of TRPV4 in 53 axonal CMT probands identified two heterozygous ankyrin repeat domain variants, R232C (segregating in a kindred in which only the proband had vocal cord paresis) and a de novo R316H, in 2 of 17 CMT2C families and none of 36 undifferentiated CMT2 cases. In transfected cells both mutants trafficked normally to the plasma membrane, raised basal intracellular calcium (HEK293) and increased cell death in HeLa cells, which ruthenium red prevented. The authors interpret this as a constitutive gain of function causing calcium cytotoxicity, while allowing that other factors may contribute.

   Easier

   The team checked the TRPV4 gene in 53 people with an inherited nerve disease of the CMT2 type and found changes in only two families, both with the CMT2C form. In one family a man had a paralyzed vocal cord, but his mother and sister, who carried the same change and had mild nerve damage on testing, did not. In another man the change was new and not inherited from his parents. In cells grown in the lab, the faulty channel let in extra calcium, and in one cell type more cells died unless the channel was blocked with a chemical.

   Simple

   TRPV4 is like a door that lets calcium into cells. In cells grown in a dish, the faulty doors found in two families let in too much calcium, and in one kind of cell this harmed the cells unless the doors were blocked. People in one family with the same faulty door had different symptoms.

   Why these ratings

   Relevance 5/5: It reports people with CMT2C and TRPV4 changes, including how vocal cord involvement can differ within one family.

   Evidence 3/5: A small genetic series with careful clinical testing plus cell experiments, but only two families carried TRPV4 changes and the lab work used standard cell lines rather than nerve cells.

   Funding and conflicts of interest

   No overall study funding statement appears in the text. Most authors report no disclosures. Dr. Siddique lists NIH advisory and editorial roles, a patent on human alpha-tocopherol transport protein, and research support from the NIH and several ALS charities. Dr. Dyck receives research support from the NIH and the FDA. No company funding of this work is mentioned.

   PubMed 21288981 · DOI

10. #10Full paperTwo-family genetic report with structural modeling and cell experiments

    Novel mutations highlight the key role of the ankyrin repeat domain in TRPV4-mediated neuropathy

    Sullivan JM, Zimanyi CM, Aisenberg W et al. · 2015 · Neurology. Genetics

    Relevance 5/5Evidence 3/52 moderate · 4 minor problems

    This paper adds two more CMT2C-causing changes to the list, both in the same part of the TRPV4 protein where many CMT2C changes sit, and all four people studied had voice involvement. The lab results fit the idea of an overactive channel, but they come from cells in a dish, not from people.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    Two-family genetic report with structural modeling and cell experiments

    Who or what

    4 people from 2 unrelated families with the CMT2C phenotype (2 examined and tested in each family), seen at Stanford and the University of Washington. Lab work used HEK293T cells and mouse MN-1 (motor neuron-neuroblastoma fusion) cells made to produce normal or mutant TRPV4, and a computer model of the four-part human TRPV4 channel built on the rat TRPV1 structure.

    What they did

    The team examined the patients and ran CMT gene testing, finding two new TRPV4 changes at the same position. They placed these changes on a 3D model of the channel alongside known neuropathy and skeletal mutations. They then put the mutant channels into cells and measured where the protein went, calcium levels inside the cells and cell death, with and without a TRPV4-blocking drug (HC-067047).

    What they found

    • Two new single-letter changes were found at the same position, arginine 237, in the ankyrin repeat domain of TRPV4: p.Arg237Gly (c.709C>G) in family 1 and p.Arg237Leu (c.710G>T) in family 2.

      “we report 2 novel heterozygous missense mutations in the TRPV4-ARD convex face (p.Arg237Gly and p.Arg237Leu)”Abstract (Results); Results, 'Detection of novel mutations in TRPV4'

    • The 4 people had distal limb weakness, a hoarse voice and vocal fold weakness. 3 of 4 had nerve tests showing axonal neuropathy, laryngoscopy confirmed vocal fold paresis in 2, and none had skeletal abnormalities. In one person, vocal fold movement was 15% to 20% of normal.

      “In individual IV.1 of family 2, laryngoscopy revealed bilateral vocal fold paresis with vocal fold movement at 15%-20% of normal.”Results, 'Clinical findings'

    • Neither change was found in large population databases, the position is highly conserved across vertebrates, and prediction tools rated both changes as damaging.

      “Neither nucleotide substitution is present in the NHLBI Exome Sequencing Project Exome Variant Server, the 1000 Genomes Project, Exome Aggregation Consortium, or dbSNP.”Results, 'Detection of novel mutations in TRPV4'

    • Arginine 237 sits on the outer (convex) face of the ankyrin repeat domain, next to other CMT2C positions (Arg232 and Arg269). Six of the 10 exposed arginines on this face are now linked to neuropathy, and 8 of 12 known neuropathy mutations lie in this domain.

      “Six of the 10 exposed arginine residues present on the convex face of TRPV4-ARD have now been associated with neuropathy.”Discussion

    • In cells, both mutant channels reached the cell surface normally but raised resting calcium levels and caused more cell death, to a degree similar to that of a known CMT2C mutation (R269C). A TRPV4-blocking drug prevented both effects, pointing to an overactive channel (gain of function).

      “Taken together, the above results suggest that the p.Arg237Gly and p.Arg237Leu mutants traffic normally to the plasma membrane but exhibit a gain of function in channel activity.”Results, 'p.Arg237Gly and p.Arg237Leu mutations alter TRPV4 function'; Figure 3

    • In the computer model, the face of the domain where neuropathy mutations cluster points toward the inside of the cell near the membrane, where it could bind other proteins. Skeletal dysplasia mutations clustered elsewhere, near the channel gate and between subunits.

      “This orientation suggests that the ARD convex face would likely be readily accessible for protein-protein/protein-ligand interactions.”Results, 'Structural analyses localize Arg237 to the convex face of the ARD'

    What the authors concluded

    The two new mutations add to the evidence that the ankyrin repeat domain is central to TRPV4 neuropathy. Both appear to increase channel activity, and the authors suggest neuropathy may arise from altered interactions between the domain's convex face and as yet unidentified binding partners. They note that blocking the channel could be a treatment approach, where appropriate.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • ModerateFew people tested in each family

      Only two people per family were genetically tested, so the paper cannot show that the change travels with the disease through each family. The case that the changes cause CMT2C rests mainly on their absence from databases, prediction tools, conservation and cell experiments.

      “was also detected in the 2 examined individuals in family 2”Results, 'Detection of novel mutations in TRPV4'

    • MinorRelatives' status not explained

      The pedigrees mark relatives as affected, unaffected or 'unclear disease status', but only 4 people were examined and the paper does not say how others were classed. This matters only if the pedigrees are used to judge penetrance or variability; the paper itself makes no such claim.

      “white = unaffected, black = affected, gray = unclear disease status, *DNA collected”Figure 1B legend

    • ModerateCell experiments are far from human nerves

      The functional tests used cell lines (HEK293T and mouse MN-1 cells) made to produce the mutant channel, with effects measured over 24 hours. These show the channel is overactive in a dish, but cannot show how much this happens, or matters, in human nerves.

      “we performed a series of in vitro studies examining the effects of heterologous expression of these mutant channels.”Results, 'p.Arg237Gly and p.Arg237Leu mutations alter TRPV4 function'

    • MinorStructural conclusions rest on a model

      Only the ankyrin repeat domain had a measured structure; the rest of the channel was modeled on a related channel (rat TRPV1). The authors accept that the real channel may take other shapes, so the binding-partner idea is a hypothesis.

      “It is also likely that the channel adopts other conformations not yet seen experimentally”Discussion

    • MinorSmall number of cell-death repeats

      Calcium results came from more than 100 cells, but the cell-death results came from 3 independent experiments, which is a small number for judging how reliable the differences are.

      “data in E are averaged from 3 independent transfections”Figure 3 legend

    • MinorClinical table missing from the copy analyzed

      The text supplied for this analysis did not include the contents of the clinical summary table, so individual ages, severity grades and nerve test values could not be checked here.

    How “unaffected” relatives were checked

    Only 4 affected people (2 per family) were examined and genetically tested. The pedigrees mark other relatives as affected, unaffected or of unclear status ('white = unaffected, black = affected, gray = unclear disease status, *DNA collected', Figure 1B legend), and family 2's '3-generation history of limb weakness and hoarseness on the maternal side' is described from family history. The paper does not say how relatives' status was decided.

    Three ways to explain it

    Scientific

    Two novel heterozygous missense variants at the same residue, p.Arg237Gly and p.Arg237Leu, were identified in two unrelated CMT2C families and map to the convex face of the TRPV4 ankyrin repeat domain alongside other neuropathy-associated arginines. In transfected HEK293T and MN-1 cells both variants trafficked normally but raised basal intracellular Ca2+ and caused cytotoxicity that was abolished by the TRPV4 antagonist HC-067047, consistent with gain of function. A homology model suggests the convex face is accessible to as yet unidentified binding partners; segregation data are limited to two affected individuals per family.

    Easier

    The researchers found two new spelling changes in the TRPV4 gene, both at the same spot (position 237), in two families with CMT2C. This spot is in a part of the protein called the ankyrin repeat domain, where many other CMT2C changes are found. In cells grown in the lab, the changed channels let in too much calcium and more cells died, and a drug that blocks TRPV4 stopped this. Only four people were studied, and lab cells may not behave like human nerves.

    Simple

    TRPV4 is like a door that lets calcium into cells. In two families with CMT2C, the door had a change in the same small spot, and in lab cells that door let in too much calcium and harmed the cells. A blocker that shut the door helped the cells in the dish, but it was not tested in people.

    Why these ratings

    Relevance 5/5: Directly about families with CMT2C, describing their symptoms and new TRPV4 mutations, with lab work on the mechanism behind CMT2C.

    Evidence 3/5: Good laboratory work with appropriate comparison mutations and a blocking-drug control, but only four patients, limited family testing, and cell lines rather than human nerves.

    Funding and conflicts of interest

    Supported by NIH grant R21NS08757902 and MDA grant 277551 (both to C.J.S.), NIH grant R01GM081340 (R.G.) and a Jane Coffin Childs Memorial Fund postdoctoral fellowship (C.M.Z.); the disclosures give some of these numbers slightly differently (for example MDA 2777551). Several authors disclose patents, consultancies, travel funding and industry research support, mostly relating to other conditions such as spinal muscular atrophy, myotonic dystrophy and ataxia; none is described as relating to TRPV4.

    PubMed 27066566 · DOI

11. #11Full paperBrief report: family study with exome sequencing and cell experiments

    Exome sequencing identifies a novel TRPV4 mutation in a CMT2C family

    Landouré G, Sullivan JM, Johnson JO et al. · 2012 · Neurology

    Relevance 5/5Evidence 3/53 moderate · 1 minor problems

    This family shows the typical CMT2C picture of limb weakness with voice and breathing involvement, and one member had hearing loss. It also shows that in this family an older genetic test missed a TRPV4 change for technical reasons.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    Brief report: family study with exome sequencing and cell experiments

    Who or what

    One family with CMT2C (family 3 in the group's 2010 study): 9 family members evaluated, 3 affected and 6 unaffected; 200 controls. Cell experiments in transiently transfected HEK293 cells producing normal (WT), R186Q or R269C TRPV4 (n = 8 per group for calcium, n = 3 per group for cell death).

    What they did

    Earlier Sanger sequencing had not found a TRPV4 change in this family. The team read the protein-coding parts of all genes (exome sequencing) in one affected person, found a new TRPV4 change, and checked it with redesigned Sanger primers in all 9 relatives and in 200 controls. They then made lab-grown cells produce the changed channel to see whether it raised calcium levels and killed cells.

    What they found

    • Three of the 9 relatives had CMT2C features: progressive distal limb weakness and wasting, a hoarse voice and noisy breathing (stridor) on exertion. Nerve tests showed an axonal neuropathy involving the phrenic nerve, and laryngoscopy showed reduced vocal fold movement.

      “Nerve conduction studies confirmed an axonal neuropathy with phrenic nerve involvement; laryngoscopy showed reductions in vocal fold opening and closing.”Results

    • Two affected people had scoliosis but no skeletal dysplasia on X-rays, and one had hearing loss in both ears.

      “Two individuals had scoliosis, but skeletal dysplasia was not evident on bone radiographs. One individual had bilateral sensorineural hearing loss.”Results

    • A new TRPV4 change, c.557G>A (R186Q), was present in all 3 affected relatives and absent from the 6 unaffected relatives and from 200 controls. No significant variants were found in other CMT genes.

      “revealed the c.557G>A sequence change in all 3 individuals with CMT2C, but not in the 6 unaffected individuals (figure 1B), nor in 200 controls.”Results

    • The earlier Sanger test missed the change because its primers sat on common DNA variants (SNPs), which probably meant only the normal copy of the gene was read.

      “A review of the Sanger sequencing primers used to amplify this region of TRPV4 revealed that the forward and reverse primers each contained a single nucleotide polymorphism (SNP).”Results

    • R186 sits on the convex face of the ankyrin repeat domain, where other neuropathy-causing changes lie; skeletal dysplasia changes in this domain lie on the concave face.

      “R186 is an exposed arginine residue situated on the convex face of the TRPV4 ankyrin repeat domain (ARD)”Results; Figure 1D legend

    • In cells, R186Q gave only a slight rise in calcium at 24 hours but more cell death than normal TRPV4 at 36 hours; R269C had larger effects. The TRP channel blocker ruthenium red prevented both effects.

      “Approximately 10% of cells expressing R186Q-TRPV4 and 15% of cells expressing R269C-TRPV4 compared to approximately 5% of cells expressing WT-TRPV4 were dead at 36 hours post-transfection”Figure 1F legend

    What the authors concluded

    The authors conclude that R186Q is a new TRPV4 mutation causing CMT2C without skeletal dysplasia in this family, that it probably increases the channel's constant (constitutive) activity, and that its place on the convex face of the ankyrin repeat domain supports the importance of that region for nerves. They also say exome sequencing can catch mutations that Sanger sequencing misses.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • ModerateSmall family

      Only 3 affected and 6 unaffected relatives were tested. Segregation in a family this size gives limited support on its own, although absence in 200 controls and the cell results add weight.

      “We evaluated 9 family members (figure 1A), 3 of whom exhibited features consistent with the CMT2C phenotype”Results

    • ModerateCell model far from human nerves

      The cell tests used HEK293 cells grown in a dish and made to produce the channel from added DNA (transient transfection), not nerve cells from patients. They show what the change can do in this system, not how it behaves in people's nerves.

      “Calcium imaging and cell death assays were performed using transiently transfected HEK293 cells as previously described.”Methods

    • ModerateWeak calcium effect and small cell experiments

      The calcium rise with R186Q was only modest and, unlike R269C, is not marked as significant. The conclusion that R186Q makes the channel overactive rests mainly on the cell death result (3 samples per group) and the blocker experiment. The paper used unpaired t tests and does not mention correcting for several comparisons.

      “Basal calcium levels are modestly increased in HEK293 cells expressing R186Q-TRPV4 and significantly increased in cells expressing R269C-TRPV4”Figure 1E legend

    • MinorAssessment of unaffected relatives and individual details not in the main text

      The main text does not say whether the 6 unaffected relatives had nerve conduction studies or laryngoscopy, and it does not say which person had hearing loss or scoliosis; these details are in an online table (table e-1) not included here. Because none of the unaffected relatives carried the change, this does not affect any penetrance claim, but readers cannot check the clinical labels.

    How “unaffected” relatives were checked

    The paper says 9 family members were 'evaluated' and that two authors 'examined patients', but the main text does not say whether the 6 unaffected relatives had nerve conduction studies or laryngoscopy; individual details are in an online table (table e-1) not included here. All 6 unaffected relatives tested negative for the change: 'not in the 6 unaffected individuals (figure 1B), nor in 200 controls.'

    Three ways to explain it

    Scientific

    Exome sequencing in a CMT2C kindred previously negative on Sanger sequencing identified a novel heterozygous TRPV4 missense variant, c.557G>A (R186Q), segregating with disease (3 affected carriers, 6 unaffected non-carriers) and absent from 200 controls; SNPs under the original primers probably caused monoallelic amplification of the wild-type allele. R186 lies on the convex face of the ankyrin repeat domain with other neuropathy mutations. In transfected HEK293 cells, R186Q produced a modest basal calcium rise and increased cell death, both blocked by ruthenium red, which the authors interpret as increased constitutive (gain-of-function) activity.

    Easier

    In one family, three people had CMT2C, with weak hands and feet, a hoarse voice and noisy breathing. An older genetic test had missed the cause because the test's starting points sat on common, harmless DNA differences. A newer method that reads all genes found a new change in the TRPV4 gene in all three affected people, and in none of the six unaffected relatives or 200 unrelated people. In lab-grown cells, the changed channel let in a little more calcium and killed more cells, which suggests it is overactive.

    Simple

    TRPV4 is like a door that lets calcium into cells. Everyone in this family with the illness had the same tiny spelling change in the TRPV4 gene. In lab cells, the changed door seemed to let in a bit too much calcium and harmed the cells. The first test missed the change because it only read the healthy copy of the gene, like reading only one of two copies of a page.

    Why these ratings

    Relevance 5/5: Directly about a CMT2C family, with voice, breathing, hearing and skeletal findings, plus a practical point that a standard genetic test missed the TRPV4 change.

    Evidence 3/5: Good genetic evidence for a small family (segregation, absence in 200 controls, conserved residue) with supportive but limited cell work; individual clinical details are in an online table not included here.

    Funding and conflicts of interest

    Supported by intramural funds from the National Institute of Neurological Disorders and Stroke at NIH, NIH grant R01NS062869 to C.J.S., the intramural research program of the NIA (Z01-AG000949-02), and NIH grant R01GM081340 to R.G. The authors report no disclosures relevant to the manuscript.

    PubMed 22675077 · DOI

12. #12Full paperFamily study (two families) with a small gene screen

    CMT2C with vocal cord paresis associated with short stature and mutations in the TRPV4 gene

    Chen DH, Sul Y, Weiss M et al. · 2010 · Neurology

    Relevance 5/5Evidence 3/54 moderate · 4 minor problems

    In these two families, people with the same TRPV4 change differed a lot, from no vocal cord problems to needing a permanent tracheotomy, and some were shorter than average. Two families are not enough to show whether short stature is a regular part of CMT2C.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    Family study (two families) with a small gene screen

    Who or what

    Two multigenerational families with CMT2C seen at the University of Washington, Seattle. Family 1 carried a new change, S542Y (at least four affected women and two affected men are described). Family 2, first reported in 1994, carried R315W (three affected people across three generations are described: the 47-year-old index woman, her daughter and her father). The study also included 20 unrelated people with familial CMT (16 CMT2 and 4 CMT1) without vocal cord weakness, and 200 controls. The clinical table and pedigree drawings were images not included in the text used for this analysis.

    What they did

    Family members were examined in a neurogenetics clinic, with nerve conduction tests and EMG, and vocal cord examinations in an otolaryngology (ear, nose and throat) clinic. The TRPV4 gene was sequenced in affected people, and the changes found were checked in other relatives and in 200 controls. Heights were compared with US reference data, and the TRPV4 gene was also screened in 20 other people with familial CMT.

    What they found

    • Both families had a motor-predominant axonal neuropathy. Vocal cord weakness ranged from none to severe, and two people needed a permanent tracheotomy.

      “The vocal cord paresis varied from absent to severe, requiring permanent tracheotomy in 2 subjects.”Abstract, Results

    • In family 1 the four affected women examined averaged 142.2 cm tall, compared with a reference mean of 164.8 cm for non-Hispanic white American women, and the two affected men were also more than 2 standard deviations (SD) below average. Their hands, feet and limbs were in proportion to their height.

      “The 4 affected and examined women in this family have an average height of 4 feet 8 inches (142.2 cm), which is more than 2 SD below the mean”Results, Family 1

    • The new S542Y change (family 1) and R315W (family 2) were found in affected members, not in the available unaffected relatives, and not in 200 controls.

      “These 2 nucleotide changes were not detected in 200 controls (400 chromosomes).”Results, Genetic studies

    • In the index woman of family 2, nerve tests 20 years apart showed further loss of motor responses and complete loss of sensory responses that had been preserved at age 27.

      “Twenty years later, there was further reduction of CMAP amplitudes and complete loss of previously preserved SNAPs”Results, Family 2

    • None of 20 people with familial CMT who had no vocal cord weakness carried a disease-causing TRPV4 change.

      “No pathologic nucleotide changes were identified in those cases.”Results, Genetic studies

    • Some people had other features: a third cranial nerve problem and sleep apnea are reported in each family, and one woman in family 1, who had reduced phrenic nerve responses and poor diaphragm movement, died of respiratory problems and heart failure at 44.

      “Third cranial nerve involvement and sleep apnea occurred in one subject in each family.”Abstract, Results; Results, Family 1

    What the authors concluded

    CMT2C with axonal neuropathy, vocal cord weakness and short stature is a distinct syndrome caused by TRPV4 changes, and TRPV4 changes can affect bone, nerve or both. TRPV4 changes are not a common cause of CMT, but the authors say neurologists should think of TRPV4 when a person has axonal neuropathy with vocal cord weakness, especially with short stature, and that families with TRPV4-related bone dysplasia should be checked for subtle neuropathy.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • ModerateShort stature link rests on few people

      The height comparison covers a handful of affected people, one unaffected man of normal height and one unaffected woman who was short but did not carry the change. In family 2, heights of 152 cm and 156 cm are given without comparison to reference values. The authors themselves say there may be more than one cause of short stature in family 1.

      “However, the short stature of the unaffected 95-year-old woman in family 1 suggests there could be more than one type of short stature in this family.”Discussion

    • ModerateUnique syndrome claimed from two families

      Calling CMT2C with short stature a unique syndrome is a strong label for two families, one of which had 'less remarkable' short stature according to the abstract.

      “CMT2C with axonal neuropathy, vocal cord paresis, and short stature is a unique syndrome associated with mutations in the TRPV4 gene.”Abstract, Conclusion

    • ModerateNegative screen of 20 people over-interpreted

      No TRPV4 changes were found in 20 people, 4 of whom had the demyelinating type CMT1, but this is too small a sample to show that TRPV4 changes are unlikely in CMT without vocal cord weakness.

      “document that pathologic TRPV4 mutations are unlikely to be found in CMT patients in the absence of a personal or family history of vocal cord paresis”Discussion

    • ModerateHow unaffected relatives were judged is not described

      The paper says unaffected relatives had the normal gene, but not whether they were examined or had nerve tests. Without this, mildly affected relatives could be missed, which matters for any conclusion about how variable the condition is. One ancestor (I-2) was described only from family information.

      “available unaffected relatives had the wild-type sequence”Results, Genetic studies

    • MinorA relative with a different nerve test pattern and unclear genotype

      The family 2 index woman's father had a neuropathy starting around age 45 with mildly slowed nerve conduction (mixed axonal and demyelinating), unlike the axonal pattern elsewhere, and no voice or breathing problems. The text does not say whether he carried R315W (the pedigree, which shows genotypes, was not available here), so it is unclear whether his neuropathy is part of the same condition.

      “Electrophysiologic testing revealed a mixed axonal/demyelinating neuropathy with reduced CMAP amplitudes and mildly reduced NCVs.”Results, Family 2

    • MinorBone cause of short stature presumed, not shown

      Spine X-rays are reported for one woman, and the paper does not say who had the X-rays of fingers and long bones. The link to a mild bone dysplasia is described by the authors as a presumption.

      “The short stature related to mutant TRPV4 is presumed to be on the basis of a slight reduction in the height of all vertebral bodies”Discussion

    • MinorEye nerve finding not confirmed

      In family 2 a partial third nerve deficit is described with a congenital squint. For family 1 the only eye finding described in the text is possible Adie's pupils in one person, which was not confirmed by testing, yet the abstract reports third nerve involvement in each family.

      “IV-5 had large pupils that were slow to react to light and were more reactive to near accommodation, suggesting Adie's pupils. No pharmacologic testing was done.”Results, Family 1

    • MinorGenetic evidence for S542Y limited to family and controls

      The new S542Y change tracked with disease in family 1 and was absent from 200 controls, which is useful evidence. However, it was not tested in the lab, and the text does not say whether other neuropathy genes were checked in these families.

    How “unaffected” relatives were checked

    Not clearly described. Affected members were examined in the University of Washington Neurogenetics Clinic, with nerve conduction tests, EMG in three people in family 1, and vocal cord examination in some. For relatives classed as unaffected, the text says only that 'available unaffected relatives had the wild-type sequence'; it does not say whether they were examined or had nerve tests. Ancestor I-2 was not examined ('Little is known about family member I-2'). In family 1, IV-2 was counted as affected even though muscle weakness was absent (a raspy voice was noted). The clinical table and pedigrees, which may hold more detail, were not available in the text used here.

    Three ways to explain it

    Scientific

    Two autosomal dominant CMT2C kindreds carried heterozygous TRPV4 variants, the novel S542Y (transmembrane domain) and R315W, which were present in affected members, absent in available unaffected relatives and absent from 200 controls. Both showed motor-predominant axonal neuropathy with variable vocal cord paresis, from absent to tracheotomy-dependent, and family 1 had short stature more than 2 SD below population means, suggesting overlap with TRPV4 skeletal dysplasia. No TRPV4 variants were found in 20 familial CMT probands without vocal cord paresis.

    Easier

    The researchers studied two families with CMT2C, an inherited nerve condition that weakens muscles and can affect the vocal cords. Each family had its own change in the TRPV4 gene. Across the two families, vocal cord problems ranged from none to so severe that a permanent breathing opening in the neck (tracheotomy) was needed. In one family the affected women were on average much shorter than typical American women, which suggests the gene change may also affect bone growth, though this is based on few people.

    Simple

    Two families had a nerve illness called CMT2C, caused by changes in a gene called TRPV4. Some family members had weak voices or trouble breathing and others did not, and in one family many were quite short. The doctors think the gene might affect bones as well as nerves, but two families are not enough to be sure.

    Why these ratings

    Relevance 5/5: It describes two CMT2C families in detail, including vocal cord involvement, progression over time and short stature.

    Evidence 3/5: A careful clinical and genetic study, but of only two families, and the short stature link and the gene screen rest on small numbers.

    Funding and conflicts of interest

    Study funding: Department of Veterans Affairs, the Seattle Institute for Biomedical and Clinical Research, and the NIH (R01 NS069719). Several authors report links with Athena Diagnostics, Inc. (license fees for diagnostic patents relating to SCA14 and CMT1C, speaker honoraria or speakers' bureau roles). Dr. Weiss also reports consulting for Genzyme Corporation and honoraria from Talecris Biotherapeutics, and Dr. Bird serves on scientific advisory boards including the CMT Association. The disclosures listed do not mention TRPV4.

    PubMed 21115951 · DOI

13. #13Full paper (scan)Family study (single pedigree) with clinical examination, nerve conduction studies, EMG and muscle MRI

    Reduced penetrance in hereditary motor neuropathy caused by TRPV4 Arg269Cys mutation

    Berciano J, Baets J, Gallardo E et al. · 2011 · Journal of neurology

    Relevance 5/5Evidence 3/52 moderate · 5 minor problems

    In this family, people with the same TRPV4 change ranged from a severely affected child to a 70-year-old relative with no detectable signs on examination, nerve tests or leg MRI. It shows that carrying a TRPV4 change does not always mean having symptoms, but one family cannot say how often this happens.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    Family study (single pedigree) with clinical examination, nerve conduction studies, EMG and muscle MRI

    Who or what

    One three-generation family (pedigree CMT858), studied by a team in Santander, Spain, carrying the TRPV4 Arg269Cys change (c.805C>T). Two affected members were studied in detail: the proband, a woman aged 44, and her daughter aged 7. Seven other relatives were examined (aged 6 to 79 in 2007). Nine family members had TRPV4 tested; three relatives (aged 70, 40 and 9 in 2010) carried the change without signs of disease.

    What they did

    The team examined family members in 2007 and re-examined the three healthy carriers in April 2010, after the genetic results were known. They did nerve conduction studies and needle EMG, and MRI of foot and lower-leg muscles (plus thigh and shoulder MRI in the proband). The genetic testing of TRPV4 was carried out and reported in an earlier paper by the same group.

    What they found

    • The same TRPV4 change caused very different illness in a mother and daughter. The mother had moderate weakness in a pattern affecting the face, shoulder blades and lower legs, while her daughter was born with stiff joints and had severe, early-onset weakness with voice-box problems.

      “The proband presented with a moderate facio-scapulo-peroneal syndrome, whereas her symptomatic daughter suffered from severe congenital spinal muscular atrophy with arthrogryposis, laryngomalacia, and vocal cord paresis.”Abstract, p. 1413

    • Three relatives carrying the change (aged 70, 40 and 9) had no signs of nerve disease when examined, and their nerve tests and lower-leg MRI scans were normal.

      “In all three mutation carriers results from clinical and electrophysiological examination, and MRI of foot and lower-leg musculature were normal.”Abstract, p. 1413

    • In the two affected members, nerve tests showed loss of motor nerve fibers (low muscle response sizes with normal conduction speeds), worse in the legs than the arms, with normal sensory nerve results.

      “There was amplitude reduction of compound muscle action potentials (CMAP) in spinal accessorius, ulnar, peroneal and tibial nerves with normal motor nerve conduction velocities (MCV).”Results, Electrophysiological findings, p. 1416

    • MRI of the proband showed widespread fatty replacement of the lower-leg muscles, only mild change in the thigh (vastus lateralis), and fatty wasting of the serratus muscles that hold the shoulder blades against the chest.

      “The outstanding finding on T1-weighted MRI images of the shoulder-girdle was bilateral fatty atrophy of serratus muscles (Fig. 4).”Results, MRI findings, p. 1416

    • The daughter needed a tracheotomy at 18 months for paralyzed vocal cords and breathing difficulty; by age 5 vocal cord movement had improved enough for it to be removed. A second tracheotomy was needed at age 6, when she could not be weaned off a ventilator after a serious illness, and was later removed again.

      “A permanent tracheotomy was performed after being diagnosed of bilateral vocal cord paresis in abduction.”Results, Clinical findings: patient III-5, p. 1415

    What the authors concluded

    The authors conclude that non-penetrance (carrying the change without any signs of disease) may be a built-in feature of TRPV4 neuropathies. They say that this, together with the wide variation in severity, should be taken into account in genetic counseling, and that TRPV4 families could help researchers look for modifier genes.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • ModeratePenetrance conclusion rests on three carriers, one aged 9

      The claim that non-penetrance may be an integral feature of TRPV4 disease is drawn from one family. The authors themselves note that only two of the three carriers were older than the average age of onset, so the 9-year-old could still develop signs. One family cannot show how often carriers stay unaffected.

      “In our pedigree we found three gene mutation carriers (Arg269Cys), of which two at the age beyond the average age of disease onset and not showing any clinical signs of the disease.”Discussion, p. 1419

    • ModerateOldest carrier had two below-normal leg nerve values described as normal

      In Table 1, the 70-year-old carrier (case I-2) had muscle response sizes of 1.4 mV (extensor digitorum brevis, normal 2 or more) and 4.2 mV (tibialis anterior, normal 5 or more). The authors mark these as borderline, while the text says the carriers' nerve studies were normal. These values may reflect age, but they could also be very mild involvement, which matters when the paper's main claim is that this person is entirely unaffected.

      “Asterisks indicate borderline values at age 70 years.”Table 1 footnote, p. 1417; text on p. 1416

    • MinorCarriers were not checked in every area the disease affected

      Carriers had a full examination, nerve conduction studies and lower-leg MRI, which is thorough. But EMG was done in only two muscles, MRI did not include the shoulders (the proband's most striking MRI finding was in the serratus muscles), and no laryngoscopy of the carriers is reported. Very subtle involvement elsewhere cannot be ruled out.

      “In cases I-2, II-5 and III-4, EMG of tibialis anterior and trapezius muscles was normal.”Results, Electrophysiological findings, p. 1416

    • MinorOne relative classed as affected from family history only

      Case I-3, in the oldest generation, is shown as affected in the pedigree but was not examined and has no genotype shown. Her status is uncertain. This does not change the main findings but the family tree should be read with it in mind.

      “Case I-3 was affected according to the history but no clinical record was available.”Results, Clinical findings: other members of the pedigree, p. 1416

    • Minor'Pure motor' label despite a mild sensory sign

      The abstract calls the neuropathy pure motor, and sensory nerve tests were normal. However, the proband's examination found mild loss of vibration sense in a stocking and glove pattern, and several sensory tests were not done in the daughter. This is a small point but it means sensory involvement was not fully excluded.

      “Mild stocking and glove vibration hypoesthesia was noted.”Results, Clinical findings: patient II-3, p. 1415

    • MinorOnly TRPV4 analyzed; modifiers proposed but not studied

      The paper reports sequencing of TRPV4 (and earlier SMN testing and karyotype in the patients) but no testing of other neuropathy genes or possible modifier genes. The suggestion that modifier genes or epigenetic factors explain the differences is a reasonable idea, not a finding.

      “With informed consent, the coding region and exon-intron boundaries of TRPV4 gene were analyzed as reported elsewhere [31].”Patients and methods, Molecular genetic analysis, p. 1414

    • MinorSmall wording inconsistencies

      The daughter's airway problem is called laryngomalacia in the results but tracheomalacia in the discussion. There are also typing slips (TPRV4, TRPV) and the pedigree legend says 'codon 805' for what the genetics section calls c.805C>T. None of these change the conclusions.

      “In our case III-5 we noted a combination of tracheomalacia and regressive vocal cord paresis”Discussion, p. 1418

    How “unaffected” relatives were checked

    Relatives were examined in person by the study team in 2007, and the three carriers were re-examined in April 2010 after genetic testing, specifically looking for early signs such as pes cavus, toe clawing, difficulty heel walking, absent ankle reflexes and small foot or hand muscles. Carriers also had nerve conduction studies, EMG of tibialis anterior and trapezius, and MRI of foot and lower-leg muscles. Quote: 'In April 2010, following molecular genetic findings (see below), we re-examined all three TRPV4 gene mutation carriers ... and again we found no signs of polyneuropathy.' One relative (I-3) was classed as affected from family history only, without examination.

    Three ways to explain it

    Scientific

    In one pedigree with the heterozygous TRPV4 c.805C>T (p.Arg269Cys) variant, an ankyrin-region change, the two affected members showed strikingly different phenotypes: an adult facio-scapulo-peroneal motor neuropathy and a congenital distal SMA with arthrogryposis and vocal cord paresis, with electrophysiology indicating a length-dependent motor axonopathy. Three carriers aged 9, 40 and 70 had normal examination, nerve conduction, EMG and lower-leg MRI, which the authors interpret as non-penetrance. The evidence comes from a single family, and the authors note only two carriers were beyond the average age of onset.

    Easier

    This study looked at one family with a change in the TRPV4 gene called Arg269Cys. A mother had weakness in her face, shoulder blades and lower legs, while her daughter was born with stiff joints and had serious breathing and voice-box problems. Three other relatives carried the same change but showed no signs of nerve disease, even on careful nerve tests and muscle scans. The authors call this reduced penetrance, meaning some people with the change do not develop the condition, but this is based on one family.

    Simple

    TRPV4 is like a door that lets calcium into cells. In this family, five people had the same change in the instructions for the door, but only two were ill and three seemed completely well when doctors checked them. So the same gene change can affect people in very different ways.

    Why these ratings

    Relevance 5/5: A detailed study of a family with a TRPV4 ankyrin-region change (Arg269Cys) that directly addresses penetrance and variability, which matter to people with CMT2C and their relatives.

    Evidence 3/5: Carriers were examined twice and had nerve tests and MRI, which is careful work, but it is one family with three non-penetrant carriers, one of them a child.

    Funding and conflicts of interest

    Supported by CIBERNED and FIS grant PI07/132E, the University of Antwerp, the Fund for Scientific Research (FWO-Flanders), the Medical Foundation Queen Elisabeth, the Association Belge contre les Maladies Neuromusculaires, the Interuniversity Attraction Poles P6/43 program of the Belgian Federal Science Policy Office and the Methusalem excellence grant of the Flemish Government; two authors held FWO-Flanders PhD fellowships. Conflict of interest: none.

    PubMed 21336783 · DOI

14. #14Full paperMulticenter genetic screening study with a clinical description of eight families (family study)

    Dominant mutations in the cation channel gene transient receptor potential vanilloid 4 cause an unusual spectrum of neuropathies

    Zimoń M, Baets J, Auer-Grumbach M et al. · 2010 · Brain: a journal of neurology

    Relevance 5/5Evidence 3/51 major · 2 moderate · 3 minor problems

    This paper showed that TRPV4 changes can cause very different illnesses, even within one family, from problems present at birth to weakness starting in adulthood, often with vocal fold involvement. It also reported relatives who carried the change without known symptoms, but the paper does not describe how they were checked, so it remains uncertain how often carriers stay unaffected.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    Multicenter genetic screening study with a clinical description of eight families (family study)

    Who or what

    145 unrelated index patients with various hereditary neuropathies, referred by neurologists from European countries and the USA. Of these, 83 were chosen because they had unusual extra features (such as congenital onset, scapular or proximal weakness, contractures, vocal fold paralysis, breathing problems or pronounced scoliosis) and 62 had axonal HMSN2 without extra features. TRPV4 mutations were found in eight families from the UK, Switzerland, Spain, the USA, Italy and Croatia. Clinical data cover 27 affected people (23 with a genetically confirmed mutation and 4 affected by history only); ages at last examination in Table 3 range from 4 to 71 years. Six mutation carriers were described as unaffected. 280 control individuals of European descent were tested for two of the mutations.

    What they did

    The team sequenced all protein-coding parts of the TRPV4 gene in each index patient, confirmed any changes with a second test, and checked whether the changes tracked with disease in each family. They used DNA markers to see whether families with the same change shared a common ancestor, and paternity testing to confirm that one change was new (de novo). They then described the symptoms, nerve conduction results and, in one family, laryngoscopy (camera examination of the voice box) of affected relatives, and mapped the changed amino acids onto a 3D structure of the chicken TRPV4 ankyrin repeat domain.

    What they found

    • Screening 145 unrelated patients found five different TRPV4 changes in eight families. In seven families the change was passed down in a dominant pattern. In one isolated Croatian patient the change (Val620Ile, previously reported in a bone condition called brachyolmia) was not inherited (de novo).

      “We performed DNA sequencing of the coding regions of transient receptor potential vanilloid 4 in a cohort of 145 patients with various types of hereditary neuropathy and identified five different heterozygous missense mutations in eight unrelated families.”Abstract; Results, Genetic findings

    • One change, Arg232Cys, had not been reported before. It was found in three families (two of them probably distantly related, as they share the same haplotype) and was absent from 280 controls. Four of the five changes affect arginine building blocks that are strictly conserved across species and sit on the same outer (convex), positively charged surface of the ankyrin repeat domain.

      “The mutant residues Arg232, Arg269 and Arg315, and previously identified Arg316 (Auer-Grumbach et al., 2010; Deng et al., 2010) are situated on the convex surface of ARD in the loops of finger 2, 3 and 4, respectively”Results, Genetic findings; Table 2; Figure 3

    • The illness varied strikingly between and within families, from congenital distal SMA with club feet or arthrogryposis, to scapuloperoneal SMA, HMSN2C, distal motor neuropathy with vocal fold paresis and plain axonal HMSN2. Age of onset ranged from birth to late adulthood. Most people had a pure or mainly motor neuropathy, and nerve conduction studies showed an axonal pattern in all those tested.

      “The clinical phenotype of the patients described in the current study is highly variable both between and within families. The majority of patients seem to fall in the category of pure or predominantly motor nerve disorders.”Results, Clinical findings; Tables 3 and 4

    • Vocal fold problems were seen in five of the eight families, often affecting only one side or the two sides unequally, with the left side more affected. Severity ranged from no symptoms to breathing failure needing a tracheostomy. In the screening cohort, 5 of the 14 patients who first presented with vocal fold paralysis had a TRPV4 mutation (36%).

      “In five out of eight families in our series, vocal fold dysfunction was observed, often unilateral or asymmetric with the left more affected than the right vocal fold”Discussion; Tables 3 and 5

    • Six carriers were described as unaffected: three in the Spanish family CMT-858 (aged 79, 36 and 6), one in the UK family CMT-455 (aged 69) and two in family F1, taken from an earlier report. The authors read this as reduced penetrance, meaning not everyone who carries the change becomes ill.

      “Reduced penetrance was observed in six asymptomatic mutation carriers, for the Arg232Cys mutation in family CMT-455 and for the Arg269Cys mutation in families CMT-858 and F1”Results, Genetic findings; Discussion

    • TRPV4 mutations were found mainly in people with extra features: 7 of 83 (8.4%) in the group with complex neuropathies, 1 of 62 (1.6%) in the group with plain axonal HMSN2, and none of the 26 patients with demyelinating neuropathy (HMSN1).

      “The mutation frequency can therefore be estimated at 8.4% in the subgroup of the complex neuropathies (7 out of 83) and only 1.6% in the subgroup of the HMSN2 neuropathies without additional features (1 out of 62).”Discussion

    What the authors concluded

    The authors conclude that dominant TRPV4 mutations cause an exceptionally broad spectrum of mostly motor, axonal neuropathies, from congenital distal SMA to adult-onset HMSN2C, and that the separate disease names are really parts of one spectrum. They found no clear link between a particular mutation and a particular form of the disease. They consider reduced penetrance relatively common and not limited to one mutation. They propose aiming TRPV4 testing at people with a pure motor, or motor and sensory, axonal neuropathy plus vocal fold paralysis, proximal arm weakness, pronounced scoliosis or contractures, and say variability and reduced penetrance should be taken into account in genetic counseling. They leave the disease mechanism open.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • MajorHow 'unaffected' carriers were assessed is not described

      The penetrance claim depends on six carriers being truly unaffected. For the four new carriers the paper says only that they were 'clinically unaffected' at a given age. It does not say who examined them, what the examination covered, or whether they had nerve conduction studies, and none of the four appears in the nerve conduction table (Table 4). The other two carriers are taken from an earlier paper. The paper's own tables show that some features can be present without symptoms (vocal fold paresis is marked 'asymptomatic' in two affected people), so mild or hidden signs in these carriers cannot be ruled out from what is reported.

      “These individuals (CMT-858.02, CMT-858.07 and CMT-858.08) were clinically unaffected at the age of 79, 36 and 6 years, respectively.”Results, Genetic findings; Tables 3 and 4

    • ModeratePenetrance conclusion rests on few carriers, some of them young

      Six carriers in three families, involving two mutations, is a small basis for saying reduced penetrance is 'relatively common' and 'not mutation-specific'. Onset in this series reached late adulthood (one affected person, F1 III.2, first had symptoms at 44 years), so carriers aged 6 and 36 could still develop signs later. The two older carriers (aged 79 and 69) give stronger support. The paper does not estimate how often carriers stay unaffected.

      “In TRPV4-associated neuropathy, however, reduced penetrance seems to be a relatively common feature that is not mutation-specific.”Discussion; Table 3

    • ModerateSelected referral cohort, so the frequencies describe this group only

      Patients were referred by neurologists experienced in rare neuromuscular disorders, and 83 of the 145 were chosen because they had the unusual features already linked to TRPV4. The cohort also included family F1, which was already known to carry a TRPV4 mutation. Figures such as 8.4%, 1.6% and 36% (5 of 14 patients with vocal fold paralysis) therefore describe this selected group, rest on small numbers, and do not show how common TRPV4 changes are among all people with CMT2 or with vocal fold paralysis. The statement that TRPV4 is 'likely to be a major gene' for neuropathy with vocal fold paralysis rests on these 5 patients.

      “For a subgroup of 83 patients, criteria for inclusion were based on the unusual clinical characteristics of the phenotypes recently described in families with TRPV4 mutations”Patients and methods, Patient cohort; Results, Genetic findings; Discussion

    • MinorClinical data are one-time and not standardized

      Patients were referred from several countries, and findings are reported at one time point (the last examination). The paper does not describe a common examination protocol or whether examiners knew the genetic result. Detailed laryngoscopy was done only in family F1, so vocal fold involvement in the other families may be under-recorded. For 26 of the 83 patients in the first group there was no detailed electrophysiology, and many entries in the nerve conduction table were not measured.

      “Detailed laryngoscopy assessment of vocal fold function was performed in the F1 family showing that the vocal fold involvement was often asymmetric (Table 5).”Results, Clinical findings; Tables 3 to 5

    • MinorLimited genetic work-up beyond TRPV4

      Only TRPV4 was sequenced, and the paper reports testing of other neuropathy genes only for the de novo patient PN-1394.1. In the families, the TRPV4 change was present in all available affected relatives, which supports it as the cause, although some families are small (two affected people each in CMT-1100 and CMT-858). The new Arg232Cys change is supported by segregation, absence from 280 European controls and conservation, but was not tested in the lab. The suggestion that Val620Ile, a known brachyolmia change, causes a neuropathy and skeletal overlap rests on one patient, and the authors present it cautiously.

      “Genetic testing for other more common causes of hereditary neuropathy (CMT1A duplication and point mutations in PMP22, MPZ, GJB1 and SH3TC2) was unremarkable in this patient.”Results, Clinical findings; Table 1; Discussion

    • MinorInconsistencies between text and tables

      The text gives the Val620Ile change as c.1958G>A, while Table 1 gives c.1858G>A. The text says 23 of the 27 affected people had a genetically confirmed mutation, but Table 3, titled as patients with proven mutations, lists 25 people, and Table 4 adds CMT-455.08, who is not in Table 3. Table 3 lists 'F1 V8' (age 18) while Table 4 lists 'F1 IV8' (age 19), and one Table 4 row has no patient label in the text version available (this may be a formatting loss). None of this changes the main conclusions, but it makes some individual data hard to check.

      “Finally the Val620Ile (c.1958G>A) mutation in exon 12”Results, Genetic findings; Tables 1, 3 and 4

    How “unaffected” relatives were checked

    Not described in detail. The four new carriers are reported only as 'clinically unaffected' at ages 79, 36 and 6 (family CMT-858) and 69 (family CMT-455). The paper does not say who examined them, what the examination included, or whether nerve conduction studies were done, and none of the four appears in the nerve conduction table (Table 4). The two F1 carriers are cited from an earlier paper (Landoure et al., 2010). Quote: 'In family CMT-455 one individual (CMT-455.04) carrying the Arg232Cys mutation was clinically unaffected at the age of 69 years.' (Results, Genetic findings).

    Three ways to explain it

    Scientific

    Sequencing of TRPV4 in 145 unrelated index patients with hereditary neuropathy, 83 of them selected for atypical features, identified five heterozygous missense mutations (including the novel Arg232Cys) in eight families, four of the five at strictly conserved arginines on the convex surface of the ankyrin repeat domain. Phenotypes ranged from congenital distal SMA and scapuloperoneal SMA to HMSN2C, distal HMN with vocal fold paresis and generic axonal HMSN2, with marked intra- and interfamilial variability and axonal electrophysiology in all patients studied. Six clinically unaffected carriers were taken to indicate reduced penetrance, but how these carriers were assessed is not described and some were young.

    Easier

    The researchers checked the TRPV4 gene in 145 people from Europe and the USA with inherited nerve disease and found changes in eight families. Even within one family, the illness could look very different from person to person: some were born with club feet or stiff joints, while others first had weakness as adults. Many had extra problems such as weakness of the vocal folds (in the voice box), hearing loss or a curved spine. Six relatives carried the gene change but were described as unaffected, which suggests not everyone with the change becomes ill. However, the paper does not say how carefully these relatives were checked.

    Simple

    TRPV4 is a gene that tells the body how to build a tiny door that lets calcium into cells. In eight families, a small spelling mistake in this gene caused nerve problems, but the problems were very different from one person to the next, even in the same family. A few relatives had the mistake but seemed well, though the paper does not say how closely they were checked.

    Why these ratings

    Relevance 5/5: A clinical and genetic series of eight families with TRPV4 neuropathy, including the American HMSN2C family F1, describing variability, vocal fold involvement and reduced penetrance.

    Evidence 3/5: The genetic work is careful (confirmation, segregation, controls, haplotypes, paternity testing), but this is a small, selected referral series with one-time clinical data, and the penetrance claim rests on six carriers whose assessment is not described.

    Funding and conflicts of interest

    Funded by the University of Antwerp, the Fund for Scientific Research (FWO-Flanders), the Medical Foundation Queen Elisabeth (GSKE), the Association Belge contre les Maladies Neuromusculaires (ABMM), the Interuniversity Attraction Poles P6/43 program of the Belgian Federal Science Policy Office, a Methusalem excellence grant of the Flemish Government, the Austrian Science Fund (P19455-B05), a McKnight Scholar Award and an NIH grant (R01GM081340), FWO PhD fellowships, the Medical Research Council and the Muscular Dystrophy Campaign, the National Institute for Health Research Biomedical Research Centres funding scheme (via UCLH/UCL), and NINDS intramural funds. No company funding is listed. Competing interests: not stated in this version.

    PubMed 20460441 · DOI

15. #15Detailed summary onlyGenetic linkage and clinical study of two families

    Confirmation of a hereditary motor and sensory neuropathy IIC locus at chromosome 12q23-q24

    McEntagart ME, Reid SL, Irrthum A et al. · 2005 · Annals of neurology

    Relevance 5/5Evidence 3/53 moderate · 3 minor problems

    In these CMT2C families, symptoms began anywhere from birth to age 62, severity varied a lot within a family, and two people developed breathing failure and needed a machine to help them breathe. Some relatives who felt well had signs of the condition when examined and tested.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    Genetic linkage and clinical study of two families

    Who or what

    Two families with CMT2C: a large new family from New Zealand (family A5, with seven affected members, three of whom had nerve tests) and a British family first reported in 1999 (family D3: the proband, his mother and his older sister).

    What they did

    The team tested 12 DNA markers across the chromosome 12q23-24 region and ran linkage analysis assuming dominant inheritance with full penetrance. They sequenced all 25 exons of ataxin-2, a candidate gene in the region, and described the clinical features, nerve tests and, in the A5 proband, quantitative sensory testing. This analysis is based on a detailed secondary summary of the paper written by another reader, not on the paper itself.

    What they found

    • Each family had a shared stretch of DNA in the chromosome 12 region that traveled with the disease. Together they gave a combined multipoint LOD score of 2.1, which the authors call supportive rather than proof on its own.

      “Combined, the families gave a multipoint LOD score of 2.1, the most that two families of this size can produce.”Secondary summary, 'Genetic results'

    • Recombination events narrowed the location of the CMT2C gene to a 3.9 Mb region containing 66 known genes. No mutation was found in ataxin-2.

      “Recombination events narrowed the gene's location to 3.9 Mb holding 66 known genes.”Secondary summary, 'Genetic results'

    • In family A5, age at onset ranged from birth to 62 and severity varied widely within the family.

      “Age at onset in family A5 ran from birth to 62, and severity varied widely within the family.”Secondary summary, 'Authors' conclusions'

    • Two members of family A5 developed breathing failure needing bi-level airway pressure support. The proband, affected from birth, needed it at 66, had paralysis of both vocal cords, and had needed a wheelchair at 55.

      “At 66 he needed bi-level airway pressure support for breathing failure with raised carbon dioxide and obstructive sleep apnoea.”Secondary summary, 'The A5 proband in detail' and table for family A5

    • At least four of the seven affected A5 members had rounded, sloping shoulders, which the authors flag as a feature of CMT2C not documented before.

      “At least four of seven affected A5 members had rounded, sloping shoulders.”Secondary summary, 'Authors' conclusions'

    • Relatives who reported no symptoms had clear signs when examined: in family D3 the mother and sister had weakness, absent reflexes and an axonal neuropathy on nerve tests, and in family A5 one symptom-free member (IV-6) had mild weakness and reduced reflexes.

      “His mother and older sister had no symptoms. Both had absent reflexes, reduced vibration sense in the legs, weakness of the hands and feet on examination, and an axonal neuropathy on nerve testing.”Secondary summary, 'Family D3 from Britain' and table for family A5

    What the authors concluded

    Two more families support the CMT2C location on chromosome 12q23-24 found earlier by Klein, and narrow it to 3.9 Mb with 66 genes, excluding ataxin-2. Clinically, onset and severity vary widely, breathing failure is an important complication, rounded shoulders may be a feature, and congenital distal SMA, scapuloperoneal neuropathy and CMT2C may be caused by one gene.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • ModerateLinkage evidence below the usual threshold

      A LOD score of 2.1 does not reach the usual cut-off for declaring linkage, so on its own this paper supports, but does not prove, the chromosome 12 location. It leans on Klein's earlier result.

      “A LOD score of 2.1 is below the usual threshold of 3 for declaring linkage.”Secondary summary, 'Limitations'

    • ModerateLinkage model assumed full penetrance

      The analysis assumed that everyone carrying the gene change shows the disease. The summary writer adds that later work disproved this; the paper itself found symptom-free relatives with signs. A wrong assumption can misclassify relatives and affect the linkage result.

      “The model assumed full penetrance, which later work disproved.”Secondary summary, 'Limitations'

    • ModerateNerve tests in only some affected members

      Only three of the seven affected A5 members had nerve tests, done at different ages and years before the clinical examinations reported, which limits the comparison between people.

      “Nerve tests were available for three of the seven affected A5 members”Secondary summary, 'Limitations'

    • MinorOverlap with a neighboring locus not fully excluded

      In family A5 the linked region could extend into the nearby locus for distal hereditary motor neuropathy type II.

      “In family A5 the linked region could extend into the neighbouring distal HMN II locus.”Secondary summary, 'Limitations'

    • MinorOnly one candidate gene sequenced

      Of 66 genes in the region, only ataxin-2 was checked, so the paper narrowed the search but did not find the gene.

      “Only one candidate gene was sequenced out of 66 in the region.”Secondary summary, 'Limitations'

    • MinorSmall second family

      Family D3 has only a few members and its clinical details were published elsewhere.

      “Family D3 has only a handful of members, and its clinical details were published elsewhere.”Secondary summary, 'Limitations'

    How “unaffected” relatives were checked

    Relatives were examined, not just asked. In family D3 the mother and older sister had no symptoms but on examination had absent reflexes, reduced vibration sense and weakness, and nerve tests showed an axonal neuropathy. In family A5, member IV-6 reported no symptoms but had mild weakness at the shoulders and lower legs and reduced reflexes on examination. The summary does not describe how relatives classed as unaffected in the linkage analysis were assessed. Quote: “His mother and older sister had no symptoms. Both had absent reflexes, reduced vibration sense in the legs, weakness of the hands and feet on examination, and an axonal neuropathy on nerve testing.”

    Three ways to explain it

    Scientific

    Linkage analysis in two further families supported the CMT2C locus on chromosome 12q23-24 (combined multipoint LOD 2.1, below the conventional threshold of 3) and narrowed the interval to 3.9 Mb containing 66 genes; ataxin-2 was excluded by sequencing. Clinically, onset ranged from birth to 62 with marked intrafamilial variability, two members developed respiratory failure requiring bi-level ventilatory support, and asymptomatic relatives had signs of axonal neuropathy on examination and nerve conduction studies.

    Easier

    Researchers studied two more families with CMT2C to check where on our chromosomes the faulty gene lay. Both families pointed to the same part of chromosome 12, though the statistical result was supportive rather than conclusive on its own. The search area shrank to 66 genes. The age when symptoms began ranged from birth to 62, two people needed machines to help them breathe, and some relatives who felt fine had signs of the condition when examined and tested.

    Simple

    Scientists were looking for where the CMT2C gene hides, like narrowing down which street a house is on. Two more families pointed to the same neighborhood on chromosome 12. In these families some people were affected as babies and others only as older adults. Some relatives who felt well still showed small signs when a doctor checked their nerves.

    Why these ratings

    Relevance 5/5: It is directly about CMT2C families, with detailed clinical descriptions of variable onset, breathing failure and relatives with signs but no symptoms.

    Evidence 3/5: Careful clinical description of two families with examined relatives and nerve tests, but the linkage result is below the usual threshold and this analysis rests on a secondary summary.

    Funding and conflicts of interest

    Not stated in the summary.

    PubMed 15668982 · DOI

16. #16Full paperFamily case report with literature review

    A TRPV4 mutation caused Charcot-Marie-Tooth disease type 2C with scapuloperoneal muscular atrophy overlap syndrome and scapuloperoneal spinal muscular atrophy in one family: a case report and literature review

    Chen H, Sun C, Zheng Y et al. · 2023 · BMC neurology

    Relevance 5/5Evidence 2/51 major · 2 moderate · 3 minor problems

    This family report shows that the same TRPV4 change can look quite different in a parent and a child, and that nerve tests can find changes in someone without symptoms. It is one family, and the father's diabetes may have added to his nerve findings.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    Family case report with literature review

    Who or what

    One Chinese family: a 58-year-old man and his 27-year-old son, both carrying the TRPV4 change p.R316C (c.946C>T), and the man's wife, who was genotyped and did not carry it. The paper also includes a review of published CMT2C patients (Table 2) and SPSMA patients (supplementary table).

    What they did

    The authors describe the father's and son's symptoms, examination, blood tests, muscle MRI, nerve conduction and EMG results, and the father's sural nerve biopsy under light and electron microscopy. They compare the two and summarize features reported in earlier CMT2C and SPSMA papers.

    What they found

    • The father developed limb weakness from age 48 and numbness from age 55, with severe shoulder-blade muscle wasting, distal weakness and absent sensory nerve responses in the legs. He was labeled as having CMT2C overlapping with scapuloperoneal muscular atrophy.

      “Sensory nerve action potentials could not be evoked in bilateral sural or superficial peroneal nerves.”Abstract; Case presentation, Patient 1

    • The father's sural nerve biopsy showed a severe loss of myelinated nerve fibers with clusters of regrowing fibers and 'pseudo-onion bulbs', which the authors read as signs of axon regrowth.

      “The scattered pseudo-onion bulbs formed by regenerating clusters were visible, which were probably a reflection of axonal regeneration”Case presentation, Patient 1; Figure 2

    • Muscle and nerve MRI in the father showed wasting of the thigh and calf muscles with fatty replacement, and an enlarged brachial plexus (the nerve network supplying the arm) that appeared bright on both sides.

      “The brachial plexus was symmetrically hypertrophy and hyperintense”Case presentation, Patient 1; Figure 1

    • The son, who carries the same change, was born with a clubfoot and had no weakness or sensory symptoms, but nerve tests showed an absent motor response in one leg nerve and EMG changes typical of long-standing nerve damage. The authors called this early SPSMA.

      “Although there was no obvious weakness or sensory symptoms, early SPSMA could be considered for him.”Abstract; Case presentation, Patient 2; Table 1

    • The change was found in the son but not in his mother, so it came from the father.

      “This mutation was absent in his wife but was heterozygous in his son”Case presentation, Patient 1; Figure 3A

    • In the authors' review of published CMT2C patients, vocal cord paralysis was common, and hearing loss and scoliosis each affected roughly a quarter to a third.

      “vocal cord paralysis (37/48, 77%), hearing loss (12/42, 29%), and scoliosis (10/37, 27%)”Discussion; Table 2

    What the authors concluded

    The same TRPV4 change can produce different pictures within one family, here an overlap of CMT2C and scapuloperoneal muscular atrophy in the father and early SPSMA in the son. The authors say this broadens the known range of TRPV4 neuropathy and adds nerve biopsy detail, and that features such as vocal cord paralysis, scoliosis and hearing loss deserve attention.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • MajorFather's long-standing diabetes not discussed

      The father had diabetes for more than twenty years and a raised glycated hemoglobin (a measure of long-term blood sugar) of 7.1%. Diabetes can itself damage sensory nerves. His numbness, absent sensory responses and nerve biopsy changes are all attributed to TRPV4, and the paper does not discuss this. That weakens both the 'CMT2C' (sensory) part of his label and the claim that the biopsy shows TRPV4 neuropathy.

      “He has been diagnosed with diabetes for more than twenty years.”Case presentation, Patient 1

    • ModerateSon labeled SPSMA without weakness

      The son has no weakness or sensory symptoms. The SPSMA label rests on EMG changes, one absent leg motor response and a clubfoot at birth. The title presents SPSMA in the family as a firm diagnosis, which goes further than these findings support. Follow-up over time would be needed.

      “who presented chronic neurogenic damage in EMG without obvious muscle weakness was considered early SPSMA”Discussion and conclusions

    • ModerateGenetic testing method and wider family not described

      The paper does not say how the change was found (panel, exome or targeted test) or whether other neuropathy genes were checked. Only three people were genotyped. Nothing is said about the father's parents or other relatives, and the pedigree legend in this text is cut off.

      “Genetic analysis revealed that this patient had a heterozygous c.946C > T mutation in the TRPV4 gene”Case presentation, Patient 1

    • MinorWrong amino acid named

      The text says the change swaps arginine for leucine, but p.R316C means arginine to cysteine. This is a clear slip that readers should be aware of.

      “resulting in amino acid substitutions of arginine to leucine (p.R316C)”Case presentation, Patient 1

    • MinorLiterature review without methods

      There is no description of how papers were searched for or chosen, so the percentages may not represent all published patients. As extracted here, the per-study vocal cord paralysis counts in Table 2 do not appear to add up to the 48 given in the text, although the difference is small.

      “a literature review of the clinical characteristics of CMT2C patients”Discussion and conclusions; Table 2

    • MinorVoice and hearing not formally tested

      Both men are listed as having no vocal cord paralysis or hearing loss, but no laryngoscopy or hearing test is described; the father's assessment mentions only normal hearing and no hoarseness. Mild vocal cord or hearing problems could be missed this way, which matters because the paper stresses these features.

      “The hearing was normal and there were no findings of hoarseness, pes cavus or scoliosis.”Case presentation, Patient 1; Table 1

    How “unaffected” relatives were checked

    Only three family members are described. The son was examined and had nerve conduction and EMG tests, which found abnormalities despite no weakness. The wife was genotyped and did not carry the change; her clinical status is not described. The father's parents and other relatives are not described, and the pedigree legend in this text is cut off after mentioning a 'mildly affected mutation carrier'. Quote: 'This mutation was absent in his wife but was heterozygous in his son' (Case presentation).

    Three ways to explain it

    Scientific

    This report describes a Chinese father and son heterozygous for TRPV4 p.R316C with discordant phenotypes: the father had a sensorimotor axonal neuropathy with scapuloperoneal atrophy (labeled CMT2C and scapuloperoneal muscular atrophy overlap) and a sural biopsy showing severe myelinated fiber loss with regenerating clusters and pseudo-onion bulbs. The son had congenital clubfoot, chronic neurogenic EMG changes and an absent fibular CMAP without weakness (labeled early SPSMA). The authors use this, with a literature review, to illustrate intrafamilial variability. The father's long-standing diabetes is a possible contributor to his sensory and biopsy findings that the paper does not address.

    Easier

    A father and son in China both carry the same TRPV4 change, but their illness looks different. The father, aged 58, has weak and wasted muscles around the shoulders and legs and lost feeling in his hands and feet; a sample of his leg nerve showed many lost nerve fibers. His 27-year-old son was born with a clubfoot and has no weakness, but nerve tests show early signs of nerve damage. The father also has long-standing diabetes, which can affect nerves, and the paper does not discuss this.

    Simple

    A dad and his son have the same small mistake in the TRPV4 gene. The dad has weak muscles and numb feet, while the son only had a twisted foot at birth and some changes on nerve tests. It shows the same mistake can look different in different people in one family. The dad also has diabetes, which can hurt nerves too, so not all of his problems may come from TRPV4.

    Why these ratings

    Relevance 5/5: It describes a family with a known CMT2C-causing TRPV4 change and different presentations within the family, which speaks directly to variability in CMT2C.

    Evidence 2/5: A single family of two affected people, with an unreported genetic testing method, no discussion of the father's diabetes (an important possible cause of his nerve findings), and a literature review without stated methods.

    Funding and conflicts of interest

    Supported by the Shanghai Municipal Science and Technology Major Project (No.2018SHZDZX01) and ZJLab. The authors declare no competing interests.

    PubMed 37391745 · DOI

17. #17Full paper (scan)Case report (letter to the editor)

    Charcot-Marie-Tooth disease type 2C and scapuloperoneal muscular atrophy overlap syndrome in a patient with the R232C TRPV4 mutation

    Koutsis G, Lynch D, Manole A et al. · 2015 · Journal of neurology

    Relevance 5/5Evidence 2/51 moderate · 4 minor problems

    This single case shows that one person with a TRPV4 variant can have features of CMT2C and scapuloperoneal muscular atrophy together, with serious breathing and voice involvement, and that nerve test results can change over the years. It also reports a sister with the same variant described as unaffected, though with few details.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    Case report (letter to the editor)

    Who or what

    One Greek woman aged 43 with the TRPV4 R232C variant (c.C694T, exon 4). Her sister was also tested and carried the variant. Her father, who died aged 51, was described from family report and was not tested.

    What they did

    The authors described her symptoms, muscle strength (MRC grades), breathing tests, chest and spine X-rays, laryngoscopy and blood tests. They compared nerve conduction studies and needle EMG done at age 20 with those done at age 43. Targeted exome sequencing found the TRPV4 variant, which was confirmed by Sanger sequencing, and her sister was tested for it.

    What they found

    • Symptoms began in childhood with unsteady walking, breathlessness on exertion and voice problems, and slowly worsened; by age 40 she depended on a wheelchair.

      “Symptom onset was in childhood with gait unsteadiness, exertional dyspnea and dysphonia. Disease course was slowly progressive leading to wheelchair-dependence and restricted upper-limb use by age 40.”Page 1972, case description

    • She had features of both CMT2C (foot drop, pes cavus, vocal cord weakness, sensory loss in the legs) and scapuloperoneal muscular atrophy (shoulder girdle wasting, scapular winging, peroneal wasting). Strength was MRC 0 to 1 in the distal legs and 3 to 3+ proximally.

      “Our patient, therefore, had features suggestive of an overlap syndrome combining CMT2C and SPMA.”Page 1973, discussion; Table 1

    • Nerve test results changed over time. At age 20 they showed a purely motor neuropathy with normal sensory responses (sural amplitude 15 μV, ulnar sensory 18 μV). At age 43 they showed a severe sensorimotor axonal neuropathy (sural 1.5 μV, ulnar sensory 2.0 μV) with denervation in shoulder muscles.

      “At 43, findings were consistent with a severe sensorimotor axonal neuropathy, with additional EMG evidence of mostly chronic denervation in scapular and shoulder muscles”Page 1972; Table 2

    • She had a severe restrictive breathing defect with a paralyzed diaphragm, partial vocal cord paralysis, scoliosis without signs of vertebral dysplasia, and a mildly raised level of the muscle enzyme CPK (222 U/L).

      “Lung function tests revealed a severe restrictive respiratory defect; chest and abdominal radiographs diaphragmatic paralysis and scoliosis without evident vertebral body dysplasia”Page 1972; Figure 1f, g

    • Her sister carried the same variant but was described as unaffected on clinical and nerve testing, which the authors take as reduced penetrance.

      “The mutation was also identified in the clinically and electrically unaffected sister”Page 1972; Figure 1a

    What the authors concluded

    The patient had an overlap of CMT2C and scapuloperoneal muscular atrophy, and her picture may have shifted over the years from a purely motor neuropathy with vocal cord paresis to this overlap. R232C showed reduced penetrance in this family. The authors speculate this could relate to a dominant-negative effect, modifier genes or epigenetic factors, and say that overlapping TRPV4 syndromes have not received enough attention.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • ModerateReduced penetrance rests on one sister with few details

      The wording suggests the sister was examined and had nerve tests, which is better than relying on family history. But her age, examination findings and nerve test results are not given. Without her age, readers cannot tell whether she might still develop signs later, and one carrier cannot show how often the variant causes no illness.

      “The mutation was also identified in the clinically and electrically unaffected sister”Page 1972; Figure 1a

    • MinorFather's illness known only by report

      The father was not examined by the authors or tested, so his diagnosis and whether he carried the variant are assumed from family report.

      “He was reported to have a similar but milder clinical picture compared to his daughter, with characteristic dysphonia”Page 1972

    • MinorPhenotype shift inferred from two time points with incomplete early testing

      The suggestion that her condition changed type over time rests on tests at only two ages, and the shoulder muscles were not tested at age 20. Shoulder involvement may therefore have been present earlier but not checked. The authors present the shift as possible rather than proven.

      “although shoulder girdle muscles were not tested with needle EMG”Page 1972

    • MinorOther genes not reported

      The letter does not say whether the other genes on the sequencing panel were checked and found normal, so a second genetic factor cannot be ruled out from the letter alone.

      “targeted exome sequencing revealed a c. C694T point mutation in exon 4 of TRPV4”Page 1972

    • MinorSingle case in a short letter

      A letter has room for few details. One case can show that a combination of features is possible, but not how common it is, and the general comments on penetrance and overlap rely on earlier studies.

      “This is a case study and hence no ethical approval was obtained from the hospital ethics committee.”Page 1974, Ethical standards

    How “unaffected” relatives were checked

    The sister who carried R232C is described as 'clinically and electrically unaffected' ('The mutation was also identified in the clinically and electrically unaffected sister'), which implies a clinical examination and nerve conduction testing. However, her age, examination findings and nerve test results are not reported. The father was not tested and his milder illness was reported by others after his death. Other relatives shown in the pedigree are not described or genotyped.

    Three ways to explain it

    Scientific

    A 43-year-old woman heterozygous for TRPV4 p.R232C had childhood-onset, slowly progressive weakness with vocal cord paresis, diaphragmatic paralysis, scapular winging and scoliosis, consistent with a CMT2C and SPMA overlap syndrome. Nerve conduction studies showed a purely motor axonal neuropathy at 20 and a severe sensorimotor axonal neuropathy at 43, which the authors interpret as a possible phenotypic shift. A heterozygous sister was reported as clinically and electrophysiologically unaffected, which the authors take as evidence of reduced penetrance, although supporting details are not provided.

    Easier

    This letter describes one Greek woman with the TRPV4 change R232C. Her problems started in childhood and slowly got worse. She had weak legs and shoulders, a weak voice box and a paralyzed breathing muscle, and needed a wheelchair by 40. Her nerve tests showed only movement nerves affected at 20, but feeling nerves were affected too by 43. Her sister has the same gene change but was described as having no signs, which suggests the change does not always cause illness. However, few details about the sister are given.

    Simple

    This is the story of one woman whose TRPV4 gene change made her muscles, voice and breathing weak. Her illness slowly changed over the years. Her sister has the same gene change but seemed well, a bit like two houses with the same faulty door where only one has noticed a problem so far.

    Why these ratings

    Relevance 5/5: A detailed description of a person with CMT2C features and a well-known TRPV4 variant, including nerve tests years apart.

    Evidence 2/5: A single case in a short letter; useful for showing what can happen, but not how often.

    Funding and conflicts of interest

    Supported by the Medical Research Council, The Wellcome Trust and The Brain Research Trust, and by the National Institute for Health Research (NIHR) University College London Hospitals (UCLH) Biomedical Research Centre. The authors report no conflict of interest.

    PubMed 26048687 · DOI

18. #18Full paperCase report

    Severe Multifocal Noninfectious Wound Dehiscence After Cavovarus Reconstruction in TRPV4-Associated Charcot-Marie-Tooth Disease Type 2C: A Case Report

    Elhaddad M, Stibelman J, Carrillo-Kashani A et al. · 2026 · Foot & ankle specialist

    Relevance 5/5Evidence 2/54 moderate · 2 minor problems

    This is one child's experience, so it cannot show that people with CMT2C heal worse after surgery. It raises a question that the authors say needs further research.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    Case report

    Who or what

    One 10-year-old girl with genetically confirmed CMT2C due to the TRPV4 variant c.806G>A (p.Arg269His) and no known family history of neuromuscular disease, treated at Cedars-Sinai Medical Center, Los Angeles. The report also compares one skin-cell (fibroblast) culture from her with one control culture.

    What they did

    The authors describe her severe, stiff, turned-in feet (worse on the left) and a left knee that would not fully straighten (by about 25°), and the surgery she had: a growth-plate procedure at the left knee and a complex 6-hour left foot and ankle reconstruction. They then describe how several of her wounds opened up within weeks, how this was treated, and the result at 15 months. They also report a skin-cell culture test done in Sweden.

    What they found

    • Within weeks of surgery, wounds opened up (dehiscence) at several separate sites, plus a larger wound on the outer hindfoot with a small area of exposed tendon.

      “within weeks she developed multifocal wound dehiscence involving the distal anterior tibia (1 × 1.5 cm), plantar foot (1 × 0.5 cm), proximal medial instep (3 × 2 cm), and distal medial instep (1 × 2 cm)”Page 3, Case Presentation

    • There was no sign of infection, and wound swabs grew no germs.

      “there was no clinical evidence of infection; wound cultures showed no growth.”Page 4, Case Presentation

    • After readmission, repeated cleaning of the wounds, vacuum (negative-pressure) dressings, nutritional support and a biodegradable skin-substitute matrix, all wounds healed, and the foot was flat and stable at 15 months.

      “At 15-month follow-up, patient-provided photographs demonstrated complete healing of all incision and dehiscence sites and a stable, plantigrade foot resting flat on the ground, without recurrent breakdown”Page 4, Case Presentation

    • Skin cells grown from her in the lab spread out from the tissue sample more slowly and stuck down less well early on than a control culture, though they multiplied normally later.

      “Compared with parallel control, the TRPV4-variant culture demonstrated delayed explant outgrowth with reduced early adherence/spread, while proliferation after initial passage appeared preserved.”Page 4, Case Presentation

    • The authors compare her course with a published series in which wound problems after CMT foot surgery were uncommon.

      “in a retrospective cohort of 89 patients undergoing CMT reconstructive surgery, the overall complication rate was approximately 8%, including 3 postoperative infections and 1 superficial wound dehiscence.”Page 4, Discussion

    What the authors concluded

    Severe wound breakdown at several sites, without infection, after surgery that the authors judged technically appropriate, raises the possibility that TRPV4-associated CMT2C makes wound healing harder. They state that one case cannot prove this, and suggest heightened monitoring, early involvement of several specialist teams and consideration of staged surgery for similar high-risk patients.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • ModerateOne case cannot show cause

      The surgery was very extensive (a 6-hour procedure with many incisions on a severely deformed foot), so wound problems could have other explanations. The authors say this themselves. Their suggestions for other patients, such as staged surgery, are drawn from this one experience.

      “That said, this report cannot establish causality; it can only highlight a plausible association and the need for heightened vigilance.”Page 5, Discussion

    • ModerateSurgery judged appropriate by the team who did it

      The argument that the wounds reflect the patient's biology rests on the surgery being technically sound. That judgment comes from the treating team, and the consensus statement and algorithms cited for it (references 7 to 9) list 'Pfeffer GB' as first author, who appears to be the senior author of this report. It is not an independent review.

      “The procedure was technically executed without intraoperative mishap”Page 5, Discussion; references 7 to 9

    • ModerateSkin-cell test is qualitative and poorly described

      The fibroblast result compares one culture with one control, gives no numbers or methods, and was done elsewhere and 'reviewed' by the authors. The authors call it hypothesis-generating, and it should not be read as evidence that TRPV4 impairs healing.

      “these qualitative findings are hypothesis-generating and require targeted functional validation before any causal inference.”Page 5, Discussion

    • ModerateOther possible contributors not fully reported

      The operation went through existing scar tissue, but no earlier operation or injury is described and her history is called 'otherwise unremarkable'. The plastic surgeons recommended nutritional optimization, but no nutrition test results are given. It is unclear whether these factors played a part.

      “the Achilles tendon through a longitudinal incision over prior scarring. Extensive scar tissue necessitated Achilles Z-lengthening”Page 3, Case Presentation

    • MinorFew genetic details

      The report does not say what kind of genetic test was done, whether other genes were checked, or whether her parents were tested. This matters little for the wound question but limits what can be said about the genetics.

      “Prior genetic testing identified a TRPV4 variant, c.806G>A (p.Arg269His).”Page 2, Case Presentation

    • MinorLong-term result partly from family photographs

      Healing at 15 months was judged from photographs provided by the family, alongside X-rays. An in-person examination at that time is not described.

      “At 15-month follow-up, patient-provided photographs demonstrated complete healing”Page 4, Case Presentation

    Three ways to explain it

    Scientific

    A 10-year-old girl with CMT2C due to TRPV4 c.806G>A (p.Arg269His) developed multifocal, culture-negative wound dehiscence within weeks of extensive cavovarus reconstruction that the authors considered technically appropriate. The wounds healed after serial debridement, negative-pressure wound therapy and a biodegradable temporizing matrix, with complete healing at 15 months. A qualitative fibroblast explant culture showed delayed outgrowth and reduced early adherence compared with one control. The authors raise the hypothesis of a TRPV4-related wound-healing vulnerability but state that a single case cannot establish causality.

    Easier

    This report describes one girl with CMT2C who had major surgery to straighten a very stiff, turned-in foot. Within weeks, several of her surgical cuts opened up, even though there was no infection. With months of specialist wound care everything healed, and 15 months later her foot was flat and stable. Skin cells grown from her in a lab spread out more slowly than cells from one comparison sample. The surgeons wonder whether her TRPV4 change makes healing harder, but one case cannot prove this.

    Simple

    A girl with CMT2C had a big operation on her foot, and her cuts opened up afterward even though there were no germs. After a lot of careful treatment, her foot healed well. The doctors wonder if her TRPV4 change made healing harder, but with only one person they cannot tell for sure.

    Why these ratings

    Relevance 5/5: Directly about a child with CMT2C and a possible surgical complication relevant to people with CMT2C considering foot surgery.

    Evidence 2/5: A single case, which can raise a question but cannot show that TRPV4 caused the poor wound healing.

    Funding and conflicts of interest

    The authors received no financial support for the research, authorship or publication. The authors declared no potential conflicts of interest.

    PubMed 42446062 · DOI

19. #19Full paperSingle-patient longitudinal case report (hearing study with an intervention)

    Remediation of Perceptual Deficits in Progressive Auditory Neuropathy: A Case Study

    Rance G, Tomlin D, Yiu EM et al. · 2024 · Journal of clinical medicine

    Relevance 5/5Evidence 2/53 moderate · 3 minor problems

    In one child with a TRPV4 change, the hearing nerve and the ability to follow speech in noise got worse over 3 years even though a standard hearing test stayed normal. A remote-microphone system helped him. As this is a single case, it does not show how common this is in CMT2C or whether others would respond the same way.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    Single-patient longitudinal case report (hearing study with an intervention)

    Who or what

    One boy with a heterozygous TRPV4 variant (c.947G>A, p. R316>H), described as having CMT2C, with congenital arthrogryposis, weakness mainly in the legs, and nerve tests showing an axonal neuropathy that affected motor more than sensory nerves. His hearing was tested at about 6-month intervals from age 6 years 2 months to 9 years 6 months. A remote-microphone listening system was fitted at 7 years 7 months. There was no control group; results were compared with published norms and with his own earlier results.

    What they did

    The team repeated a set of hearing tests over a 3-year period: standard hearing thresholds, auditory brainstem responses (scalp recordings of activity in the hearing nerve and brainstem), a gap detection test of how well brief silences are heard, and a test of understanding speech against competing speech from the same or a different direction. After teachers and parents reported severe communication difficulties, he was given a remote-microphone system, in which the teacher wears a microphone that sends their voice to a receiver behind his ear. The team measured his speech understanding with and without it and asked him to rate everyday listening difficulty with a questionnaire.

    What they found

    • His ability to detect quiet sounds stayed normal in both ears at every test.

      “On each test occasion, hearing threshold levels were within normal limits (≤15 dBHL) bilaterally.”Section 2.1, Sound Detection Levels

    • The size of the auditory brainstem response (wave V) fell steadily over the study. Its timing showed no overall change, although the left ear showed a clinically significant delay (more than 0.3 ms) near the end.

      “Wave V’s response amplitude decreased significantly over the recording period ( r = -0.993, p < 0.001).”Section 2.2, Electrophysiology; Figure 1

    • His ability to notice brief silences in a noise, normal at first, worsened over the study. By the end he needed a gap about twice as long as at the start.

      “The gap detection threshold worsened (from normal levels at baseline) over the study period, indicating that the patient’s auditory pathway was less able to encode brief changes in the acoustic signal ( r = 0.814, p = 0.049)”Section 2.3, Auditory Temporal Processing; Discussion; Figure 2

    • Understanding speech in noise when the speech and noise came from the same direction stayed stable. When they came from different directions, which relies on comparing what the two ears hear, performance worsened markedly over the first 18 months and then returned to normal levels after the device was fitted.

      “those conditions that involved the spatial separation of the target and background noise (DV90 and SV90) showed significant deterioration through the first 18 months of the study before recovering to normal levels following the provision of auditory intervention.”Section 2.4, Speech Perception; Figure 3

    • With the remote-microphone system, his score for understanding speech in background noise rose from 38% to 78% at fitting. After 4 weeks he rated everyday listening difficulty as substantially lower when using it. He wore it 3 to 5 hours a day.

      “the patient’s discrimination score improving from 38% in the unaided condition to 78% when wearing the remote-microphone system.”Section 2.5, Device Fitting; Figure 4

    • The authors report that his spatial listening improved across the trial period even when tested without the device. They interpret this as a change in the brain's hearing pathways (neuroplasticity) and suggest it happened beyond the brainstem, possibly in attention-related processing in the auditory cortex, because the brainstem measures kept getting worse.

      “Furthermore, his binaural perception (spatial processing ability) improved throughout the trial period, even when not wearing the device ( Figure 3 ).”Discussion; Figure 3

    What the authors concluded

    In this child, the function of the hearing nerve and timing-based hearing got worse over time even though he could still detect quiet sounds normally, which the authors link to a progressive axonal neuropathy (axonopathy). A remote-microphone listening system improved his speech understanding and everyday communication. The authors suggest that long-term use produced changes in the brain (neuroplasticity) that improved his listening even when he was not wearing the device.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • ModerateStrong claim about brain change from one uncontrolled case

      The abstract says the device 'appeared to produce' neuroplastic changes, but the Discussion says it 'clearly' did. With one child and no comparison group, the later improvement without the device cannot be firmly linked to the device. Other explanations, such as growing familiar with a test repeated about every 6 months, are not discussed. The authors do note that single cases need caution and that no matched control was used.

      “Clearly, sustained exposure to the acoustically enhanced signal provided by the device resulted in some form of neuroplastic change, but the mechanism is unclear.”Discussion; Study Limitations

    • Moderate'First to describe the mechanism' goes beyond the data

      The paper says it is the first to describe the mechanism behind hearing problems in TRPV4-linked CMT2C. The data show a shrinking brainstem response and worsening timing in one child, which fits nerve damage. But the authors themselves offer two possible explanations (loss of nerve fibers, or less well-timed nerve firing) and did not test between them. One child's results cannot show what happens in CMT2C in general.

      “This case report is the first to describe the mechanism (progressive axonopathy) underlying auditory dysfunction in TPRV4-linked Charcot-Marie-Tooth disease (type 2C).”Discussion

    • Moderate'No change in overall disease severity' is not measured and sits uneasily with the case history

      The authors say his general condition did not change during the study and use this to suggest that hearing changes may reveal hidden (subclinical) progression. But no measure of overall disease severity is reported. The case history says he lost the ability to walk at 9 years of age, within the study period, although this is put down to recurring contractures. A later nerve test at 10 years was done because of new cramping and muscle twitches (fasciculations) in the left arm. So the idea that hearing worsened while the rest of the disease stayed stable is not well supported.

      “he showed no discernible change in overall disease severity throughout the study period.”Detailed Case Description; Table 1 footnote; Discussion

    • MinorLimited statistics and reporting

      Changes over time are tested with simple correlations across the test sessions, and the statistical methods are not described. The gap detection result is borderline (p = 0.049) and is for the left ear only. The word 'significant' is used for the speech-in-noise decline and the device benefit without any test being reported. The speech-in-noise and questionnaire results are shown only as figures, with no numbers in the text, and the questionnaire was self-rated, not blinded and done after only 4 weeks.

      “Speech perception testing (at fitting) showed a significant perceptual advantage when wearing the device.”Sections 2.3 to 2.5; Figures 2 to 4

    • MinorBrief genetic and clinical background

      The TRPV4 variant was found on a clinical gene panel. The paper does not say whether his parents were tested or whether anyone else in the family is affected. It calls the condition CMT2C, but the boy had congenital arthrogryposis and a mainly motor neuropathy, and the variant is described as previously linked to both CMT2C and scapuloperoneal SMA. The paper does not discuss where his condition sits within the range of TRPV4 disorders, so how far his hearing course applies to other people with CMT2C is unclear.

      “identified a heterozygous c.947G>A (p. R316>H) pathogenic variant in exon 6 of the TRPV4 gene which has previously been associated with Charcot-Marie-Tooth disease (Type 2C) and scapuloperoneal spinal muscular atrophy”Detailed Case Description

    • MinorSmall inconsistencies

      The text says nerve tests were done at 5 and 7 years, but Table 1 gives 4.5, 7 and 10 years. The text describes the gap test as using a 500 ms noise burst, while the Figure 2 legend says a 500 Hz burst of noise. The gene name is misspelled ('TPRV4', 'TPR4') in the Discussion, and the timing of the left-ear delay ('between the ninth year and 9 years and 5-month data points') is unclear. These do not change the main findings.

      “Nerve conduction studies and electromyography performed at 5 and 7 years of age (and repeated at 10 years of age due to upper limb symptoms)”Detailed Case Description; Table 1; Section 2.3; Figure 2 legend; Discussion

    Three ways to explain it

    Scientific

    Longitudinal assessment of a child with a heterozygous TRPV4 R316H variant and a motor-predominant axonal neuropathy showed stable normal pure-tone thresholds but a progressive decline in ABR wave V amplitude and worsening gap detection, consistent with a progressive auditory neuropathy. Binaural (spatially separated) speech-in-noise perception deteriorated over the first 18 months and returned to normal after fitting of a remote-microphone system, with improvement reported even in unaided testing. The authors attribute this to neuroplasticity, but as a single uncontrolled case the cause of the improvement and its generalizability to CMT2C remain uncertain.

    Easier

    This study followed the hearing of one boy with CMT2C over a 3-year period. On a standard hearing test he could hear quiet sounds normally. But recordings from the hearing nerve got weaker, and he found it harder to pick out speech in background noise, which suggests the hearing nerve itself was being affected. A small microphone worn by his teacher, sending their voice straight to his ear, helped a lot, and the researchers report his listening in noise later improved even without it. Because this is only one child with no comparison group, we cannot be sure why he improved or whether the same would happen for others.

    Simple

    This boy could hear quiet sounds well. But the nerve that carries sound to his brain slowly worked less well, a bit like a phone line getting crackly, so voices got muddled when it was noisy. A special microphone that sent his teacher's voice straight to his ear helped him understand. But he is only one boy, so we do not know if it would work the same for everyone.

    Why these ratings

    Relevance 5/5: It follows the hearing of a child with a TRPV4 variant described as CMT2C over time and tests a hearing intervention, which bears directly on hearing problems in CMT2C.

    Evidence 2/5: A carefully measured single case followed over time, but with one patient and no control, so it cannot show what caused the improvement or how common these hearing changes are in CMT2C.

    Funding and conflicts of interest

    G.R. was supported by the Graeme Clark Chair in Audiology & Speech Science. The authors declare no conflicts of interest. The device used was a commercial system (Sonova AG, Phonak Roger); no company funding is stated.

    PubMed 38610891 · DOI

20. #20Detailed summary onlyLetter to the editor describing a single patient (case report)

    Case of Charcot-Marie-Tooth Type 2C Due to a TRPV4 Gene Mutation With Isolated Sudomotor Autonomic Dysfunction

    Baker J, Kimpinski K · 2018 · Journal of clinical neuromuscular disease

    Relevance 5/5Evidence 2/53 moderate · 2 minor problems

    In at least one person with CMT2C, the nerves that control sweating failed while heart rate and blood pressure control stayed normal, and she found it hard to keep cool. This single report cannot say how many people with CMT2C have this.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    Letter to the editor describing a single patient (case report)

    Who or what

    One 57-year-old woman with CMT2C caused by a TRPV4 mutation (the summary says the exact mutation is not stated). She has vocal cord paresis and a tracheostomy, and was seen at an autonomic testing laboratory in London, Ontario.

    What they did

    The team ran a standard autonomic reflex screen: a sweat test at the forearm, upper leg, lower leg and foot, heart rate responses to deep breathing and to a Valsalva maneuver (forced breathing out against resistance), and heart rate and blood pressure during a head-up tilt. They then searched EMBASE, PubMed and Scopus for earlier reports of sweating or autonomic problems in CMT2C. This analysis is based on a detailed secondary summary of the letter written by another reader, not on the letter itself.

    What they found

    • Sweat output was far below normal at all four sites tested: 0.06 microliters at the forearm, 0.018 at the upper leg, 0.012 at the lower leg and 0.042 at the foot.

      “A 57-year-old woman with CMT2C had almost no measurable sweating at four body sites”Secondary summary, opening summary and 'Test results' table

    • Her heart rate and blood pressure reflexes were normal. Heart rate change with deep breathing was 14.1 beats per minute (normal at least 6.2), the Valsalva ratio was 1.36 (normal at least 1.30), and on tilt her heart rate rose 5.2 beats per minute with a 1.8 mmHg change in systolic pressure.

      “So the nerves that drive sweating were failing at every site tested, while the nerves controlling heart rate and blood pressure worked normally.”Secondary summary, 'Test results'

    • Her main complaint was severe difficulty controlling her temperature, with frequent feelings of heat, a raised temperature and no sweating.

      “She managed it herself with ice packs and by drinking large amounts of ice water.”Secondary summary, 'The patient'

    • The authors' literature search found no earlier report of isolated sweating problems with troublesome overheating in CMT2C.

      “Their literature search found no earlier report of isolated sweating dysfunction with troublesome overheating in CMT2C.”Secondary summary, 'What the authors say about why'

    • As a possible explanation, the authors point out that TRPV4 is switched on by moderate warmth, and cite rat studies linking it to body temperature control.

      “The channel is activated by moderate warmth, between 24 and 42 degrees C, and animals lacking it have altered temperature sensing.”Secondary summary, 'What the authors say about why'

    What the authors concluded

    The authors describe failure of the nerves that drive sweating, with normal heart rate and blood pressure control, in a woman with CMT2C, and say they found no earlier report of this. They suggest it may relate to TRPV4's role in sensing warmth and controlling body temperature, which has mainly been shown in rodents.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • ModerateSingle patient

      One person cannot show how common sweating problems are in CMT2C, or prove that CMT2C is what caused them.

      “One patient. Nothing shows how common this is in CMT2C.”Secondary summary, 'Limitations'

    • ModerateOther causes of poor sweating not discussed

      According to the summary, other possible causes such as medicines, other illness or age were not discussed, so it is not certain the problem comes from CMT2C.

      “Other causes of reduced sweating, such as medication, other illness or age, are not discussed.”Secondary summary, 'Limitations'

    • ModerateNo direct check of sweat gland nerves or body temperature

      There was no skin biopsy to count the small nerves to the sweat glands and no recorded body temperatures, so neither the nerve damage nor the overheating was measured directly.

      “No skin biopsy, sweat gland nerve count or body temperature measurements are reported.”Secondary summary, 'Limitations'

    • MinorMutation and nerve test details missing

      The summary says the specific TRPV4 mutation, her neurological examination and her nerve test results are not given, which makes her case hard to compare with other people with CMT2C.

      “It is a two-page letter, so her neurological examination and nerve test results are not given.”Secondary summary, 'Limitations'

    • MinorProposed explanation rests on animal studies

      The idea that TRPV4's role in temperature control explains her symptoms comes from rodent research. It is a reasonable idea, not something shown in people.

      “The link to TRPV4's role in temperature sensing rests on rodent studies.”Secondary summary, 'Limitations'

    Three ways to explain it

    Scientific

    In a single patient with TRPV4-related CMT2C, quantitative sweat testing showed markedly reduced sweat output at all four sites, while heart rate responses to deep breathing and Valsalva and the response to head-up tilt were normal, indicating isolated sudomotor dysfunction. The authors propose a link to TRPV4's role as a warmth-activated channel involved in temperature regulation, based largely on rodent data. As a single case, it shows the feature can occur but not its frequency or cause.

    Easier

    Doctors tested the nerves that control sweating, heart rate and blood pressure in one woman with CMT2C. She made almost no sweat at any of the four places tested, but her heart rate and blood pressure responses were normal. She had a lot of trouble staying cool. The authors think the TRPV4 channel, which responds to warmth, may be involved, but this idea comes mostly from animal studies.

    Simple

    One woman with CMT2C hardly sweated at all, so she got too hot easily, a bit like a car whose cooling system is not working. The parts of her body that control her heartbeat and blood pressure were fine. Because this is only one person, we do not know how many people with CMT2C have this.

    Why these ratings

    Relevance 5/5: It describes a person with CMT2C and a problem, poor sweating with overheating, that the summary notes is not on the usual CMT2C symptom lists.

    Evidence 2/5: A single case in a two-page letter, read here only through a secondary summary, so it shows the problem can occur but not how often or why.

    Funding and conflicts of interest

    The summary says the authors report no conflicts of interest. Funding is not mentioned in the summary.

    PubMed 29465618 · DOI

21. #21Detailed summary onlyResearch letter describing a single family

    Striking phenotypic variability in familial TRPV4-axonal neuropathy spectrum disorder

    Aharoni S, Harlalka G, Offiah A et al. · 2011 · American journal of medical genetics. Part A

    Relevance 5/5Evidence 2/51 major · 3 moderate · 2 minor problems

    In this one family, the same TRPV4 change caused very different severity between siblings, from needing a breathing tube from early life to almost no signs at age 14. Whether the 14-year-old brother will stay unaffected is not known, because he was young and did not have nerve tests.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    Research letter describing a single family

    Who or what

    One family of Ashkenazi and Sephardi Jewish origin seen in Israel and London. Five members carry TRPV4 c.943C>T (R315W): a mother aged 39 and four children aged 9, 11, 13 and 14. The father and a 3-year-old brother do not carry it; an infant sister was not tested.

    What they did

    The team sequenced every coding exon and splice site of TRPV4 in the proband, then tested the parents and four siblings with a restriction enzyme test. They examined family members, carried out nerve tests in those who agreed, reviewed chest, spine and limb X-rays for skeletal dysplasia, and compiled published reports of TRPV4 neuropathy families. This analysis is based on a detailed secondary summary of the paper written by another reader, not on the paper itself.

    What they found

    • Five carriers of the same R315W change ranged from a girl with paralysis of both vocal cords and a tracheostomy to a 14-year-old brother who plays sports and had a normal voice and laryngoscopy.

      “Five members of one family carry the same R315W mutation and range from a child who needed a tracheostomy at birth to a 14-year-old who plays sport with no symptoms.”Secondary summary, opening summary and table 'The family, person by person'

    • Vocal cord involvement varied: the proband and her 11-year-old brother had paralysis of both vocal cords and tracheostomies, the 13-year-old had one paralyzed and one weak cord, the mother had a weak left cord and had been hoarse since childhood, and the 14-year-old had normal vocal cords.

      “Normal voice and normal laryngoscopy.”Secondary summary, table 'The family, person by person'

    • The proband was first thought to have a purely motor neuropathy, but on repeat testing her sural sensory response had disappeared, so the diagnosis changed to a motor and sensory neuropathy. The mother also had a motor and sensory axonal neuropathy.

      “When her nerve tests were repeated, the sural sensory response had gone, and the diagnosis became a motor and sensory neuropathy.”Secondary summary, 'What changed over time'

    • The scoliosis, clubfoot and contractures in this family were judged to result from nerve and muscle weakness, as X-rays showed no primary bone disorder.

      “Scoliosis, clubfoot and contractures in this family come from nerve and muscle weakness. X-rays showed no primary bone disorder.”Secondary summary, 'Authors' conclusions'

    • From the literature, the authors report that at least one person had documented vocal cord paralysis in 16 of 20 published TRPV4 neuropathy families (80%), and that non-penetrance had been seen in four families.

      “At least one person had documented vocal cord paralysis in 16 of 20 published TRPV4 neuropathy families (80%).”Secondary summary, 'What the authors pull from the wider literature'

    • The authors conclude that families should be counseled about the wide range of severity and the possibility that a carrier stays well, and that TRPV4 should be the first gene tested in a mainly motor axonal neuropathy with vocal cord paralysis.

      “Families should be counselled about both the wide range of severity and the possibility that a carrier stays well.”Secondary summary, 'Authors' conclusions'

    What the authors concluded

    The same TRPV4 change can cause very different severity even between siblings, and a carrier may stay well. Skeletal problems in this family came from weakness rather than a bone disorder. The authors recommend TRPV4 as the first gene to test in a mainly motor axonal neuropathy with vocal cord paralysis, and counseling families about variable severity.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • MajorSymptom-free carrier had no nerve tests

      The idea that a carrier can stay well rests largely on the 14-year-old brother. He was examined and had a normal laryngoscopy, but declined nerve tests, his ankle reflexes were hard to obtain, and he is young, so mild or later disease cannot be ruled out.

      “The symptom-free carrier was 14 and declined nerve testing, so mild or later disease cannot be ruled out.”Secondary summary, 'Limitations'

    • ModerateOther carriers declined nerve and hearing tests

      Two affected brothers also declined nerve tests and the family declined hearing tests, so the statement that nobody in the family reported hearing loss rests on self-report.

      “Two affected brothers also declined nerve testing, and the family declined hearing tests.”Secondary summary, 'Limitations'

    • ModerateThe 80% figure is about families, not individuals

      It counts families with at least one person with vocal cord paralysis, not the chance that an individual carrier develops it. Families with this feature may also be more likely to be recognized and published.

      “The 80% figure counts families with at least one affected member.”Secondary summary, 'Limitations'

    • ModerateOne family, five carriers

      General advice about counseling and testing is drawn from a single family plus a literature review.

      “One family and five carriers.”Secondary summary, 'Limitations'

    • MinorHoarse relatives never examined

      Four relatives on the mother's side were said to be hoarse but were not examined or tested, so the true number of carriers in the family is unknown.

      “None was available for examination.”Secondary summary, 'The family, person by person'

    • MinorMother's X-rays not available

      The conclusion that there was no primary bone disorder could not be checked in the mother.

      “The mother's x-rays were not available.”Secondary summary, 'Limitations'

    How “unaffected” relatives were checked

    The father and a 3-year-old brother, who do not carry the change, are described as unaffected. The symptom-free carrier, a 14-year-old brother, was examined (normal apart from ankle reflexes that were hard to obtain, with a normal voice and laryngoscopy) but declined nerve conduction tests. Four maternal relatives said to be hoarse were not examined. Quote: “The symptom-free carrier was 14 and declined nerve testing, so mild or later disease cannot be ruled out.”

    Three ways to explain it

    Scientific

    In a single family, five heterozygous carriers of the ankyrin repeat TRPV4 variant R315W showed striking intrafamilial variability, from bilateral vocal cord paralysis requiring tracheostomy to a 14-year-old with an essentially normal examination, consistent with variable expressivity and possibly reduced penetrance. The proband's phenotype evolved from motor to sensorimotor axonal neuropathy on repeat testing, and skeletal deformities were attributed to weakness rather than primary dysplasia. Non-penetrance in this family is not firmly established, because the apparently unaffected carrier was young and declined nerve conduction studies.

    Easier

    Five people in one family carry the same TRPV4 change, R315W, but are affected very differently. Two children needed a breathing tube in the neck because their vocal cords did not move, while a 14-year-old brother plays sports and had an almost normal examination. He did not have nerve tests and is still young, so it is not certain he will stay unaffected. The authors say families should know that severity can vary a lot.

    Simple

    Think of TRPV4 as a door that lets calcium into cells. In this family everyone with the same faulty door was affected differently: some children needed help to breathe, while one brother plays sports and seems fine for now. Doctors cannot yet tell who will be more or less affected.

    Why these ratings

    Relevance 5/5: It describes a family with TRPV4 axonal neuropathy and vocal cord paralysis, directly illustrating the variability and possible reduced penetrance that matter to people with CMT2C.

    Evidence 2/5: A research letter on one family in which several carriers declined nerve tests, read here only through a secondary summary.

    Funding and conflicts of interest

    The summary says the work was funded by the Muscular Dystrophy Campaign and the St George's Charitable Foundation. Competing interests are not mentioned in the summary.

    PubMed 22065612 · DOI

22. #22Abstract onlyReview

    TRPV4 axonal neuropathy spectrum disorder

    McEntagart M · 2012 · Journal of clinical neuroscience: official journal of the Neurosurgical Society of Australasia

    Relevance 5/5Evidence 1/51 moderate problem

    This review describes TRPV4 neuropathy as mainly affecting the motor nerves, often with vocal cord weakness, and sometimes with hearing loss and bladder urgency. It notes that severity varies and that some people who carry a TRPV4 change may not show the condition.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    Review

    Who or what

    No new patients. A review of the TRPV4 axonal neuropathy spectrum, its overlap with TRPV4 skeletal dysplasias, and how mutations affect the channel.

    What they did

    The author reviewed the clinical features of the TRPV4 neuropathy spectrum, the emerging overlap disorders combining neuropathy and skeletal dysplasia, and knowledge of how TRPV4 mutations affect channel function. This analysis is based only on the abstract, so the methods cannot be judged.

    What they found

    • The TRPV4 neuropathy spectrum is mainly a motor axonal neuropathy, often with vocal cord paralysis, and sometimes with sensorineural hearing loss and bladder urgency and incontinence.

      “a predominantly motor axonal peripheral neuropathy, frequently in association with vocal cord paralysis, and occasionally accompanied by sensorineural hearing loss and bladder urgency and incontinence.”Abstract

    • These disorders are inherited in a dominant way, vary in severity, and do not always show in people who carry the mutation (reduced penetrance).

      “These disorders show autosomal dominant inheritance, variable disease expression and reduced disease penetrance.”Abstract

    • The review also covers disorders that combine neuropathy and skeletal dysplasia, and what was known about how mutations change channel function.

      “This article reviews the clinical features of the neuropathy spectrum, the emerging neuropathy/skeletal dysplasia overlap disorders and the present knowledge of the impact of mutations in this gene on channel function.”Abstract

    What the authors concluded

    TRPV4 neuropathies form a spectrum of dominantly inherited, mainly motor axonal neuropathies, often with vocal cord paralysis, showing variable expression and reduced penetrance, and overlapping with TRPV4 skeletal dysplasias. The biological function of the channel was described as uncertain.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • ModerateMethods cannot be judged from the abstract

      Only the abstract was available, so it is not possible to see which studies support statements such as reduced penetrance or how often each feature occurs. As a review it presents no new data, and the abstract describes the channel's biological function as uncertain at the time.

      “TRPV4 encodes a calcium-permeable non-selective cation channel of uncertain biological function.”Abstract

    Three ways to explain it

    Scientific

    This review characterizes the TRPV4 axonal neuropathy spectrum as a predominantly motor axonal neuropathy, frequently with vocal cord paralysis and occasionally with sensorineural hearing loss and bladder urgency and incontinence, inherited in an autosomal dominant manner with variable expressivity and reduced penetrance. It also covers neuropathy and skeletal dysplasia overlap disorders and the effects of mutations on channel function. The supporting evidence cannot be judged from the abstract.

    Easier

    This article reviews nerve conditions caused by TRPV4 changes, which mainly weaken muscles through damage to the motor nerves. Many people also have weak or paralyzed vocal cords, and some have hearing loss or bladder urgency. The condition passes from parent to child, its severity varies, and some people with the change may show few or no signs.

    Simple

    A change in the TRPV4 gene can make the nerves to the muscles and the voice box weak. It can run in families, and it can be strong in one person and hardly noticeable in another.

    Why these ratings

    Relevance 5/5: It is a review focused directly on TRPV4 neuropathy, the condition that includes CMT2C, covering its features, inheritance and penetrance.

    Evidence 1/5: Rated 1 because only the abstract was available, and a review summarizes earlier work rather than adding new data.

    Funding and conflicts of interest

    Not stated in the abstract.

    PubMed 22617546 · DOI

23. #23Abstract onlyFamily study with genetic linkage exclusion

    Charcot-Marie-Tooth disease type 2C: a distinct genetic entity. Clinical and molecular characterization of the first European family

    Santoro L, Manganelli F, Di Maio L et al. · 2002 · Neuromuscular disorders: NMD

    Relevance 5/5Evidence 1/51 moderate problem

    In this family, how badly the vocal cords were affected did not match how weak the arms and legs were, and the people with the weakest limbs had few breathing symptoms. The abstract shows CMT2C was a separate condition from the other CMT2 types known at the time.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    Family study with genetic linkage exclusion

    Who or what

    One family described as the first European family affected by CMT2C. The abstract does not give the number of family members studied.

    What they did

    The authors described the clinical features of the family and tested whether the condition was linked to the known gene regions for CMT2A, B, D, E and F and for distal hereditary motor neuronopathy type VII. This analysis is based only on the abstract, so the methods cannot be judged.

    What they found

    • The condition in this family was not linked to any of the CMT2 gene regions known at the time, or to the distal hereditary motor neuronopathy type VII region.

      “Genetic analysis excluded linkage to locus of Charcot-Marie-Tooth disease type 2A, B, D, E and F, and to locus of distal hereditary motor neuronopathy type VII.”Abstract

    • Most people who inherited the condition showed it, severity varied, and limb weakness appeared most severe in the youngest generation.

      “In this family the disease has high penetrance, variable severity and apparently the most severe limb muscle involvement in the youngest generation.”Abstract

    • Vocal cord paralysis was not related to the degree of limb weakness, and those with the most severe muscle involvement had few or no breathing symptoms.

      “Vocal cord paralysis is unrelated to the degree of muscular weakness and patients with the most severe muscle involvement have absent or minimal respiratory symptoms.”Abstract

    What the authors concluded

    CMT2C is clinically and genetically different from CMT2A, B, D, E and F, and is not caused by the same gene as distal hereditary motor neuronopathy type VII.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • ModerateMethods cannot be judged from the abstract

      Only the abstract was available. It does not say how many relatives were studied, how they were classed as affected or unaffected, or what the linkage scores were. The claims of high penetrance and more severe disease in the youngest generation cannot be checked, and the word 'apparently' shows the authors were cautious about the second point.

      “apparently the most severe limb muscle involvement in the youngest generation”Abstract

    How “unaffected” relatives were checked

    Cannot be judged: the abstract reports high penetrance but does not say whether relatives were examined by a neurologist or had nerve conduction tests.

    Three ways to explain it

    Scientific

    In the first European family reported with CMT2C, linkage to the CMT2A, B, D, E and F loci and to the distal hereditary motor neuronopathy type VII locus was excluded, supporting CMT2C as a genetically distinct entity. The disease showed high penetrance and variable expressivity, with apparently more severe limb involvement in the youngest generation, and vocal cord paralysis did not correlate with limb weakness. Family size and assessment methods are not given in the abstract.

    Easier

    Doctors described the first European family known to have CMT2C, a type of CMT that can affect the vocal cords and the breathing muscles. Genetic tests showed the condition was not linked to any of the CMT2 gene regions known at the time, so it was a separate condition. Most people who inherited it showed signs, severity varied, and how weak someone's vocal cords were did not match how weak their arms and legs were.

    Simple

    In one family, many people had CMT2C, and it affected each of them differently. Some had a weak voice because their vocal cords did not move well, even when their arms and legs were not very weak. For others it was the other way round.

    Why these ratings

    Relevance 5/5: It describes a CMT2C family, including how vocal cord, breathing and limb problems relate to each other.

    Evidence 1/5: Rated 1 because only the abstract was available, so family size, examination methods and linkage data could not be checked.

    Funding and conflicts of interest

    Not stated in the abstract.

    PubMed 12062259 · DOI

24. #24Abstract onlyPhD thesis: genetic linkage and gene discovery in families, with cell and frog egg studies

    Clinical, laboratory, and genetic studies of families with Charcot-Marie-Tooth type 2C disease

    Landoure G · 2011 · University College London (University of London)

    Relevance 5/5Evidence 1/51 moderate problem

    This thesis describes work that linked CMT2C in families to changes in the TRPV4 gene, including R269C and R269H. In lab cells these changes made the channel more active and more harmful to cells.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    PhD thesis: genetic linkage and gene discovery in families, with cell and frog egg studies

    Who or what

    Five unrelated families with CMT2C, more than 200 controls, and TRPV4 variants expressed in HEK293 cells and Xenopus oocytes (frog eggs). The abstract does not give the number of people studied.

    What they did

    The author ran linkage analysis and SNP genotyping in the families, sequenced all genes in the linked region of chromosome 12, then tested the R269C and R269H variants in HEK293 cells and Xenopus oocytes and mapped the mutations onto the structure of the ankyrin repeat domain. This analysis is based only on the thesis abstract, so the methods cannot be judged.

    What they found

    • Two of the five families showed significant linkage to chromosome 12q24.11, and this region was excluded in one other family, suggesting CMT2C can have more than one genetic cause.

      “We studied five unrelated families with CMT2C, of which two showed significant linkage to chromosome 12q24.11.”Abstract

    • Sequencing found TRPV4 mutations R269C and R269H in two families, both absent from more than 200 controls. R186Q, R232C and a change of uncertain significance (D584N) were found later.

      “two heterozygous missense mutations were identified in the transient receptor potential, subfamily V, member 4 (TRPV4), at positions c.805C>T and c.806G>A in two families, predicting the amino acid substitutions R269C and R269H, respectively.”Abstract

    • In HEK293 cells and frog eggs, R269C and R269H reached the cell surface normally but increased cell toxicity and channel currents compared with the normal channel. Neuropathy mutations sat on the opposite face of the ankyrin repeat domain from skeletal dysplasia mutations.

      “R269C and R269H TRPV4 trafficked normally to the cell surface, but caused increased cell toxicity and increased constitutive and activated channel currents compared to wild-type TRPV4.”Abstract, later part

    What the authors concluded

    TRPV4 mutations cause a degenerative disorder of peripheral nerves, and different mutations in this channel produce strikingly different diseases.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • ModerateMethods cannot be judged from the abstract

      Only the abstract was available, so it is not possible to check how many people were studied, how relatives were classed as affected or unaffected, the linkage scores, or how the cell experiments were done. The channel tests used HEK293 cells and frog eggs (Xenopus oocytes), not nerve cells. It is a PhD thesis rather than a journal article.

      “When expressed in HEK293 cells and Xenopus oocytes”Abstract, later part

    How “unaffected” relatives were checked

    Cannot be judged: the abstract does not say how family members were classed as affected or unaffected, or whether they were examined or had nerve tests.

    Three ways to explain it

    Scientific

    Linkage of two of five CMT2C families to 12q24.11 and sequencing of the interval identified heterozygous TRPV4 missense mutations R269C and R269H, with R186Q, R232C and a variant of uncertain significance (D584N) found subsequently; one family excluded the locus, indicating genetic heterogeneity. The neuropathy residues lie in the intracellular N-terminal ankyrin repeat domain, on the opposite face from skeletal dysplasia mutations. R269C and R269H trafficked normally but increased cytotoxicity and constitutive and activated currents, consistent with gain of function. Methods and data cannot be assessed from the abstract.

    Easier

    This PhD research studied five families with CMT2C. In two of them the condition was traced to a stretch of chromosome 12, where changes in the TRPV4 gene were found. In one other family, the condition did not trace back to that region, so CMT2C may have more than one cause. In lab-grown cells, two of the TRPV4 changes made the channel pass more current and harmed the cells. The nerve-disease changes sat on a different side of one part of the protein from changes that cause bone disease.

    Simple

    TRPV4 is like a door that lets calcium into cells. This research found that in some families with CMT2C the door has a small fault. In lab cells, the faulty door let through more calcium than normal and hurt the cells. One family's illness did not seem to come from this door.

    Why these ratings

    Relevance 5/5: It describes work identifying TRPV4 as a cause of CMT2C in families and how the R269C and R269H changes affect the channel.

    Evidence 1/5: Rated 1 because only the abstract was available, so the family data, linkage scores and lab methods could not be checked; it is also a thesis rather than a journal article.

    Funding and conflicts of interest

    Not stated in the abstract.

25. #25Abstract onlyClinical description of two families, with a genetic linkage exclusion in one

    Hereditary motor and sensory neuropathy with diaphragm and vocal cord paresis

    Dyck PJ, Litchy WJ, Minnerath S et al. · 1994 · Annals of Neurology

    Relevance 5/5Evidence 1/51 moderate · 1 minor problems

    This is the paper that first named the condition now called CMT2C. It described weakness of the limbs, vocal cords and breathing muscles that varied a lot between people, and sensory loss that people did not notice themselves.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    Clinical description of two families, with a genetic linkage exclusion in one

    Who or what

    Two families (kindreds) with an inherited nerve disorder. The abstract does not give the number of people examined.

    What they did

    The authors described the symptoms, examination findings and nerve test results in two families, and named the condition HMSN IIc (now called CMT2C). In one large family they checked whether the condition was linked to the gene regions known for HMSN IA and IB. This analysis is based only on the abstract, so the methods cannot be judged.

    What they found

    • The condition was passed down in a dominant pattern and caused weakness of the limbs, vocal cords and the muscles between the ribs, to a varying degree.

      “an autosomal dominant inherited disorder characterized by a variable degree of muscle weakness of limbs, vocal cords, and intercostal muscles and by asymptomatic sensory loss”Abstract

    • The authors found sensory loss that people had not noticed themselves, which means it was picked up by examination or testing rather than by asking.

      “asymptomatic sensory loss”Abstract

    • In severely affected people, it began in infancy or childhood, and breathing failure shortened their lives.

      “Life expectancy in severely affected patients is shortened because of respiratory failure.”Abstract

    • Nerve conduction speeds were normal, so the authors classed it as an axonal type 2 neuropathy and gave it the name HMSN IIc.

      “Because nerve conduction velocities are normal and it is an inherited axonal neuropathy, we classify the disorder as a variety of hereditary motor and sensory neuropathy type II (HMSN II) (HMSN IIc).”Abstract

    What the authors concluded

    This is a distinct type of inherited axonal neuropathy (HMSN IIc), separate from the HMSN IA and IB forms, which adds to the evidence that type II neuropathy has several different causes.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • ModerateMethods cannot be judged from the abstract

      Only the abstract was available. It does not say how many people were studied, how many were severely affected, or how it was decided which relatives were affected and which were not. The statement about shortened life expectancy cannot be checked, and it describes severely affected people in two families in 1994, not everyone with CMT2C.

      “Life expectancy in severely affected patients is shortened because of respiratory failure.”Abstract

    • MinorWritten before the gene was known

      The paper predates the 2010 discovery that TRPV4 changes cause this condition, so it cannot say which gene change these families carried. Later papers, not this one, link HMSN IIC to TRPV4.

      “no linkage to DNA markers known to map near the HMSN IA locus on chromosome 17p or the HMSN IB locus on chromosome 1q was demonstrated”Abstract

    How “unaffected” relatives were checked

    Partly visible: the abstract reports 'asymptomatic sensory loss', which can only be found by examining or testing people, and normal nerve conduction velocities, which shows nerve tests were done. It does not say whether every relative was examined.

    Three ways to explain it

    Scientific

    Dyck and colleagues described two autosomal dominant kindreds with an axonal hereditary motor and sensory neuropathy featuring variable limb, vocal cord and intercostal weakness and asymptomatic sensory loss, onset in infancy or childhood in severe cases, and reduced life expectancy from respiratory failure in severely affected patients. Normal conduction velocities placed it within HMSN II, and they designated it HMSN IIc. Linkage to the HMSN IA (17p) and IB (1q) loci was not demonstrated in one large pedigree.

    Easier

    This 1994 paper is the first description of the condition now called CMT2C. In two families, it caused weakness in the arms and legs, the vocal cords and the muscles used for breathing, and how severe this was varied a lot between people. Some people had lost feeling without noticing it. In the most severely affected, it started in childhood and breathing failure shortened their lives. Nerve signal speeds were normal, which showed the nerve fibers themselves (the axons) were affected, so the authors named it HMSN IIc.

    Simple

    This is the first paper about CMT2C. Doctors met two families where the condition made arms, legs, voice and breathing muscles weak, more in some people than others. Some people had lost a little feeling without knowing, like a smoke alarm with a quiet battery that only a tester notices.

    Why these ratings

    Relevance 5/5: This is the paper that first described and named HMSN IIc, the condition now called CMT2C.

    Evidence 1/5: Rated 1 because only the abstract was available, so family sizes, how people were examined and the linkage data could not be checked.

    Funding and conflicts of interest

    Not stated in the abstract.

    PubMed 8179305 · DOI

26. #26Abstract onlyFamily report

    Varying occurrence of vocal cord paralysis in a family with autosomal dominant hereditary motor and sensory neuropathy

    Donaghy M, Kennett R · 1999 · Journal of Neurology

    Relevance 5/5Evidence 1/51 moderate · 1 minor problems

    In this family, two relatives with no symptoms at all had the neuropathy when their nerves were tested. It is an early example of why 'no symptoms' is not the same as 'not affected'.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    Family report

    Who or what

    One white British family: a person with symptoms, their mother and their sister.

    What they did

    The authors described one family member who developed vocal cord paralysis at 41, and tested the mother and sister with nerve conduction studies even though they had no symptoms. This analysis is based only on the abstract, so the methods cannot be judged in detail.

    What they found

    • One family member developed vocal cord paralysis (recurrent laryngeal nerve palsy) at age 41, after 4 years of nerve symptoms, and also had a palsy of one of the nerves that move the eye.

      “developed a recurrent laryngeal nerve palsy at the age of 41 years, in addition to 4 years of symptomatic polyneuropathy and an abducens nerve palsy”Abstract

    • The mother and sister had no symptoms, but nerve tests showed they had the neuropathy too.

      “Neither of the other family members (the mother and sister) with electrophysiologically confirmed polyneuropathy had any neuropathic symptoms in the limbs or laryngeal or respiratory muscle involvement.”Abstract

    • The authors say this shows the value of examining and nerve-testing close relatives, rather than relying on whether they report symptoms.

      “The discovery of asymptomatic family members attests to the diagnostic value of clinical and electrophysiological study of first-degree relatives”Abstract

    • Unlike in the two earlier families, vocal cord problems here started in adulthood rather than in the first two decades of life.

      “This family differs from the two previously reported pedigrees in which laryngeal or diaphragm weakness had commenced within the first two decades.”Abstract

    What the authors concluded

    This is the second report of HMSN type IIC. Laryngeal involvement can begin in adulthood, and relatives without symptoms can still have the neuropathy, so first-degree relatives should be examined and nerve-tested.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • ModerateMethods cannot be judged from the abstract

      Only the abstract was available, so the examination findings, nerve test values and family tree cannot be checked.

    • MinorNo gene test was possible in 1999

      The diagnosis of HMSN IIC rests on the clinical picture, because TRPV4 had not been found yet. The family has not been confirmed to carry a TRPV4 change in this paper, and the authors say inheritance is only 'likely' dominant.

      “An autosomal dominant pattern of inheritance is likely.”Abstract

    How “unaffected” relatives were checked

    Examined and tested: the mother and sister had no symptoms but had nerve conduction studies, which confirmed polyneuropathy. Quote: “the mother and sister with electrophysiologically confirmed polyneuropathy”.

    Three ways to explain it

    Scientific

    In a British family with autosomal dominant axonal HMSN, the proband developed recurrent laryngeal nerve palsy at 41 years with an abducens palsy, while the mother and sister had electrophysiologically confirmed but asymptomatic polyneuropathy. The authors interpret this as the second HMSN IIC kindred, notable for adult-onset laryngeal involvement, and argue for clinical and electrophysiological screening of first-degree relatives. No molecular confirmation was available.

    Easier

    Doctors described a British family where one person lost the use of a vocal cord nerve at age 41, after having nerve symptoms in the limbs for 4 years. Their mother and sister felt completely fine, but nerve tests (which measure how well signals travel along the nerves) showed they had the same nerve condition. The doctors concluded that close relatives should be examined and tested, because feeling well does not prove someone is unaffected.

    Simple

    One person in a family had a weak voice from an inherited nerve condition, probably CMT2C. Their mom and sister felt fine, but a nerve test showed they had it too, like a car that drives well but whose check-up still finds a fault.

    Why these ratings

    Relevance 5/5: It is one of the earliest CMT2C family reports and directly shows relatives who felt well but had the neuropathy on testing.

    Evidence 1/5: Rated 1 because only the abstract was available, and it is a single small family without genetic confirmation.

    Funding and conflicts of interest

    Not stated in the abstract.

    PubMed 10463355 · DOI

27. #27Full paperGenetic screening study of a patient cohort, with family segregation, population controls and limited lab follow-up

    Comprehensive analysis of the TRPV4 gene in a large series of inherited neuropathies and controls

    Fawcett KA, Murphy SM, Polke JM et al. · 2012 · Journal of neurology, neurosurgery, and psychiatry

    Relevance 4/5Evidence 4/53 moderate · 4 minor problems

    In this large UK group, TRPV4 changes were a rare cause of axonal CMT and motor neuropathy, and some rare TRPV4 variants turned out to be harmless. The authors stress that a new TRPV4 variant needs careful checking before it is reported as the cause of someone's neuropathy.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    Genetic screening study of a patient cohort, with family segregation, population controls and limited lab follow-up

    Who or what

    422 unrelated patients with inherited neuropathy from a London specialist center and UK referrals, all without a cause found in the common genes. They were reported as 271 with axonal CMT (CMT2) and 151 with distal hereditary motor neuropathy (dHMN); 368 were British and the rest came from the Far East, Pakistan, India and other European countries. At least 345 population controls from the 1958 British birth cohort plus Pakistani controls. Relatives of four variant carriers, and blood-derived cells (lymphoblasts) from one patient and one control.

    What they did

    They sequenced the protein-coding parts of the TRPV4 gene in each patient. New variants were checked in healthy controls, in relatives where possible, and with five prediction programs. For one variant that creates an early stop signal (Y567X), they measured TRPV4 RNA and protein in the patient's blood cells.

    What they found

    • Five new TRPV4 variants were found that were absent from controls: G20R, E218K, N302Y, Y567X and T701I.

      “13 missense and nonsense variants were identified, of which five were novel and absent from controls (G20R, E218K, N302Y, Y567X and T701I).”Abstract, Results

    • Only three of these (G20R, N302Y and T701I) were judged possibly disease-causing, so TRPV4 changes explained less than 1% of CMT2 and dHMN in this cohort.

      “In this comprehensive analysis of the TRPV4 gene, we identified mutations in <1% of patients with CMT2/dHMN.”Abstract, Conclusions

    • G20R was found in a woman with dHMN and in her father, who had no symptoms and normal strength but some EMG changes. The authors suggest the variant may cause disease of variable severity, but this is uncertain.

      “G20R was detected in a patient with dHMN and her asymptomatic father and is possibly pathogenic with variable expressivity.”Abstract, Results

    • Y567X, which cuts the protein short, traveled with severe CMT2 in one family that also carried an MFN2 change. The faulty RNA was destroyed, yet the amount of TRPV4 protein in blood cells looked normal. So the authors judged Y567X unlikely to cause disease and concluded that losing one working copy of TRPV4 (haploinsufficiency) is not enough to cause neuropathy.

      “Although Y567X caused nonsense mediated mRNA decay, the amount of TRPV4 protein on western blotting of patient lymphoblasts was no different to control. Y567X is therefore unlikely to be pathogenic.”Abstract, Results

    • Rare TRPV4 variants, including ones that stop the protein early, are not always harmful. Some were seen in a healthy control or did not travel with disease in the family.

      “this study demonstrates that TRPV4 is a highly polymorphic gene in which rare missense and nonsense variants do not necessarily cause disease.”Discussion

    • Citing earlier studies, the authors note that TRPV4 changes were more common in CMT2 with extra features such as vocal cord or diaphragm weakness, and suggest routine TRPV4 testing could focus on such patients.

      “suggesting routine TRPV4 screening could be restricted to patients with such features”Discussion

    What the authors concluded

    TRPV4 mutations are a rare cause of CMT2 and dHMN, found in less than 1% of patients in this cohort. TRPV4 carries many rare harmless variants, so a new variant should be checked with family studies, prediction tools and, above all, control screening before results are given to patients. Losing one copy of TRPV4 does not appear to be enough to cause neuropathy.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • MinorPatient and control numbers do not match across the paper

      The introduction and methods describe 279 CMT2 and 173 dHMN patients, but the abstract and results report sequencing 271 and 151 (422 in total). Control numbers are also given differently (at least 345, 350 Caucasian, 96 or 91 Pakistani). The difference is not explained. It does not change the headline frequency much, but it is unclear exactly who was tested.

      “we screened TRPV4 coding exons and splice junctions for mutations in 279 unrelated CMT2 and 173 unrelated HMN patients”Introduction; compare Methods, Results and Abstract

    • ModerateDisease role of the three 'potentially pathogenic' variants not shown

      No lab tests of channel function were done. Segregation could not be checked for N302Y or T701I. Prediction programs disagreed: four of five called G20R neutral and only one of five called N302Y damaging. These variants should be seen as unproven.

      “Although not enough family members were available for segregation analyses, N302Y and T701I are potentially pathogenic mutations requiring further functional validation.”Discussion

    • ModerateHow relatives were judged 'unaffected' is mostly not described

      Several conclusions depend on relatives being unaffected. E218K was ruled out because two 'unaffected' siblings carried it, and Y567X was said to segregate using an 'unaffected' mother and brother. The paper does not say whether these relatives were examined or had nerve conduction studies. The N302Y patient's son was not examined. Only the G20R father is described as examined, and his EMG suggested he might be mildly affected.

      “The variant was also present in two unaffected siblings (figure 2B) and is predicted to be benign by three out of five web based prediction algorithms.”Results (E218K)

    • ModerateHaploinsufficiency conclusion rests on very little data

      The claim that losing one copy of TRPV4 does not cause neuropathy is based on one western blot of blood-derived cells from one patient compared with one unrelated control, plus a second stop variant seen in a control. Blood cells are not nerve cells, and no measurement or statistics of the blot are described.

      “Lymphoblasts from the proband with the TRPV4 Y567X mutation and an unrelated control were extracted from peripheral blood”Methods, Cell culture and western blotting

    • MinorLimited clinical information for about half the patients

      Only 139 (50%) of the CMT2 patients and 118 (68%) of the dHMN patients were seen in the authors' hospital. The rest were DNA samples sent in with limited clinical details, so some diagnoses may be less certain. One patient referred as CMT2 was later found to have no neuropathy.

      “For external patients, more limited information was available and diagnosis was based on the information received.”Methods, Patient cohort

    • MinorSelected, pre-screened cohort

      Patients came from a specialist center and had already tested negative for common genes. The frequency estimate applies to this kind of group, not to all people with CMT2.

      “who had not been found to have a mutation in the common genes causing CMT2 and dHMN or where a variant had been identified that did not explain the phenotype”Methods, Patient cohort

    • MinorOur copy lacks the table, figures and supplement

      The archived text used here does not include Table 1, the pedigrees or the supplementary tables, so details such as the full variant list could not be checked.

    How “unaffected” relatives were checked

    Mixed and mostly not described. The G20R father was examined and had EMG, which raised the possibility that he is mildly affected: “Her father was asymptomatic with normal muscle strength but EMG of the tibialis anterior showed occasional higher amplitude giant potentials (5-7 mV)”. The N302Y patient's son was not examined: “Her 35-year-old son was asymptomatic but has not been examined.” For the 'unaffected' siblings (E218K), mother and brother (Y567X) and sister (T701I), the paper does not say whether they were examined or had nerve conduction studies.

    Three ways to explain it

    Scientific

    In 422 patients with CMT2 or dHMN negative for common genes, TRPV4 sequencing found five novel variants absent from controls, of which only G20R, N302Y and T701I were considered potentially pathogenic, giving a TRPV4 mutation frequency of less than 1%. Pathogenicity was not established functionally or by segregation. The nonsense variant Y567X caused nonsense-mediated decay without reducing lymphoblast TRPV4 protein, from which the authors infer that haploinsufficiency is not sufficient to cause neuropathy and that TRPV4 harbors many rare benign variants.

    Easier

    The researchers checked the TRPV4 gene in 422 people with inherited nerve disease whose cause had not been found. Less than 1% had a TRPV4 change that might cause disease, and even those changes were not proven to be harmful. Some rare TRPV4 changes, including one that cuts the protein short, seemed to be harmless, so the authors say new TRPV4 changes must be checked carefully before patients are told.

    Simple

    Scientists checked TRPV4, the instructions for a tiny gate in our cells, in 422 people with nerve problems. Only a very few had a change that might cause trouble, and some changes looked like harmless typos, like a spelling slip that does not change what a sentence means.

    Why these ratings

    Relevance 4/5: It measures how often TRPV4 changes cause axonal CMT and motor neuropathy and shows that many rare TRPV4 variants are harmless, which matters for anyone given a TRPV4 result, though no confirmed CMT2C cases were found.

    Evidence 4/5: A large screen with population controls supports the low frequency, but the disease role of each new variant rests on prediction tools and small families without functional tests.

    Funding and conflicts of interest

    Funded by the Medical Research Council, the Muscular Dystrophy Campaign, NINDS/ORD, an Alzheimer's Research UK fellowship, the Wellcome Trust and NIHR Biomedical Research Centre funding. Competing interests: one author (RQ) was a principal investigator for a drug trial sponsored by PTC Therapeutics, and has received research grants from the Muscular Dystrophy Campaign, Action Research and AGSD, as well as lecture fees from Genzyme. The paper does not link these to TRPV4.

    PubMed 22851605 · DOI

28. #28Full paper (scan)Genetic linkage (exclusion) study in one large family

    Hereditary motor and sensory neuropathy type 2C is genetically distinct from types 2B and 2D

    Nagamatsu M, Jenkins RB, Schaid DJ et al. · 2000 · Archives of neurology

    Relevance 4/5Evidence 4/53 moderate · 2 minor problems

    This paper is part of the history of how CMT2C was defined. It showed that the gene in a large CMT2C family did not lie where two other CMT2 genes had been mapped, but it did not find the gene and gives little clinical information.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    Genetic linkage (exclusion) study in one large family

    Who or what

    One large family with HMSN 2C (CMT2C) studied at the Mayo Clinic Peripheral Neuropathy Research Center: 33 subjects, including 12 affected individuals and 11 individuals at risk. DNA markers tested: 8 markers in chromosome region 3q13-q22 (the HMSN 2B locus) and 7 markers near 7p14 (the HMSN 2D locus).

    What they did

    The team took blood from family members and tested DNA markers in two regions of the genome where other forms of CMT2 (2B and 2D) had been mapped. They used linkage analysis, a statistical method that asks whether a stretch of DNA is passed down together with the disease more often than chance would allow. They also ran simulations to check how much information the family could provide.

    What they found

    • The family showed dominant inheritance, with weakness and wasting of the lower-leg (peroneal) and hand muscles, and 9 of the 12 affected people had weakness or paralysis of the diaphragm or vocal cords.

      “Of 12 affected subjects, 9 had clinical evidence of diaphragmatic or vocal-cord paresis or paralysis.”Methods, Kinship, p. 670

    • Severity varied between affected family members and did not differ between men and women.

      “The severity of clinical symptoms in affected persons was variable; phenotypes did not differ by sex.”Methods, Kinship, p. 670

    • The HMSN 2B region on chromosome 3 was excluded: all two-point LOD scores were negative and multipoint scores were below minus 2 across the region.

      “The multipoint LOD scores in the region of these markers were less than -2, excluding the HMSN 2C locus from this region.”Results, p. 670

    • The HMSN 2D region on chromosome 7 was also excluded.

      “the multipoint LOD scores in the region of these markers were less than -2 between points 9.7 cM proximal to D7S1801 and 10.1 cM distal to D7S435, which excludes the HMSN 2C locus from this region.”Results, p. 670

    • Simulations showed the family carried enough information for linkage studies, with a maximum attainable LOD score of 7.7.

      “The maximum attainable LOD score at a recombination fraction of 0 with fully informative linkage markers including all persons at risk was 7.7 (average LOD score, 6.1).”Methods, Linkage analysis, p. 671

    What the authors concluded

    The authors conclude that HMSN 2C is genetically distinct from HMSN 2A, 2B and 2D, so at least 4 genetically different forms of autosomal dominant HMSN 2 exist. They state that a linkage study using markers across many chromosomes would be needed to find the HMSN 2C locus.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • ModerateOne family used to speak for the whole disease type

      Only the larger of the two HMSN 2C families the group had described was tested, and exclusion of HMSN 2A came from an earlier study. The title says HMSN 2C in general is distinct from 2B and 2D, but strictly the result applies to this family. Another family with a similar picture could have a different genetic cause.

      “We have reported 2 unrelated HMSN 2C families; the larger of these kindred was genetically distinct from HMSN 2A.”Introduction, p. 669

    • ModerateModel assumed near-complete penetrance

      The analysis assumed a penetrance of 0.99 and no phenocopies. If a relative counted as unaffected actually carried the faulty gene, or an affected person had neuropathy for another reason, the model would count this as evidence against linkage. The authors reduced this risk by leaving out at-risk unaffected relatives under 26, but did not report analyses under other penetrance values.

      “no phenocopies were assumed, and penetrance was assumed to be 0.99 for heterozygotes and 1.0 for homozygotes.”Methods, Linkage analysis, p. 671

    • ModerateHow unaffected relatives were judged is not described here

      The pedigree marks people as affected by examination, affected by history, or unaffected. This paper refers back to an earlier publication for clinical details and does not say whether unaffected relatives had a neurological examination or nerve conduction studies. Some affected status also comes from history alone. Because linkage depends on correct labels, this matters for how far the exclusion can be trusted.

      “black and white symbols, affected by history; solid black symbols, affected by clinical examination; white symbols, unaffected”Figure 1 legend, p. 670

    • MinorExclusion only; the gene was not found

      The study shows where the HMSN 2C gene is not, in two regions. It does not locate the gene, as the authors say themselves.

      “A linkage study employing DNA markers from multiple autosomes will be needed to map the HMSN 2C locus.”Comment, p. 672

    • MinorErrors in terms and marker lists

      The paper calls LOD 'limit of detection', although its own Methods describe the score as the logarithm of a likelihood ratio. The chromosome 7 marker names also differ between the Methods (which list D7S1869), the Table (which lists D7S516 instead) and Figure 3 (which shows D7S1801). These slips do not change the result but reduce clarity.

      “Pairwise limit of detection (LOD) scores (Z values) were calculated”Methods, Linkage analysis, p. 671

    How “unaffected” relatives were checked

    Not described in detail in this paper. The Methods say the 'neuropathic status of 33 subjects' was investigated and refer to an earlier publication for clinical and electrophysiologic details. The pedigree legend separates 'affected by history', 'affected by clinical examination' and 'unaffected'. Young at-risk relatives were left out: 'Clinically unaffected offspring who were at risk but younger than 26 years were excluded.' Whether unaffected relatives had nerve conduction studies is not stated.

    Three ways to explain it

    Scientific

    In a large autosomal dominant HMSN 2C kindred (12 affected, 11 at risk), two-point and multipoint linkage analysis excluded the HMSN 2B (3q13-q22) and HMSN 2D (7p14) loci, with multipoint LOD scores below -2 across both regions under a model of 0.99 penetrance and no phenocopies. Together with earlier exclusion of HMSN 2A, the authors infer at least 4 genetically distinct autosomal dominant HMSN 2 types. The HMSN 2C locus itself was not mapped.

    Easier

    The researchers studied one large family with CMT2C, an inherited nerve disease that can also weaken the vocal cords and the main breathing muscle (the diaphragm). They tested whether the disease was passed down together with two stretches of DNA already linked to other types of CMT2. It was not, so the CMT2C gene had to be somewhere else in the genome. This study did not find where.

    Simple

    Scientists were looking for the gene behind one family's nerve illness, a bit like hunting for a lost key in a big house. They searched two rooms where keys for similar illnesses had been found, and the key was not there. They still had to search the rest of the house.

    Why these ratings

    Relevance 4/5: Directly about a large HMSN 2C (CMT2C) family with vocal cord and diaphragm weakness, but it only rules out two gene locations and gives little clinical detail, so its value today is mainly historical.

    Evidence 4/5: Strongly negative LOD scores in a large, clinically characterized and informative family make the exclusion of these two regions convincing, but it is one family, only two regions were tested, and the model assumed near-complete penetrance.

    Funding and conflicts of interest

    Supported in part by research grants from the Muscular Dystrophy Association of America (grant 12), the National Institute of Neurological Disorders and Stroke (grant 14304) and the Kawamura fund, Gifu City, Japan. Competing interests: not stated.

    PubMed 10815132 · DOI

29. #29Full paperLaboratory study in fruit flies and cultured mouse nerve cells

    TRPV4 disrupts mitochondrial transport and causes axonal degeneration via a CaMKII-dependent elevation of intracellular Ca2

    Woolums BM, McCray BA, Sung H et al. · 2020 · Nature communications

    Relevance 4/5Evidence 3/53 moderate · 3 minor problems

    In flies and mouse nerve cells, a CMT2C-linked TRPV4 mutation made the channel overactive and damaged axons through calcium, an enzyme called CaMKII and stalled mitochondria (the cell's power stations). A TRPV4 blocker reduced the damage in flies. This points to possible treatment targets, but nothing here was tested in people.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    Laboratory study in fruit flies and cultured mouse nerve cells

    Who or what

    Fruit flies (Drosophila) engineered to make human TRPV4 in their nerve cells at low, moderate or high levels: normal TRPV4, the neuropathy mutation R269C, R269C with an added pore-blocking change (M680K), and R232C for one test. A genetic screen of 692 fly lines covering 502 genes. Trigeminal nerve cells from mice aged 6 to 10 weeks, grown in dishes and given extra human TRPV4 (normal or R269C) by a virus. An immortalized rat sensory nerve cell line for one experiment.

    What they did

    They measured wing expansion, climbing, nerve cell shape, electrical activity, calcium levels and the movement of mitochondria (the cell's power stations) along axons in flies making normal or mutant TRPV4. They tested a TRPV4-blocking drug (GSK2193874, also called GSK219), screened for genes that change the damage, and checked key calcium results in mouse nerve cells grown in dishes.

    What they found

    • In flies, R269C caused nerve cell dysfunction and axon degeneration that grew worse with higher amounts of the protein. The damage disappeared when the channel pore was blocked genetically or with a drug, pointing to too much channel activity (gain of function).

      “in vivo expression of a neuropathy-causing TRPV4 mutant (TRPV4 R269C ) causes dose-dependent neuronal dysfunction and axonal degeneration, which are rescued by genetic or pharmacological blockade of TRPV4 channel activity”Abstract; Results, Figures 1 and 2

    • Feeding fly larvae the TRPV4 blocker GSK219 reduced axon and dendrite damage caused by R269C.

      “Remarkably, GSK219 treatment of TRPV4 R269C (high) flies reduces axonal degeneration and dendritic branching defects as compared to vehicle treatment alone”Results, TRPV4 antagonists suppress TRPV4-mediated neurodegeneration

    • A screen identified the enzyme CaMKII as the strongest modifier. Lowering CaMKII almost abolished the R269C wing problem and also reduced axon loss, climbing problems and nerve overactivity in flies.

      “Knockdown of the single Drosophila CaMKII using four independent RNAi lines on three distinct genetic backgrounds reduces the penetrance of the TRPV4 R269C -mediated wing phenotype to 0-10%”Results, CaMKII is required for TRPV4 R269C-mediated neurotoxicity

    • In cultured mouse nerve cells, R269C gave a larger calcium response to a TRPV4-activating drug than normal TRPV4, and a CaMKII inhibitor (KN-93) blunted TRPV4 calcium responses.

      “The maximum response of TRPV4 R269C (1.2 ± 0.016) is greater than that of TRPV4 WT (0.91 ± 0.014)”Results, Figure 5

    • R269C slowed the transport of mitochondria along fly axons before any visible degeneration, suggesting this is an early step.

      “we observed mitochondrial transport defects downstream of neuropathogenic TRPV4 activity prior to the onset of observable degeneration, suggesting that mitochondrial transport impairments are an early pathological event.”Discussion; Results, Figures 6 and 7

    • The mitochondrial transport protein Miro made R269C damage worse, but only when Miro could bind calcium.

      “Miro-mediated enhancement of TRPV4 R269C -dependent axonal degeneration is dependent on the ability of Miro to bind Ca 2+”Results, Miro modifies TRPV4 R269C-mediated toxicity

    What the authors concluded

    Neuropathy-causing TRPV4 mutations make the channel overactive (gain of function). This raises calcium inside nerve cells in a way that needs CaMKII, disrupts the transport of mitochondria along axons through the protein Miro, and leads to axon degeneration. Because TRPV4 blockers prevented the damage in flies, the authors see TRPV4 antagonists as a promising treatment strategy for patients.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • ModerateArtificial models, far from people, mostly one mutation

      The human gene was added to flies at levels the researchers chose, and the mouse cells were normal nerve cells given extra TRPV4 by a virus, not cells carrying the mutation in their own gene. Almost all experiments used one mutation, R269C; R232C was tested only in the wing assay. Results may not translate directly to people with CMT2C or to other mutations.

      “we generated transgenic Drosophila lines that express human TRPV4 under the control of the GAL4/UAS binary expression system”Results, first section; Methods

    • ModerateDamage depended on very high expression, and normal TRPV4 was also toxic at high levels

      Axon degeneration in sensory neurons was seen with high but not moderate R269C expression, and high normal TRPV4 also caused mild damage. Some effects may therefore reflect overproduction rather than the mutation alone.

      “Of note, high expression (but not moderate expression) of TRPV4 WT causes a mild wing expansion phenotype and a mild loss of axonal projections and dendritic branching”Results, TRPV4 R269C causes axonal and dendritic degeneration

    • ModerateTreatment claims go beyond the data

      The abstract and discussion speak of promise for patients, but the blocker was only given to fly larvae in food and applied to fly brain preparations. No mammal was treated and no person took part.

      “These phenotypes are prevented by TRPV4 antagonists, which hold promise as a therapeutic strategy for the treatment of patients with TRPV4-associated neuropathies.”Discussion, final paragraph

    • MinorText overstates one mouse cell result

      The text says mouse neurons with R269C respond 'more rapidly' than normal, but the figure legend shows the difference in speed was not statistically significant (p = 0.2). Only the size of the response differed clearly.

      “t 1/2max = 16 ± 3.8 s ( p = 0.2, unpaired two-tailed t test)”Figure 5 legend; compare Results text

    • MinorDrug dose stated differently and partial drug resistance

      For the mitochondrial experiments, the methods say larvae were raised on 10 μM of the blocker, while the Figure 7 legend says 100 μM. The methods also note that R269C was relatively insensitive to the drug in electrical recordings.

      “larvae were raised on food with 10 μM GSK2193874 due to the observed insensitivity of TRPV4 R269C to this compound in electrophysiologic recordings”Methods, Live cell imaging of axonal transport; Figure 7 legend

    • MinorSmall groups and limited blinding

      Many experiments used 4 to 10 larvae, flies or cells per group, and two wing-assay groups had only 2 and 3 flies. Only the counting of calcium transients is described as blinded; wings were scored by eye without blinding being mentioned.

      “scored their wing phenotype by eye using a stereomicroscope”Methods, Wing expansion assay; figure legends

    Three ways to explain it

    Scientific

    Expression of human TRPV4 R269C in Drosophila neurons caused dose-dependent, pore-dependent neuronal dysfunction and axonal and dendritic degeneration, consistent with a gain-of-function mechanism, and the antagonist GSK2193874 reduced degeneration. A modifier screen identified CaMKII as required for R269C-induced hyperexcitability, Ca2+ elevation and toxicity, and CaMKII inhibition reduced TRPV4-mediated Ca2+ responses in cultured mouse trigeminal neurons. R269C impaired axonal mitochondrial transport before degeneration, and toxicity was modulated by Miro in a Ca2+-binding-dependent manner; all data come from overexpression models.

    Easier

    The researchers put a human TRPV4 gene carrying a neuropathy mutation into fruit flies and into mouse nerve cells in a dish. The faulty channel let in too much calcium, made nerve cells overactive, slowed the movement of mitochondria (the cell's power stations) along nerve fibers, and damaged the fibers. Blocking TRPV4 with a drug, or turning down an enzyme called CaMKII, reduced the damage in flies, but this has not been tested in people.

    Simple

    TRPV4 is like a door that lets calcium into nerve cells, and this mutation makes the door open too easily. In flies, too much calcium jammed the delivery trucks that carry power packs along the nerve. A medicine that closes the door helped the fly nerves, but it has not been tried in people.

    Why these ratings

    Relevance 4/5: It studies how a TRPV4 neuropathy mutation damages nerve cells and tests a TRPV4 blocker, but in flies and mouse cells rather than people.

    Evidence 3/5: Careful lab work with several methods giving consistent results, but it relies on overproducing human TRPV4 in flies and cultured mouse cells, with small groups, far from patients.

    Funding and conflicts of interest

    Funded by NIH NINDS grants, a Muscular Dystrophy Association grant and Inherited Neuropathy Consortium fellowships. The authors declare no competing interests.

    PubMed 32471994 · DOI

30. #30Full paperCell, protein and fruit fly study

    Neuropathy-causing TRPV4 mutations disrupt TRPV4-RhoA interactions and impair neurite extension

    McCray BA, Diehl E, Sullivan JM et al. · 2021 · Nature communications

    Relevance 4/5Evidence 3/52 moderate · 5 minor problems

    This lab study suggests one way CMT2C mutations might harm nerves: by breaking TRPV4's hold on RhoA, a protein that shapes nerve fibers. It was done in cells and flies, so it points to ideas for future research rather than anything shown in people.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    Cell, protein and fruit fly study

    Who or what

    Normal and mutant human TRPV4 studied in cultured cells: HEK293T human kidney-derived cells (including a stable line that makes normal or R269C TRPV4 when switched on), MN-1 mouse motor neuron-neuroblastoma hybrid cells and COS7 cells; purified proteins for NMR; mouse choroid plexus tissue; and fruit flies expressing human TRPV4 R269C. Four neuropathy mutations (R232C, R237L, R269C, R315W) were compared with two skeletal dysplasia mutations (I331F, D333G). Fly experiments used 9 to 17 neurons, 3 to 9 larvae, and 162 and 68 flies for the wing test.

    What they did

    The team searched for proteins that bind TRPV4 and found RhoA, a switch protein that controls the cell's internal scaffolding (the actin cytoskeleton). They tested how TRPV4 and RhoA affect each other, and whether neuropathy and skeletal dysplasia mutations change this, using binding tests, calcium imaging, RhoA activity sensors and measurements of nerve-like branches (neurites) in MN-1 cells. They then blocked the fly version of RhoA in flies carrying a human TRPV4 neuropathy mutation.

    What they found

    • The ankyrin repeat domain of TRPV4 binds directly to RhoA when RhoA is in its inactive (GDP-bound) state.

      “We demonstrate direct interaction of the TRPV4 ARD with inactive, GDP-bound RhoA”Discussion, paragraph 1

    • All four neuropathy mutations tested almost completely stopped TRPV4 from binding RhoA, while the skeletal dysplasia mutations kept binding to a variable degree. Blocking channel activity did not restore binding.

      “Remarkably, all tested neuropathy mutations resulted in nearly complete disruption of TRPV4-RhoA interaction, whereas the skeletal dysplasia mutations displayed variably preserved interaction with RhoA”Results, 'TRPV4 neuropathy mutations disrupt RhoA interaction'

    • When mutant and normal TRPV4 were made together in cells, the mutant reduced the normal channel's binding to RhoA, which the authors link to the dominant inheritance of TRPV4 neuropathy.

      “These results suggest that TRPV4 neuropathy mutations can act in trans to disrupt TRPV4-RhoA interactions, even in the presence of a WT TRPV4 allele”Discussion, 'TRPV4-RhoA interactions are specifically disrupted by neuropathy mutations'

    • Normal TRPV4 and RhoA hold each other back: RhoA reduced calcium entry through normal TRPV4 but not through mutant TRPV4, and normal TRPV4 reduced RhoA activation while the R269C mutant did not.

      “Together, these data suggest that TRPV4-RhoA interactions modulate TRPV4 channel activity, and that this modulatory effect is disrupted by neuropathy-causing mutations.”Results, 'RhoA inhibits TRPV4 ion channel function'

    • When the channel was switched on with a drug, mutant TRPV4 activated RhoA faster, for longer and more strongly than normal TRPV4.

      “RhoA FRET was significantly greater with agonist stimulation of neuropathy mutant TRPV4, with more rapid onset, longer duration, and higher peak RhoA activation”Results, 'TRPV4-mediated calcium influx activates RhoA'

    • Normal TRPV4 promoted nerve-like branches in MN-1 cells but the neuropathy mutant did not, even with the channel blocked. In flies, blocking the fly version of RhoA protected nerve fibers and dendrites from damage caused by mutant TRPV4.

      “we find that TRPV4 neuropathy mutant-induced axonal and dendritic degeneration can be rescued in vivo by genetic inhibition of the Drosophila RhoA ortholog Rho1.”Discussion, 'TRPV4-RhoA interactions are specifically disrupted by neuropathy mutations'

    What the authors concluded

    RhoA is a key partner through which TRPV4 shapes cells. Neuropathy mutations break the TRPV4-RhoA link, which leads to too much RhoA activity, too much channel activity and poor nerve branch growth. This may help explain why these mutations harm nerves while skeletal dysplasia mutations do not, and the authors suggest that targeting excess TRPV4 and RhoA activity may hold therapeutic promise.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • ModerateCells and flies, far from human nerves

      Most experiments used human kidney-derived cells and a mouse hybrid motor neuron-like cell line, with TRPV4 added artificially. No patient nerves or patient-derived nerve cells were studied. The fly model expresses human TRPV4 in fly nerve cells, and the rescue used a genetic change in Rho1 (the fly version of RhoA) rather than a drug. The results point to a mechanism but do not show it happens in people with CMT2C.

      “a mouse motor neuron-neuroblastoma fusion cell line”Results, 'TRPV4 interacts with the small GTPase RhoA'

    • MinorOverproduced proteins can have side effects

      Many experiments relied on cells making large amounts of TRPV4 and RhoA. The authors note that adding extra RhoA may itself change cell shape and calcium levels, which makes some results harder to interpret. They did use a stable cell line with only modest TRPV4 overproduction for key tests.

      “which may reflect a nonspecific effect of altered cytoskeletal dynamics with overexpression of RhoA”Results, 'RhoA inhibits TRPV4 ion channel function'

    • ModerateFew mutations tested in the functional experiments

      Binding was tested with four neuropathy and two skeletal dysplasia mutations. The later functional steps (calcium, RhoA activity, nerve branch growth and flies) used mainly one neuropathy mutation at a time (R237L or R269C). It is not shown that every CMT2C mutation behaves the same way.

      “we examined four neuropathy mutants that affect conserved arginines within the convex surface of the ARD (R232C, R237L, R269C, R315W) and two skeletal dysplasia mutants”Results, 'TRPV4 neuropathy mutations disrupt RhoA interaction'

    • MinorSmall numbers of repeats for some experiments

      Several results are shown as representative blots from two or three independent experiments, and some fly measurements used only 3 to 9 larvae. This is common in lab work but means some individual results rest on few repeats.

      “representative blot from two independent experiments”Methods, 'Statistics and reproducibility' (Fig. 1g)

    • MinorSmall reporting inconsistencies

      The figure legend for Fig. 2h gives n = 7 for lanes 1, 2 and 3, but the Methods give lanes 1, 2 and 5, listing lane 5 twice. These look like typing errors and do not change the main findings.

      “or 7 (lanes 1, 2, and 5) independent experiments”Methods, 'Statistics and reproducibility' compared with Fig. 2 legend

    • MinorProtein screen used an older counting method

      The first screen that picked out RhoA ranked proteins by spectral counts, which the authors say has largely been replaced by better methods. This matters little because RhoA binding was then confirmed by several other methods.

      “due to inherent limitations with quantification based on spectral counts, this approach has now largely been replaced by quantification based on precursor signal intensity”Methods, 'Liquid chromatography-mass spectrometry'

    • MinorTreatment idea not tested

      The authors suggest targeting TRPV4 and RhoA may help patients. This is worded cautiously, but no drug was tested in patients or in nerve models here.

      “Our results suggest that targeting excessive TRPV4 and RhoA pathway activity may hold therapeutic promise for patients with TRPV4 mutations”Discussion, 'Disrupted TRPV4-RhoA interaction and relevance to human disease'

    Three ways to explain it

    Scientific

    The TRPV4 ankyrin repeat domain binds inactive, GDP-bound RhoA directly, and this binding mutually restrains TRPV4 channel activity and RhoA activation. Neuropathy mutations at surface arginines of the ARD (R232C, R237L, R269C, R315W) nearly abolish the interaction independently of channel activity, whereas skeletal dysplasia mutations (I331F, D333G) show variably preserved binding. Loss of binding plus calcium-driven RhoA activation impairs neurite outgrowth in MN-1 cells, and dominant-negative Rho1 rescues axonal and dendritic degeneration in a Drosophila model expressing TRPV4 R269C. All data come from cell lines, purified proteins and flies.

    Easier

    TRPV4 is a channel on the surface of cells. This study found that it holds on to another protein, RhoA, which controls the cell's internal scaffolding. The TRPV4 mutations that cause nerve disease stop TRPV4 from holding RhoA, so RhoA becomes too active and the nerve-like branches of cells grown in dishes do not grow properly. In flies carrying a human TRPV4 neuropathy mutation, blocking the fly version of RhoA protected their nerve fibers. This was all done in cells and flies, so it is not yet known whether the same happens in people.

    Simple

    Think of TRPV4 as a door in a nerve cell that also holds the leash of a dog called RhoA, which can pull the cell's branches back in. The CMT2C changes make the door drop the leash, so RhoA pulls too much and the branches stay short. Calming RhoA helped in flies, but this has not been tested in people.

    Why these ratings

    Relevance 4/5: It studies a possible mechanism by which CMT2C and other TRPV4 neuropathy mutations, including R269C and R315W, damage nerves.

    Evidence 3/5: Careful lab work using several independent methods and an animal model, but all in cell lines and flies rather than people.

    Funding and conflicts of interest

    Funded by NIH (NINDS) grants including K08 NS102509, F31 NS105404, R01 NS094239, NS082563 and R01 NS115475, the Muscular Dystrophy Association (629305), an Inherited Neuropathies Consortium Fellowship, an American Academy of Neurology fellowship, and several PhD fellowships and German research center funds. The authors declare no competing interests.

    PubMed 33664271 · DOI

31. #31Full paperStructural biology and cell study (cryo-electron microscopy, electrophysiology, computer simulation and cell experiments)

    TRPV4-Rho GTPase complex structures reveal mechanisms of gating and disease

    Kwon DH, Zhang F, McCray BA et al. · 2023 · Nature communications

    Relevance 4/5Evidence 3/52 moderate · 3 minor problems

    This study gives a detailed picture of how a partner protein called RhoA may normally hold TRPV4 closed, and why CMT2C changes near that contact point could leave the channel too open. It is laboratory work on purified protein and cell lines, so it suggests a mechanism and a drug target rather than showing any effect in people.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    Structural biology and cell study (cryo-electron microscopy, electrophysiology, computer simulation and cell experiments)

    Who or what

    Purified full-length human TRPV4 made in human embryonic kidney (HEK293S GnTI-) cells; the purified channel carried along the cells' own Rho protein (assigned as RhoA). Four cryo-EM structures were solved (no drug, the blocker GSK279, and the activators GSK101 and 4α-PDD) at 3.30 to 3.75 Å. Function was tested in HEK293 and HEK293T cells, Xenopus frog eggs and a mouse motor neuron-neuroblastoma fusion cell line (MN-1). No patients or patient cells were studied.

    What they did

    The team purified human TRPV4 and used cryo-electron microscopy to see its 3D shape with and without drugs that open or close it. They found the partner protein RhoA attached to each of the channel's four subunits and mapped where known neuropathy changes sit. They then changed amino acids at the TRPV4-RhoA contact surface and measured binding and calcium entry in cultured cells.

    What they found

    • RhoA binds the ankyrin repeat region of each TRPV4 subunit and is anchored to the cell membrane by its tail, so it behaves like a built-in extra part of the channel.

      “RhoA appears to act as an auxiliary subunit, interacting with the ARD through three loops connecting AR2-AR3, AR3-AR4, AR4-AR5, and with the membrane bilayer through the prenylated C-terminal tail.”Results, structures of human TRPV4 in complex with Rho

    • Most amino acids changed in TRPV4 neuropathy sit at the contact surface with RhoA.

      “Strikingly, most residues mutated in TRPV4-mediated neuropathy (R237, R269, R315, and R316) are clustered at the interface with RhoA”Results, neuropathy mutations disrupt RhoA binding to TRPV4

    • Engineered changes at the contact surface, in either TRPV4 or RhoA, reduced binding between them and increased calcium entry through TRPV4 in cultured cells, similar to the neuropathy mutant R269C. Treating cells with a bacterial enzyme that blocks RhoA (C3 transferase) also increased calcium entry.

      “With expression of TRPV4 mutants that fail to interact with RhoA (TRPV4 E183A/C/K or D263A/L/K/N), we found increased basal and hypotonic saline-induced calcium influxes, similar to the neuropathy mutant R269C”Results, neuropathy mutations disrupt RhoA binding to TRPV4

    • The more strongly a RhoA variant still bound TRPV4, the more it held back TRPV4 activity, which the authors take as evidence that losing this contact underlies the overactivity caused by neuropathy changes.

      “These data suggest that TRPV4-RhoA interaction strength correlates with TRPV4 channel activity, and that disruption of this interaction underlies the gain of function due to neuropathy mutations within the ARD.”Results, neuropathy mutations disrupt RhoA binding to TRPV4

    • RhoA appeared more tightly held in the closed channel than in the open one (contact area about 752 versus 684 square ångströms). The authors propose RhoA acts like a clamp that stops the movement needed for opening.

      “When membrane-anchored RhoA binds to TRPV4 at the cytoplasmic ARD, it exerts forces on the ARD analogous to a clamp, which suppresses ARD motion associated with TRPV4 activation, resulting in TRPV4 inhibition”Results, RhoA-mediated TRPV4 inhibition

    • The blocker GSK279 (GSK2798745) and the activators bind the same pocket, and the authors note that no known disease mutation sits in the GSK279 binding site.

      “Notably, none of the TRPV4 disease mutations 68 overlap with the GSK279 binding site, making it a promising target for future TRPV4 inhibitors.”Discussion (the number 68 in the quote is a reference citation)

    What the authors concluded

    RhoA acts as an auxiliary subunit and natural brake for TRPV4: by holding the ankyrin repeat region still, it keeps the channel closed. Because many neuropathy-causing TRPV4 changes sit where RhoA binds, the authors propose that weakening this contact lets the channel open too much and can lead to TRPV4-related neuromuscular disease. They say the drug-bound structures should help develop TRPV4-blocking medicines.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • ModerateNo patients; overproducing cell lines

      The calcium experiments used a mouse hybrid cell line and human kidney cells made to produce extra TRPV4 and RhoA, and the structures come from protein purified in detergent. These are far from human motor and sensory nerves, so the results show a possible mechanism, not what happens in people with CMT2C.

      “MN-1 cells were transfected with TRPV4-GFP (WT or mutant) constructs or co-transfected with TRPV4-GFP and RhoA-Myc (WT or mutant) constructs using Lipofectamine LTX.”Methods, calcium imaging

    • ModerateDisease link partly relies on earlier work and normal-channel structures

      The structures are of normal TRPV4, not of neuropathy mutants. In this paper the new binding experiments mainly used engineered changes at the contact surface; the evidence that patient mutations themselves weaken RhoA binding comes largely from the group's earlier study. The headline that disrupting this contact 'can lead to' disease is a reasonable hypothesis rather than something shown directly here.

      “We previously showed that neuropathy-causing mutations (R232C, R237L, R269C, R315W) in TRPV4 disrupt the interaction with RhoA, consistent with our structures”Results, neuropathy mutations disrupt RhoA binding to TRPV4

    • MinorIdentity of the bound Rho protein is tentative

      RhoB and RhoC also came along with TRPV4 during purification, and the authors say they cannot exclude that some of the bound protein is RhoB or RhoC. The assignment of RhoA is described as tentative.

      “For this manuscript, we tentatively assign RhoA as the primary candidate based on the cryo-EM map and the previous identification of RhoA as a TRPV4 binding partner through an unbiased screen”Results, structures of human TRPV4 in complex with Rho

    • MinorClamp model rests on indirect evidence

      The weaker RhoA signal in the open structure could mean RhoA is more mobile or that less of it is bound; the authors cannot tell which from the maps alone. Some maps were also lower quality in places. The clamp model draws on these density differences and on computer simulations, so it is a well-supported proposal rather than a direct observation.

      “Because we include GSK101 before freezing, this data suggests that either bound RhoA becomes more flexible, or its occupancy decreases during GSK101-dependent TRPV4 activation”Results, RhoA-mediated TRPV4 inhibition

    • MinorDrug implications not tested for neuropathy

      The safety of GSK2798745 cited in the paper comes from trials in other conditions. The paper does not report testing whether this drug calms neuropathy mutant channels or helps in any CMT2C model.

      “has been safe in clinical trials of pulmonary edema, chronic cough, and diabetic macular edema”Introduction

    Three ways to explain it

    Scientific

    Cryo-EM structures of full-length human TRPV4 co-purified with RhoA (3.30 to 3.75 Å) show membrane-anchored RhoA bound to ankyrin repeats AR2 to AR5 of each subunit, restraining the rigid-body rotation of the ankyrin repeat domain that accompanies ligand-dependent gating. Most neuropathy residues (R237, R269, R315, R316) lie at this interface, and interface mutations in either protein reduced binding and increased TRPV4-mediated calcium influx in cell lines, supporting loss of RhoA-mediated inhibition as a mechanism for gain of function. Agonists and the clinical antagonist GSK279 share a binding pocket in the voltage-sensor-like domain that does not overlap known disease mutations.

    Easier

    TRPV4 is a channel on the surface of the cell that lets calcium in. Using a very powerful microscope, the researchers saw that another protein, RhoA, grips the inner part of TRPV4 and seems to stop it from opening too easily. Many of the gene changes that cause TRPV4 nerve disease sit exactly where RhoA grips, and breaking this grip in lab-grown cells made the channel let in more calcium. They also saw where a TRPV4-blocking drug attaches, which could help design medicines, but this was not tested in CMT2C.

    Simple

    Think of TRPV4 as a door that lets calcium into a cell, and RhoA as a hand that holds the door partly shut. In CMT2C, the mistakes in the door seem to be right where the hand holds on, so the door may swing open too much. This was seen in lab experiments, not yet in people.

    Why these ratings

    Relevance 4/5: Offers a structural explanation of how CMT2C-causing changes might make TRPV4 overactive, and maps where a candidate blocking drug binds.

    Evidence 3/5: High-quality structural and cell work, but the disease link is inferred from where normal amino acids sit and from cell-line experiments, not from patients or from structures of the mutant channel.

    Funding and conflicts of interest

    Supported by NIH grants R35NS097241, R35NS122306, R01GM138472 and K08NS102509, Muscular Dystrophy Association grant 629305, and NIH grant U24GM129547 for cryo-EM facility work. The authors declare no competing interests.

    PubMed 37353484 · DOI

32. #32Full paperLaboratory study: protein microarray screen with follow-up in cultured cell lines

    Profiling the TRPV4 ankyrin repeat domain interactome and its disruption by neuromuscular disease-causing mutations

    Loder AK, Kosmanopoulos GP, Aisenberg WH et al. · 2026 · The Journal of biological chemistry

    Relevance 4/5Evidence 3/52 moderate · 4 minor problems

    This laboratory study adds to evidence that disease-causing TRPV4 changes alter which partner proteins hold on to the channel's ankyrin region, and points to a protein called ARHGEF10 as one partner that may normally help calm the channel. It is early-stage work on chips and in cultured cells and does not test any treatment.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    Laboratory study: protein microarray screen with follow-up in cultured cell lines

    Who or what

    Purified pieces of human TRPV4 made in bacteria: the whole N-terminal region (amino acids 1 to 397; 2 microarray slides) and the ankyrin repeat domain (ARD, amino acids 149 to 397) in normal form and with the R269C or R315W neuromuscular disease changes or the D333G skeletal dysplasia change (3 slides each). These were tested against a HuProt microarray carrying more than 17,000 human proteins. Follow-up experiments used HEK293T cells (a standard laboratory cell line) and MN-1 cells (a mouse motor neuron-neuroblastoma fusion line). No patients or animals were studied.

    What they did

    The team washed TRPV4 pieces over a chip spotted with thousands of human proteins to see which ones they stuck to, and compared the lists for the normal and disease-causing versions. They then checked a few of the hits in cultured cells, by pulling TRPV4 out of the cells and seeing which proteins came with it, by measuring ubiquitin tags on TRPV4, and by calcium imaging to measure channel activity.

    What they found

    • The normal TRPV4 ankyrin repeat domain bound 78 candidate partner proteins on the chip, including proteins involved in tagging proteins with ubiquitin and in small GTPase signaling.

      “Probing of the microarray with TRPV4 WT -ARD revealed 78 interactors, including proteins related to ubiquitination and small GTPase signaling, such as the ubiquitin ligase NEDD4L and the RhoGEF ARHGEF10.”Abstract

    • Two known TRPV4 partners, PACSIN2 and PSD95, were picked up by the screen, which the authors use as a check that the method works.

      “identified PACSIN2 as a positive binding partner”Results, Characterizing the TRPV4 interactome by protein microarray screening

    • In HEK293T cells, adding NEDD4L increased ubiquitin tagging of TRPV4, while adding OTUB2 (which binds the disordered start of the protein) reduced it.

      “Together, these data identify novel interactors of the TRPV4 WT ARD (NEDD4L) and IDR (OTUB2) with opposing effects on TRPV4 ubiquitination status.”Results, Coexpression of novel TRPV4 interactors alters TRPV4 ubiquitination

    • Twenty-one partners that bound the normal and skeletal dysplasia versions were not detected with either neuromuscular disease version (R269C, R315W). No new partners appeared with the disease versions.

      “Comparison of the ARD interactomes of WT and mutant TRPV4 revealed 21 interactions affected by disease-causing mutations.”Abstract; Results, Identification of protein interactions disrupted by neuromuscular disease-causing mutations

    • One of the lost partners, ARHGEF10, is itself linked to inherited neuromuscular disease. In mouse motor neuron-like cells it bound less to the disease versions, and it dampened calcium entry through normal TRPV4 but not through the R269C or R315W versions.

      “calcium imaging studies demonstrated that ARHGEF10 overexpression suppressed TRPV4 WT channel activity, but that this inhibition is abrogated by disease-causing mutations.”Abstract; Results, Figure 5

    What the authors concluded

    The authors conclude that the TRPV4 ankyrin repeat domain interacts with many proteins, including ones involved in ubiquitination and small GTPase signaling, and that neuromuscular disease mutations remove some of these interactions. They suggest that losing ARHGEF10's damping effect may contribute to the increased channel activity of the mutant channels, and offer their lists as a resource for future studies.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • ModerateFar from patients

      All work used isolated protein pieces on a chip or cell lines (HEK293T cells and mouse MN-1 motor neuron-like cells) made to overproduce TRPV4 and its partners. No patient cells, nerve tissue or animals were studied, so it is not known whether these interactions happen, or matter, in human nerves.

      “Unlike HEK293T cells, MN-1 cells do not express endogenous TRPV4 and thus are well suited to transfection-based studies of mutant TRPV4 channels”Results, Neuromuscular disease-causing mutations disrupt functional interactions between TRPV4 and ARHGEF10

    • Moderate'Lost' interactions defined by a cut-off, not measured

      A protein counted as a partner if its signal was 2 standard deviations above the slide average on at least two of three slides. With over 17,000 proteins, this lets through some chance hits and can miss weaker binders. The 21 'disrupted' interactions are proteins that fell below the line for the disease versions, not measured reductions in binding, and only ARHGEF10 was confirmed in cells.

      “candidate binding partners were identified as proteins for which, on at least two of the three experiments performed with each construct, the average signal intensity of the duplicate spots was ≥2 SDs above the mean value”Experimental procedures, Human protein microarray

    • MinorChip format misses known partners

      RhoA, a well-established TRPV4 partner, was not detected, showing the chip can miss real interactions. The authors suggest this is because TRPV4 prefers RhoA in its GDP-bound form, and note that cell-free tests cannot copy context-dependent binding. Cell experiments were also done with a TRPV4 blocker present, so binding in active channels was not tested.

      “Although we and others have recently demonstrated that the small GTPase RhoA is a robust interactor of the TRPV4-ARD ( 12 , 22 , 28 ), it was not identified as an interactor in this study.”Discussion

    • MinorModest effects and unclear repeat numbers

      The key ARHGEF10 results are statistically significant but only just (p values between 0.022 and 0.043), the number of repeats for the binding experiment is not stated in the figure legend, and calcium imaging used 11 to 14 wells per condition. Independent replication would make the finding more secure.

      “Kruskal-Wallis test, Dunn’s multiple comparison test, WT versus R269C, p = 0.043; WT versus R315W, p = 0.025.”Figure 5 legend

    • MinorCandidate count does not add up

      The paper says 107 candidates were found and 26 were excluded for being in the wrong part of the cell, leaving 78. But 107 minus 26 is 81. The discrepancy is not explained. It does not affect the ARHGEF10 result but shows the lists need care.

      “Utilizing these criteria, 107 candidate binding proteins were identified.”Results, Identification of protein interactors of the TRPV4 WT-ARD

    • MinorSignificance threshold applied inconsistently

      The methods set a false discovery rate below 0.01 as the threshold for significant enrichment, but the results call cytoplasm enrichment significant at FDR = 0.034, which is above that threshold. This affects only the description of the candidate list.

      “A threshold of 0.01 (FDR < 0.01) was used to identify significantly enriched GO categories or UniProt Annotated Keywords.”Experimental procedures, Human protein microarray; compare Results, Fig. 3B

    Three ways to explain it

    Scientific

    Using a human proteome microarray, the authors identified 78 candidate binding partners of the wild-type TRPV4 ankyrin repeat domain, 21 of which were not detected with the neuromuscular disease mutants R269C and R315W but were retained with the skeletal dysplasia mutant D333G. In transfected MN-1 cells, ARHGEF10 co-immunoprecipitated less with the mutants, and ARHGEF10 overexpression suppressed wild-type but not mutant TRPV4-mediated calcium responses, suggesting that loss of an inhibitory interaction may contribute to the gain of function. These are cell-free and overexpression findings that need confirmation in native tissue.

    Easier

    The TRPV4 channel has a region called the ankyrin repeat domain that works like a handle other proteins can grab. Researchers tested which of more than 17,000 human proteins grab this handle and found 78 likely partners. Disease-causing changes seen in TRPV4 nerve and muscle conditions seemed to stop 21 of them from grabbing on. One partner, ARHGEF10, appeared to calm the normal channel but could not calm the disease versions, although this was only shown in cells grown in the lab.

    Simple

    TRPV4 is like a door that lets calcium into cells, and it has a handle that helper proteins can hold to stop the door from opening too much. Scientists found that with the disease versions, some helpers, such as one called ARHGEF10, may not hold on as well. This was seen in lab dishes, not in people.

    Why these ratings

    Relevance 4/5: Studies how two classic TRPV4 neuromuscular disease changes (R269C and R315W) alter the channel's protein partners, which bears on the mechanism behind CMT2C.

    Evidence 3/5: Well-controlled laboratory work with follow-up in two cell types, but it is a screen on a chip and in cell lines overproducing the proteins, with only one disease-related hit tested in depth and modest statistics.

    Funding and conflicts of interest

    Funded by US National Institutes of Health grants (R35 NS122306, F31 NS105404, K08 NS102509, R01 NS131402, R01 GM081340), an EMBO Long-Term Fellowship and the German Research Foundation (DFG). The authors declare no conflicts of interest.

    PubMed 41338459 · DOI

33. #33Full paperCell study (calcium measurement, cell death and electrical recordings in lab cell lines)

    Mutant TRPV4-mediated toxicity is linked to increased constitutive function in axonal neuropathies

    Fecto F, Shi Y, Huda R et al. · 2011 · The Journal of biological chemistry

    Relevance 4/5Evidence 3/54 moderate · 4 minor problems

    In lab cell lines, the TRPV4 changes linked to CMT2C and SPSMA made the channel open more often and let in more calcium, which harmed the cells. This supports the 'overactive channel' idea, but it was not tested in nerves or in people.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    Cell study (calcium measurement, cell death and electrical recordings in lab cell lines)

    Who or what

    Three lab cell lines (HEK293, HeLa and Neuro2a, which the authors describe as a neural cell type) made to produce normal human TRPV4 or one of three disease-linked versions: R269H, R315W and R316C. Calcium was measured in over 2000 to over 9000 cells per condition by flow cytometry; electrical recordings used small numbers of HeLa cells (for example 8 and 6 cells). No patients or animals.

    What they did

    Earlier studies disagreed on whether TRPV4 neuropathy changes make the channel work too much or too little. The authors tested the same three changes in three cell lines, looking at where the channel sat, calcium levels, cell death and the effect of the channel blocker ruthenium red. They also recorded electrical currents through whole cells and through single channels for R269H.

    What they found

    • Normal and all three mutant channels reached the cell surface in all three cell lines. Some channel was also held inside cells, but this happened equally with the normal channel.

      “We observed that wild-type and the three mutant TRPV4 channels were present on the plasma membrane in all the three cell lines tested”Results, subcellular localization

    • All three mutants raised resting calcium and gave a larger peak response to the TRPV4 activator 4αPDD than the normal channel, in all three cell lines.

      “In all three cell lines tested, mutant TRPV4 led to substantially higher basal calcium levels than wtTRPV4, suggesting an increased constitutive activity for the mutants”Results, intracellular calcium; Figures 5 to 7

    • Cells with mutant TRPV4 died more often after 48 hours, and the TRP channel blocker ruthenium red prevented both the extra calcium entry and the cell death.

      “We observed that transfection with TRPV4 mutants resulted in remarkably increased cytotoxicity in all three cell lines tested at 48 h compared with wtTRPV4”Results, cell viability

    • In HeLa cells, the TRPV4 current was roughly six times larger with R269H than with the normal channel.

      “at 100 mV the normalized ruthenium red-sensitive current was 9.2 ± 4.3 and 52.4 ± 21.6 pA/pF for cells expressing the wild-type and mutated channels”Results, whole-cell recordings

    • Single R269H channels carried the same current per opening as normal channels but were open more of the time, pointing to a change in how the channel opens and closes.

      “the open probability was 0.03 ± 0.01 for wild-type and 0.17 ± 0.04 for mutant channels”Results, single-channel recordings

    What the authors concluded

    The three neuropathy-linked TRPV4 changes make the channel overactive (gain of function), letting more calcium into cells and causing cell death that can be blocked by ruthenium red. The authors argue this supports testing TRPV4-blocking drugs as a treatment.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • ModerateCell lines overproducing the channel

      All work used cell lines given extra TRPV4 for a short time, not nerve cells from patients or animals. The amount of channel varied between cells, and the authors suggest that stable cell lines with equal expression would be better. Results in this setting may not match human nerves.

      “Stable cell lines with identical levels of TRPV4 expression would be more appropriate to further address this issue.”Discussion

    • ModerateOnly some cells analyzed

      Calcium analysis was limited to cells with high marker expression, and in HeLa cells most cells did not respond and were removed from the analysis. Excluding non-responders can exaggerate differences and makes the HeLa results less representative.

      “there was a much higher fraction of non-responders in HeLa cells (79.5%) compared with HEK293 (12.5%) or Neuro2a cells (11.3%).”Results, intracellular calcium

    • ModerateAmount of channel not fully matched

      For electrical recordings, cells with similar brightness were chosen, but mutant cells looked brighter overall, and only cells that recovered from ruthenium red were included. More mutant channels in the membrane could explain part of the larger currents. The authors acknowledge that greater membrane insertion is possible.

      “Similar ZsGreen1 GFP-fluorescent cells were chosen for experiments, although the cells expressing the mutant channels seemed generally brighter.”Experimental procedures, electrophysiology

    • MinorIndividual cells counted as independent samples

      Calcium statistics treat thousands of cells as separate data points, which makes very small p-values easy to reach. The number of independent experiments behind the calcium data is not clearly given.

      “n >2000 cells for each condition”Figure 5 legend (similar in Figures 6 and 7)

    • MinorNumbers swapped between text and table

      The text gives the fast open time as 1.28 ms for mutant and 2.02 ms for normal channels, but Table 1 gives 1.28 ms for normal and 2.02 ms for mutant. The table also labels two rows 'Open τ1 fractional contribution'. The direction stated in the text (longer in mutant) matches the table, so this looks like a reporting error.

      “1.28 ± 0.17 ms, versus 2.02 ± 0.18 ms in wild-type”Results, single-channel recordings; Table 1

    • ModerateTreatment claim goes beyond the data

      The protective effect came from ruthenium red, which the paper calls a TRP channel blocker, in cell lines. Saying TRPV4 blockers could be tested 'immediately' in patients goes further than cell line data can support.

      “Our data imply that a treatment using TRPV4 antagonists could be immediately tested as a therapeutic approach for these diseases.”Discussion, final paragraph

    • MinorElectrical recordings for only one mutant

      Whole-cell and single-channel recordings were done only for R269H. R315W and R316C were tested by flow cytometry alone, so the gating explanation is shown for one change.

      “Whole-cell voltage-clamp recordings were performed from HeLa cells transiently transfected with wild-type or R269H TRPV4 cDNA”Experimental procedures, electrophysiology

    • MinorSome control data not shown

      The absence of natural TRPV4 in Neuro2a and HeLa cells is stated but not shown, and natural TRPV4 in HEK293 cells is said to vary between batches.

      “Neuro2a cells and HeLa cells do not have detectable levels of endogenous TRPV4 expression in our experiments (data not shown)”Results, endogenous expression

    Three ways to explain it

    Scientific

    In three transiently transfected cell lines (HEK293, HeLa, Neuro2a), R269H, R315W and R316C TRPV4 trafficked to the plasma membrane and produced higher basal and 4αPDD-evoked calcium and more cell death than wild type, effects blocked by ruthenium red. In HeLa cells, R269H increased ruthenium red-sensitive current density and single-channel open probability without changing single-channel conductance, consistent with altered gating and possibly greater membrane insertion. The authors conclude that these neuropathy mutations act by gain of function, a conclusion drawn from overexpression in cell lines only.

    Easier

    TRPV4 is a channel that lets calcium into cells. Earlier studies disagreed about whether disease-causing changes make it work too much or too little. In three types of lab-grown cells, this team found that three known changes made the channel open more often, let in more calcium and killed more cells, and that a channel-blocking chemical prevented this. This supports the idea that the changes make the channel overactive, but it was tested only in lab cell lines.

    Simple

    Think of TRPV4 as a door for calcium. In cells in a dish, the faulty doors stayed open more often than normal doors and let in too much calcium, which harmed the cells. A chemical that blocks the door stopped the harm, but only in the dish.

    Why these ratings

    Relevance 4/5: It tests the basic mechanism of CMT2C-linked TRPV4 changes, including R269H from the original CMT2C family, but uses only lab cell lines.

    Evidence 3/5: Consistent results across three cell lines and two methods, but all from cell lines overproducing the channel, with selective cell analysis and small numbers in the electrical recordings.

    Funding and conflicts of interest

    Supported by NINDS Grant NS050641, NHLBI Grant HL095731 and NIH Grants T32 AG20506 and T32 MH067564, plus the Les Turner ALS Foundation, the Vena E. Schaff ALS Research Fund, the Harold Post Research Professorship, the Herbert and Florence C. Wenske Foundation, the David C. Asselin MD Memorial Fund, the Help America Foundation and the Les Turner ALS Foundation/Herbert C. Wenske Foundation Professorship. Competing interests: not stated.

    PubMed 21454511 · DOI

34. #34Full paperProtein structure and biochemistry study (X-ray crystallography, test-tube assays and computer simulations)

    Structural and biochemical consequences of disease-causing mutations in the ankyrin repeat domain of the human TRPV4 channel

    Inada H, Procko E, Sotomayor M et al. · 2012 · Biochemistry

    Relevance 4/5Evidence 3/51 moderate · 5 minor problems

    This study looked closely at the shape of the part of TRPV4 where several neuropathy changes sit. It did not find a simple reason why these changes damage nerves, and it has no direct implications for symptoms or treatment.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    Protein structure and biochemistry study (X-ray crystallography, test-tube assays and computer simulations)

    Who or what

    No patients or cells. The ankyrin repeat domain of human TRPV4 (amino acids 149 to 396) was made in Escherichia coli bacteria and its structure solved with and without ATP. Of the 15 known disease-linked mutations in this domain (five linked to neuropathy and ten to skeletal dysplasia), 13 were made and tested; T295A could not be produced. Some experiments and the computer simulations used the rat TRPV1 ankyrin repeat domain.

    What they did

    They grew crystals of the TRPV4 ankyrin repeat domain and used X-rays to work out its 3D shape with and without ATP, a small molecule that adjusts the channel's activity. They measured how heat-stable the normal and mutant domains were and how well they bound ATP. They used computer simulations of the related TRPV1 domain to test whether ATP makes the domain more stable.

    What they found

    • They solved the first matched pair of structures of a TRPV ankyrin repeat domain with and without ATP. Binding ATP caused only small changes, mainly in a long, flexible loop called Finger 3.

      “Importantly, these structures represent the first cognate pair of ATP-bound and ATP-free TRPV-ARD structures.”Results, Structures of Human TRPV4-ARD

    • ATP made the domain slightly more heat-stable (melting temperature 37.1 ± 0.1 °C without ATP and 37.9 ± 0.2 °C with ATP). The melting point is close to body temperature, so the authors suggest small changes in stability could matter.

      “Several lines of evidence indicate that ATP binding increases the protein stability of both TRPV1- and TRPV4-ARD”Results, ATP Binding Stabilizes TRPV4-ARD and TRPV1-ARD; Discussion

    • Neuropathy-linked mutations (at arginines R232, R269, R315 and R316) sit on the outer, convex face of the domain, while skeletal dysplasia mutations are spread mainly over the inner, concave face.

      “Mutations at arginine residues located on the convex face of hTRPV4-ARD are associated with neuropathies”Results, Structural Analysis of TRPV4 Mutations Associated with Human Diseases

    • Most mutant domains were less heat-stable than normal. The neuropathy-linked R232C did not differ from normal, and the neuropathy-linked R269C was more stable than normal (38.6 ± 0.2 °C).

      “Most hTRPV4-ARD mutants showed significantly reduced thermal stability”Results; Discussion

    • Four mutations, including the neuropathy-linked R232C and R269H, weakened ATP binding, and two skeletal dysplasia mutations (E183K and E278K) strengthened it, but ATP binding did not match the type of disease.

      “although several mutations either impaired or enhanced ATP binding, there was no correlation between the disease phenotypes and the ATP-binding phenotype.”Results, ATP Binding by hTRPV4-ARD Mutants

    • Neither heat stability nor ATP binding explained why some mutations cause neuropathy and others cause skeletal disease.

      “it is difficult to explain the disease-specific segregation on the basis of only these two biochemical phenotypes, suggesting that additional mechanisms are likely at play in the regulation of the TRPV4 channel.”Discussion

    What the authors concluded

    The flexible Finger 3 loop may act as a switch that helps control channel activity, and ATP binding stabilizes the ankyrin repeat domain. Some disease mutations reduce the domain's stability or change ATP binding, which could play a part in disease, but these properties cannot explain why some mutations cause neuropathy and others skeletal dysplasia. Other mechanisms, possibly specific to each tissue, remain to be found.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • ModerateIsolated protein fragment made in bacteria

      Only the ankyrin repeat domain was studied, made in bacteria and tested in solution. It was not part of a whole channel, not in human cells and not in nerves. How its stability or ATP binding affects the working channel in people is not tested here.

      “Recombinant human TRPV4-ARD protein was expressed in Escherichia coli BL21(DE3)”Experimental Procedures, Protein Production and Purification

    • MinorLink between stability and channel activity is speculative

      Many of the stability changes are small, and the authors themselves say the connection to channel activity is not established. R269C, a neuropathy mutation known to increase channel activity, made the domain more stable rather than less.

      “although the relationship between the stability of ARD and channel activity remains speculative.”Discussion

    • MinorSome evidence comes from a different channel

      The computer simulations and the single-cysteine test used the rat TRPV1 domain, not TRPV4. The authors use these to support the idea that ATP stabilizes the domain in general.

      “Two sets of molecular dynamics simulations (apo and ATP) were conducted using the structure of rTRPV1-ARD bound to ATP”Experimental Procedures, Molecular Dynamics Simulation of rTRPV1-ARD

    • MinorPossible crystal artifacts

      The shape change in Finger 3 was also seen in a molecule without ATP, which the authors say might reflect how the crystals packed rather than a real effect.

      “Alternatively, the shrunken Finger 3 structure may result from crystal contacts and/or crystallization condition differences.”Results, Structural Consequences of ATP Binding

    • MinorGroup comparison pools repeat measurements

      The finding that skeletal dysplasia mutants are on average less stable than neuropathy mutants compares pooled repeat measurements (n = 24 and n = 15) rather than numbers of different mutations, and the authors note that not all mutations follow the trend.

      “although not all mutants obey this trend”Results, Structural Analysis of TRPV4 Mutations Associated with Human Diseases

    • MinorNot all mutations tested

      13 of 15 mutations were studied. T295A could not be produced, and the text does not name the second mutation that was left out.

      “we generated mutant hTRPV4-ARD corresponding to 13 of the 15 disease-causing mutations located within the ARD.”Results, Structural Analysis of TRPV4 Mutations Associated with Human Diseases

    Three ways to explain it

    Scientific

    Crystal structures of the human TRPV4 ankyrin repeat domain with and without ATP show that ligand binding mainly alters the flexible Finger 3 loop, and thermal denaturation, cysteine accessibility and steered molecular dynamics (partly on TRPV1) indicate that ATP stabilizes the domain. Neuropathy-associated arginine substitutions map to the convex face, whereas skeletal dysplasia mutations lie mainly on the concave face. Most of 13 disease mutants reduced domain thermal stability and some altered ATP binding, but neither property segregated with neuropathy versus skeletal phenotype, implying additional regulatory mechanisms.

    Easier

    The TRPV4 channel has a section called the ankyrin repeat domain, which sits inside the cell and helps control how the channel behaves. The researchers made this section in bacteria and worked out its 3D shape, with and without ATP, a small molecule that fine-tunes the channel. Changes that cause nerve disease sit on one face of this section, and bone-disease changes mostly sit on the other. Most changes made the section less stable when heated, and some changed how it held ATP, but neither result explained why some changes affect nerves and others affect bones.

    Simple

    TRPV4 is like a door that lets calcium into cells, and this study looked at the door's handle on the inside. The scientists mapped where nerve-disease and bone-disease changes sit on the handle, but they still could not tell why some changes hurt nerves and others hurt bones.

    Why these ratings

    Relevance 4/5: It studies the part of TRPV4 that carries several neuropathy-linked changes (R232C, R269C, R269H, R315W, R316C), but it is basic protein science with no patients.

    Evidence 3/5: Careful structural and biochemical work that supports its claims about the protein fragment, but it used an isolated piece of the channel made in bacteria, so any links to disease are suggestions.

    Funding and conflicts of interest

    Funded by National Institutes of Health Grant R01GM081340 (to R.G.). The X-ray work used the Advanced Photon Source, supported by grant RR-15301 from the National Center for Research Resources and by the US Department of Energy. M.S. was a Howard Hughes Medical Institute Fellow of the Helen Hay Whitney Foundation. The authors declare no competing financial interest.

    PubMed 22702953 · DOI

35. #35Full paperStructural biology, computer simulation and cell study

    TRPV4 channel activity is modulated by direct interaction of the ankyrin domain to PI(4,5)P2

    Takahashi N, Hamada-Nakahara S, Itoh Y et al. · 2014 · Nature communications

    Relevance 4/5Evidence 3/52 moderate · 4 minor problems

    This lab study suggests one way some CMT2C mutations could make TRPV4 too active: they stop a membrane fat from holding the channel down. It was done with a protein fragment and kidney-derived cells, so it is a clue to the mechanism, not something tested in patients.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    Structural biology, computer simulation and cell study

    Who or what

    Purified ankyrin repeat domain (ARD) of TRPV4 made in bacteria (human and chicken versions); a crystal structure of the chicken TRPV4 ARD; computer docking and molecular dynamics simulations; human embryonic kidney (HEK293) cells made to overproduce human TRPV4 or mutants, including the disease-linked R232C, R269H, R315W and R316H and several engineered changes.

    What they did

    They tested which membrane fats (phosphoinositides) stick to the TRPV4 ankyrin domain, solved a crystal structure of the domain bound to IP3 (the head of the membrane fat PI(4,5)P2), and used simulations to map the binding site. They then measured TRPV4 currents, calcium levels and cell death in kidney-derived cells while raising or lowering PI(4,5)P2, comparing normal and mutant channels.

    What they found

    • The TRPV4 ankyrin domain binds membrane phosphoinositides, most strongly PI(4,5)P2, and this binding suppresses channel activity.

      “phosphoinositide binding to the TRPV4 ARD leads to suppression of the channel activity”Abstract; Results, Figures 1 and 6

    • Lowering PI(4,5)P2 in the cell membrane, by blocking it or breaking it down, increased TRPV4 activity.

      “The TRPV4 channel activities were increased by titration or hydrolysis of membrane PI(4,5)P2.”Abstract; Results, Figures 6 and 8

    • The disease mutations R269H, R315W and R316H lost PI(4,5)P2 binding and sensitivity and showed raised channel activity.

      “Notably, disease-associated TRPV4 mutations that cause a gain-of-function phenotype abolished PI(4,5)P2 binding and PI(4,5)P2 sensitivity.”Abstract; Results, Figures 4 and 7

    • These three mutations made the ankyrin domain less stable, which may be why they no longer bind PI(4,5)P2.

      “However, the R269H, R315W and R316H mutations ... destabilized the protein structure”Results, Figure 4b and Figure 5

    • R232C, another CMT2C-linked mutation, behaved differently: it kept PI(4,5)P2 sensitivity and stability, so this mechanism does not explain every ankyrin domain mutation.

      “Among the disease-associated ARD mutations we tested, only ... the R232C mutation did not affect the PI(4,5)P2 sensitivity and”Discussion

    • When mutant and normal TRPV4 were made together in the same cells, as in patients who carry one normal and one mutant copy, the channels were still overactive.

      “HEK293 cells co-expressing TRPV4 ... and either the R269H, R315W or N296D-H299P mutant showed elevated basal activities and higher responses to 4a-PDD”Results, Supplementary Figure 13

    What the authors concluded

    The TRPV4 ankyrin domain binds the membrane fat PI(4,5)P2, and this binding holds channel activity down. Most of the disease mutations tested (R269H, R315W, R316H) lose this binding and become overactive, which may contribute to cell damage in CMT2C, congenital distal SMA and SPSMA. R232C works differently, so other mechanisms also exist, and the authors say mice carrying such mutations would be needed for direct proof.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • ModerateModel far from people

      The crystal structure is of a fragment of chicken TRPV4, not the whole human channel. All channel tests were in HEK293 kidney-derived cells overproducing TRPV4. No nerve cells, animals or patient cells were studied, so it is unknown whether the same happens in human nerves.

      “we prepared crystals of the chicken ... TRPV4 ARD and exposed them to a solution containing IP3”Results, Crystal structure of the ARD-IP3 complex

    • ModerateLink between the mutations and lipid binding may be indirect

      The disease mutations sit on a different surface from the binding site seen in the crystal. Their loss of binding may come from the domain becoming unstable, and the authors cannot rule out that changed interactions with other partners cause the overactivity. The title and abstract state the link more firmly than the discussion does.

      “we cannot exclude the possibility that the gain-of-function ... phenotype of these mutants is attributable to an alteration ... in interactions with other regulatory factors, caused by”Discussion

    • MinorNot all disease mutations fit

      R232C kept PI(4,5)P2 binding, and mutations outside the ankyrin domain were not tested, so this mechanism explains only part of TRPV4 neuropathy.

      “This implies that the R232C mutation elicits a ... gain-of-function phenotype through PI(4,5)P2-independent”Discussion

    • MinorDisagreement with another group's findings

      Another research group reported that PI(4,5)P2 helps TRPV4 respond to cell swelling and heat, the opposite direction. The authors suggest differences in timing and recording method, but the disagreement is not settled.

      “Recently, Garcia-Elias et al. reported that PI(4,5)P2 facilitates”Discussion

    • MinorSmall numbers and simple statistics

      Groups were small (n of 5 to 17 in the electrical recordings and 3 to 6 in the stability tests, although the methods say more than five stability experiments were shown). Many p values were close to 0.05, and many comparisons were made with Student's t-test without any stated correction for multiple testing.

      “analyses were performed using the Student’s t-test”Methods, Statistical analyses; figure legends

    • MinorSmall labeling slip

      The Figure 7 legend names a mutant 'R251E', while the text consistently calls it K251E. This does not affect the conclusions.

      “Effects of intracellular PI(4,5)P2 and IP3 on the activities of R232C, R251E, R269H”Figure 7 legend

    Three ways to explain it

    Scientific

    The TRPV4 ankyrin repeat domain binds phosphoinositides, preferentially PI(4,5)P2, at a site defined by a 1.9 Å crystal structure of the chicken ARD with IP3, and PI(4,5)P2 binding suppresses channel activity in HEK293 cells. The disease mutations R269H, R315W and R316H destabilized the ARD, lost PI(4,5)P2 binding and sensitivity, and showed gain-of-function currents and increased cell death, whereas R232C retained PI(4,5)P2 sensitivity. Whether loss of PI(4,5)P2 regulation, rather than instability-driven changes in other interactions, causes the gain of function remains unresolved.

    Easier

    TRPV4 has a section called the ankyrin domain that sits just inside the cell. This study found that this section sticks to a fat in the cell membrane called PI(4,5)P2, and that this contact keeps the channel's activity down. Three CMT2C and SMA mutations made the section unstable, stopped it from sticking and left the channel more active. Another mutation (R232C) worked differently. All tests were done in lab-grown kidney-derived cells, not nerves.

    Simple

    TRPV4 is like a door that lets calcium into a cell, and it has a handle that normally hooks onto the wall to stop the door from opening too much. Some CMT2C changes seem to make the handle wobbly so it cannot hook on, and the door opens too easily, at least in cells grown in a lab.

    Why these ratings

    Relevance 4/5: It proposes a molecular reason why several ankyrin domain mutations linked to CMT2C and SMA make TRPV4 overactive.

    Evidence 3/5: Good structural and cell work using several methods, but done on an isolated chicken protein fragment and in overproducing kidney-derived cells, not nerve cells or patients.

    Funding and conflicts of interest

    Funded by the Japan Society for the Promotion of Science (NEXT Program) and grants from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan, with supercomputer support. The authors declare no competing financial interests.

    PubMed 25256292 · DOI

36. #36Full paperLaboratory cell study (in vitro) by a drug company, including tests of its own experimental TRPV4 blocker

    The impact of TRPV4 pathogenic mutations on barrier integrity

    Sampaio G, Ismaili T, Torkamani A et al. · 2025 · Human molecular genetics

    Relevance 4/5Evidence 3/54 moderate · 3 minor problems

    In lab-grown cells, some TRPV4 changes linked to CMT2C, including R269C, R269H and R315W, weakened the seal between cells, but 15 of the 22 neuropathy changes tested did not. The experimental blocker ABS-0872, made by the company that ran the study, prevented or reversed this in cells; the paper does not show whether it would help people.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    Laboratory cell study (in vitro) by a drug company, including tests of its own experimental TRPV4 blocker

    Who or what

    Human cell lines grown in the lab; no patients or animals. Cells that line blood vessels (HUVECs), including a version with less natural TRPV4 made by CRISPR editing, and an eye cell line from the retina (ARPE-19) given extra normal or mutant TRPV4 for a short time. All 51 pathogenic or likely pathogenic TRPV4 mutations listed in ClinVar were tested: 22 linked to peripheral neuropathy, 22 to skeletal dysplasia (13 of them metatropic dysplasia) and 7 to both.

    What they did

    The team measured the electrical resistance of a layer of cells in real time (a method called ECIS), which falls when the seal between cells weakens. They first switched TRPV4 on with a chemical activator in blood vessel lining cells, then gave the eye cells each of the 51 disease-linked TRPV4 changes and recorded calcium entry and the seal. They tested the company's experimental blocker, ABS-0872, on a few of the changes that weakened the seal, and looked at a seal protein called ZO1.

    What they found

    • Switching TRPV4 on with the activator GSK101 weakened the seal formed by blood vessel lining cells. Cells with less TRPV4, or cells treated first with ABS-0872, were largely protected.

      “Upon the addition of GSK101, WT HUVEC cells exhibited a pronounced reduction in barrier strength, while HUVEC-TRPV4low cells had reduced barrier disruption caused by GSK101 agonist”Results, first section; Figure 1E

    • In the eye cells, the CMT2C change R269C raised resting calcium and weakened the seal, shown both by dye leaking through the cell layer and by falling resistance. The normal channel did not weaken the seal.

      “expression of TRPV4WT had no impact on barrier resistance relative to mock transduced cells. By contrast, expression of mutant TRPV4R269C caused a significant decrease in barrier resistance”Results, effect of R269C; Figure 2

    • Only some changes weakened the seal: 7 of 22 neuropathy changes, 8 of 13 metatropic dysplasia changes (including all those reported as lethal) and none of the other skeletal dysplasia changes. The 7 neuropathy changes that did so were R237L, R269C, R269H, R269S, E278K, R315W and P827H; the other 15 did not.

      “For the PN group, 7 out of 22 mutations (31%) caused significant barrier disruption, and most of the mutations grouped near the N-terminal ankyrin repeat domain (ARD) of TRPV4”Results, subset of mutants; Figure 3B

    • Almost every change let in extra calcium (47 of 51 raised resting calcium), but how much calcium a change let in did not predict whether it weakened the seal.

      “we did not observe a correlation between basal TRPV4 activity and barrier disruption.”Results, subset of mutants; Figure 3A

    • ABS-0872 lowered resting calcium for each of the five changes tested, at similar doses. Given before the change was added, or 8 hours after, it prevented or reversed the weakened seal; given later, it restored the seal less fully.

      “We observed that ABS-0872 inhibitor efficiently prevented barrier disruption and promoted barrier recovery for all the mutants tested”Results, inhibition with ABS-0872; Figures 4 and 5; Discussion

    • With G78W, R269C and R269H, the seal protein ZO1 moved away from the edges where cells meet, and ABS-0872 moved it back. A second seal protein, Claudin-3, behaved similarly.

      “This data indicated that TRPV4 mutants may cause barrier disruption by affecting tight junctions, and increasing paracellular transport.”Results, tight junction proteins; Figure 6

    What the authors concluded

    TRPV4 activity and gain-of-function mutations can weaken the seal between cells in lab models. Every change that did this was linked to peripheral neuropathy or metatropic dysplasia, which the authors present as a new genotype-phenotype pattern, although their discussion calls the correlations weak. They conclude that ABS-0872 prevents the damage in these cells and, given early, reverses it.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • ModerateEye cells given extra channel, not nerves or blood vessels

      The 51 changes were tested in a retinal cell line that naturally makes little TRPV4, after adding the channel for a short time, because adding it to blood vessel lining cells killed them. This shows what a mutant channel can do in one cell type in a dish, not what happens in the spinal cord or nerves of people with CMT2C. The authors note that a single cell type lacks the complexity of real tissue.

      “Transfection of WT or mutant TRPV4 into HUVEC or HUVEC-TRPV4low cells caused pronounced cell death. Therefore, we chose to work with the epithelial cell line ARPE-19.”Results, effect of R269C; Discussion

    • ModerateCompany study of its own drug

      The work was sponsored by Actio Biosciences, every author works there, and ABS-0872 is the company's compound. This does not make the results wrong, but no independent laboratory has repeated them in this paper, which matters most for the treatment findings.

      “All authors are Actio Bioscineces employees and disclose financial relationships with commercial interests that may be relevant to the submitted work.”Funding and conflict of interest statement

    • ModerateGenotype-phenotype claim stronger at the start than at the end

      The introduction says the work establishes a genotype-phenotype correlation. The discussion is more careful: only 7 of 22 neuropathy changes weakened the seal, the split of skeletal changes into metatropic and other groups was made after seeing the results, no statistical comparison between the groups is reported, and the authors doubt the results show that barrier leak explains the different diseases.

      “Overall, we interpret these results as showing yet to be understood functional differences amongst the mutations that appear to have some weak correlations with clinical presentation.”Introduction; Results; Discussion

    • ModerateBlocker tested on a few chosen changes, only in cells

      ABS-0872 was tested on five changes for calcium and four for the seal, picked because they weakened it, so 'all the mutants tested' means this small group. Doses were given to cells in a dish, later treatment worked less well, and the paper reports no animal or human data on the drug.

      “We selected a subset of PN and MD mutations that caused barrier disruption and evaluated the extent to which pharmacologic inhibition with ABS-0872 could reverse barrier dysfunction.”Results, inhibition with ABS-0872; Figures 4 and 5

    • MinorSmall changes in the seal

      A change counted as weakening the seal if the measurement was significantly lower than in untreated cells. In the bar chart, the neuropathy changes that met this test stayed above 90% of the untreated level. The paper does not say how large a drop would matter in a living body.

      “We scored a mutation as causing barrier disruption if the ECIS value was significantly less than that of mock (p < 0.05 using One-way ANOVA).”Results, subset of mutants; Figure 3B

    • MinorReporting errors

      One sentence contradicts itself about which of three changes damaged the seal. In the figures, K276E is also labeled K279E and L276E, the blocker is labeled ABS005 in Figure 5B, and the ZO1 axis label does not match the ratio described in the text. These look like slips rather than wrong results, but they make the paper harder to check.

      “mutants with similar basal activity such as 270V (SD), 269H (PN) and 276E (MD) only the former two caused barrier disruption while 270V did not cause any damage to the barrier”Discussion; Figures 4 to 6

    • MinorSome data not shown

      A few statements rest on data not shown, and several results are in supplementary figures, which were not available for this summary.

      “presented minimal functional response to the TRPV4 agonist GSK101 (data not shown)”Results, effect of R269C

    Three ways to explain it

    Scientific

    In ECIS impedance assays, pharmacological TRPV4 activation (GSK101) disrupted HUVEC barriers, an effect that partial CRISPR knockdown or the inhibitor ABS-0872 prevented. Transient expression of all 51 ClinVar pathogenic or likely pathogenic TRPV4 variants in ARPE-19 cells raised basal calcium for 47 of 51, but disrupted the barrier only for 7 of 22 peripheral neuropathy variants (mostly in the ankyrin repeat domain), 8 of 13 metatropic dysplasia variants and no other skeletal dysplasia variants, with no correlation between basal activity and disruption. ABS-0872 inhibited the mutant channels and prevented, or with early dosing reversed, barrier loss in vitro; the authors, all company employees, describe the genotype-phenotype correlation as weak.

    Easier

    Cells that line blood vessels, and some other cells, hold tightly together to form a seal. In lab-grown cells, switching TRPV4 on weakened this seal, and so did the CMT2C change R269C. When the team tested all 51 known disease-causing TRPV4 changes in an eye cell line, almost all made the channel let in extra calcium. But only some weakened the seal: 7 of 22 nerve disease changes, most changes behind one severe bone condition (metatropic dysplasia), and none of the other bone disease changes. The experimental blocker ABS-0872, made by the company that ran the study, protected the seal and helped it recover in these cells, but it was not tested in animals or people in this paper.

    Simple

    TRPV4 is like a door that lets calcium into cells. Cells in a dish normally hold hands tightly to make a wall. With some faulty doors stuck open, the cells let go a little and the wall became leaky. A new medicine that partly closes the door helped them hold on again. This was only in cells in a dish, not in people.

    Why these ratings

    Relevance 4/5: It tests the TRPV4 changes listed for CMT2C, including R269C, R269H and R315W, in a lab model of the cell seal that mouse work has linked to CMT2C, and tests an experimental TRPV4 blocker made by the authors' company.

    Evidence 3/5: A systematic test of all 51 ClinVar mutations with repeated measurements, but in an eye cell line given extra channel for a short time, with small changes in the seal, some reporting errors and no animal or human testing of the blocker; all authors work for the company that makes it.

    Funding and conflicts of interest

    Sponsored by Actio Biosciences, which developed ABS-0872. All authors are Actio Biosciences employees and disclose financial relationships with commercial interests that may be relevant to the work.

    PubMed 39964066 · DOI

37. #37Full paperLaboratory variant-testing study combined with a small clinical case series

    Combined clinical, structural and cellular studies discriminate pathogenic and benign TRPV4 variants

    Berth SH, Vo L, Kwon DH et al. · 2025 · Brain: a journal of neurology

    Relevance 4/5Evidence 3/55 moderate · 3 minor problems

    This study suggests that two things help judge whether a TRPV4 variant is likely to cause disease: whether it makes the channel overactive in lab tests, and features such as vocal cord weakness or problems from birth. The results come from cells in a dish and a small number of patients, so they are a guide but do not settle the status of an individual variant.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    Laboratory variant-testing study combined with a small clinical case series

    Who or what

    30 TRPV4 variants (as stated in the abstract), tested in cells grown in the lab that were made to overproduce the channel: MN-1 cells for calcium measurements, HEK293T cells for protein levels, cell death and protein binding, and canine kidney (MDCK) cells for where the channel sits in the cell. Clinical details are given for seven patients with TRPV4 variants of unknown significance, ranging from a 2-year-old boy to a 75-year-old man, and some of their relatives were genotyped.

    What they did

    The team made each TRPV4 variant in the lab, put it into cultured cells and measured calcium entry, cell death, protein amount, position in the cell and binding to the partner proteins RhoA and PACSIN3. They mapped each variant onto the known 3D structure of the channel. They then combined these results with clinical and family information to update each variant's formal ACMG classification.

    What they found

    • Seven known neuropathy-causing changes in the ankyrin repeat domain all made the channel overactive in the lab tests, which showed the tests can pick up known disease variants.

      “expression of seven known neurological disease-causing mutations within the ARD (R186Q, R232C, R269C, R269H, R315W, R316C, R316H; shown in red in Fig. 1B ) all caused gain of ion channel function”Results, first section

    • In the ankyrin repeat domain, overactivity was found only with changes to charged building blocks at the spot where the channel binds RhoA. Other changes in this domain gave normal or reduced activity.

      “We find that gain-of-function mutations within the TRPV4 intracellular ankyrin repeat domain target charged amino acid residues important for RhoA interaction, whereas ankyrin repeat domain residues outside of the RhoA interface have normal or reduced ion channel activity.”Abstract

    • A second cluster of overactive variants (S93F, S94L, P97R) sits in the flexible 'intrinsically disordered region'. For P97R, blocking the channel's binding to the membrane fat PIP2 removed the overactivity, suggesting a different mechanism from the RhoA-site changes.

      “In contrast, disrupting PIP 2 binding to TRPV4 was sufficient to rescue gain of function of the TRPV4 P97R variant.”Discussion, intrinsically disordered region section

    • Variants tested in the transmembrane domain and in other parts of the disordered region did not cause overactivity and were judged likely benign.

      “In contrast, assessed variants in the transmembrane domain and other regions of the intrinsically disordered region do not cause gain of function and are likely benign.”Abstract

    • In the patients studied, overactive variants went with problems from birth, vocal cord weakness and mainly motor disease. Variants judged benign came from people with later-onset, mainly sensory neuropathy.

      “Clinical features associated with gain of function and cytotoxicity include congenital onset of disease, vocal cord weakness and motor-predominant disease, whereas patients with likely benign variants often demonstrated late-onset and sensory-predominant disease.”Abstract

    • Adding the lab results changed the formal ACMG classification of 9 of 19 clinical variants.

      “inclusion of our functional data changed the ACMG classification for 9 of 19 clinical variants analysed”Discussion

    What the authors concluded

    Disease-causing TRPV4 variants make the channel overactive through at least two routes: changes at the RhoA binding surface of the ankyrin repeat domain, and changes in the central disordered region that may alter how the channel interacts with the cell membrane. Many variants elsewhere are probably harmless. Combining lab tests, protein structure and clinical features gives a framework for judging new variants, which the authors say will matter for choosing patients for future trials of TRPV4-blocking drugs.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • ModerateCell lines that overproduce the channel

      All tests used lab cell lines given extra copies of TRPV4, not nerve cells or cells from patients. The authors themselves warn this can give misleading results, so a variant's behavior in these tests may not match what happens in a person's nerves.

      “our functional assays rely on transient transfection and over-expression of TRPV4 variants, which could lead to misleading functional effects.”Conclusions (limitations)

    • ModerateTests not checked against known harmless variants

      The assays were shown to detect known disease variants, but not shown to give normal results for a set of known harmless variants. This matters because many variants were called benign precisely because they gave normal or reduced results.

      “While we validated our functional assays with several pathogenic variants, similar validation using a series of known benign variants would strengthen confidence in the assessment of variants with normal or reduced ion channel activity.”Conclusions (limitations)

    • ModerateAssumes overactivity is the only disease mechanism

      Normal or reduced channel activity was treated as evidence that a variant is benign. If some TRPV4 disease works another way, people with such variants could be wrongly reassured. The authors acknowledge this.

      “we cannot fully exclude alternative mechanisms of disease”Conclusions (limitations)

    • ModerateClinical pattern rests on few, mixed patients

      The link between overactivity and features such as vocal cord weakness comes from seven new patients plus earlier reports, with no statistics. Several patients had other possible causes for their symptoms: the G321V patient had type 2 diabetes, the M680V patient also had a FOXC1 deletion, and the S94L patient was treated for possible multiple sclerosis. Information on the V264D child was limited. The R271H patient is described as having upper limb weakness and 'motor neuropathy', yet the Discussion groups her with 'solely length-dependent' cases.

      “none of the patients with TRPV4 variants that presented with solely length-dependent polyneuropathies, such as the patients with the I545N, G321V and R271H variants, had gain-of-function toxicity of TRPV4.”Discussion, clinical phenotypes section

    • Moderate'Asymptomatic' relatives not described as examined

      Relatives who carry a variant but are called asymptomatic are used as genetic evidence, yet no neurological examination or nerve tests are described for them. TRPV4 disease can be very mild or vary within families, as the paper itself notes, so 'asymptomatic' by report is weak evidence. The brother with neuropathy who lacks the I545N variant is stronger evidence.

      “revealed that his asymptomatic sister also had the I545N mutation, while his brother, who was diagnosed with peripheral neuropathy, did not have the I545N mutation”Results, I545N section

    • MinorText calls some variants benign while the table keeps them uncertain or likely pathogenic

      The Discussion lists R271H, R271C and G321V among variants that are 'benign', but Table 1 gives their updated ACMG class as likely pathogenic (R271H) or uncertain (R271C, G321V). The lab result points toward benign, but the formal classification did not reach benign. Readers could be confused about the status of these variants.

      “the G20R, R160Q, R179C, R219H, R224Q, R248C, V264D, S319L, R271C, R271H, G321V, C480F, V515F, I545N and M680V variants are benign”Discussion, first paragraph; Table 1

    • MinorSmall internal inconsistencies and typos

      The Methods give a final salt concentration of 70 mM for the hypotonic test, but the Figure 1 legend says 30 mM. The Results say expression of all ankyrin domain variants was comparable to normal, while the Figure 4 legend reports reduced R248C and S319L expression. Several variant names are mistyped in legends (V246D, R217H, R319L, P79R). These do not change the main conclusions but show the paper needs careful reading.

      “treated with hypotonic saline (30 mM NaCl) at time = 20 s”Figure 1 legend; Methods (calcium imaging); Figure 4 legend

    • MinorCompany links

      Several authors take part in a natural history study supported by Actio Biosciences, and one author's lab work on TRPV4 is supported by the same company. This is declared openly.

      “C.J.S. is supported by Actio Biosciences for laboratory-based studies of TRPV4 channelopathy.”Competing interests

    How “unaffected” relatives were checked

    Relatives were mostly described by report, not by documented examination. The I545N carrier sister and the V264D carrier father are both called 'asymptomatic', with no neurological examination or nerve tests described for either. Quote: 'His father had the same variant and was asymptomatic.' (Results, G321V and V264D section). The S93F father is described with signs such as scapular winging and areflexia, but how he was assessed is not stated.

    Three ways to explain it

    Scientific

    In heterologous overexpression assays, known pathogenic TRPV4 variants consistently showed gain of function (raised basal and hypotonic-evoked calcium and increased cytotoxicity). Gain-of-function variants clustered at charged ankyrin repeat domain residues at the RhoA interface and in the central intrinsically disordered region (S93F, S94L, P97R), where the P97R effect depended on PIP2 binding; transmembrane domain variants and ankyrin repeat domain variants away from the RhoA interface showed normal or reduced function and were classed as likely benign. Adding the functional data changed the ACMG class for 9 of 19 clinical variants. In a small number of patients, gain of function was associated with congenital onset, vocal cord weakness and motor-predominant disease, but the approach assumes gain of function is the disease mechanism and was not validated against known benign variants.

    Easier

    TRPV4 is a channel in the cell surface that lets calcium in. The researchers put 30 different TRPV4 changes into cells grown in the lab and checked whether each made the channel overactive and harmful to the cells. Known disease-causing changes made it overactive; many changes of uncertain meaning did not, so the team judged them probably harmless. People whose changes made the channel overactive tended to have problems from birth, a weak voice box or mainly muscle weakness, but this pattern comes from only a handful of patients and from lab cells, not nerves.

    Simple

    TRPV4 is like a door that lets calcium into cells. The scientists tested many small spelling mistakes in the TRPV4 instructions. They found that the ones known to cause illness jam the door too far open. Many others leave the door working normally and are probably harmless. They tested this in cells in a dish and in only a few people, so it is a helpful guide rather than a final answer.

    Why these ratings

    Relevance 4/5: It directly addresses how to tell disease-causing from harmless TRPV4 variants in neuropathy, which matters for anyone given an uncertain TRPV4 result, but most of the work is in lab cells rather than patients.

    Evidence 3/5: Careful, blinded lab work testing many variants with several assays, but all in cell lines overproducing the channel, without testing against known harmless variants, and with clinical links drawn from only a handful of patients.

    Funding and conflicts of interest

    Funded by the NIH National Institute of Neurological Disorders and Stroke (several grants), the Merkin Peripheral Neuropathy and Nerve Regeneration Center, the German Research Foundation (DFG) and the Dr. John H. Doran Neuropathy Research Initiative. Competing interests: M.H., S.T., T.O.C., C.J.S. and B.A.M. take part in an investigator-initiated TRPV4 neuropathy natural history study (NCT05600764) supported by Actio Biosciences, and C.J.S. is supported by Actio Biosciences for lab studies of TRPV4 channelopathy.

    PubMed 39021275 · DOI

38. #38Full paperCell, mouse tissue and fly study

    Multiubiquitination of TRPV4 reduces channel activity independent of surface localization

    Aisenberg WH, McCray BA, Sullivan JM et al. · 2022 · The Journal of biological chemistry

    Relevance 4/5Evidence 3/51 moderate · 4 minor problems

    This lab study found one of the cell's natural brakes on TRPV4, small ubiquitin tags, and showed that the CMT2C change R269C carries fewer of them, apparently because it is overactive. Boosting the tags partly calmed the faulty channel in lab cells, but this has not been tested in animals with the disease or in people.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    Cell, mouse tissue and fly study

    Who or what

    TRPV4 studied in cell lines (human embryonic kidney HEK293T, the rat sensory neuron line 50B11, dog kidney MDCK cells and mouse motor neuron-like MN-1 cells), choroid plexus tissue from mice engineered to carry tagged ubiquitin, and transgenic Drosophila flies producing normal or R269C TRPV4 in their nerve cells. The neuropathy mutants tested were R269C and R237L. No patients or patient cells were studied.

    What they did

    The team tested whether TRPV4 carries ubiquitin tags (small protein labels that cells attach to proteins) and used mass spectrometry to map where they attach. They blocked key attachment sites, or added the tagging enzyme NEDD4, and measured calcium entry and how much TRPV4 sat on the cell surface. They then compared tagging of the neuropathy mutant R269C with normal TRPV4 in cells and flies, and tested whether extra NEDD4 calmed the mutant channel.

    What they found

    • TRPV4 carries several single ubiquitin tags (multiubiquitination), found at 15 lysine sites, mostly in the flexible, unstructured stretches at its two ends.

      “MS analyses of ubiquitinated TRPV4 derived from eight distinct transfections identified 15 ubiquitinated lysine residues”Results, TRPV4 is ubiquitinated at lysine residues in its cytoplasmic intrinsically disordered regions

    • Blocking tagging at five sites in the N-terminal flexible region (the 5K-IDR mutant) made TRPV4 much more active, without putting more channels on the cell surface. Blocking sites at the other end had no such effect.

      “5K-IDR-expressing cells showed a 300% greater response to 10 nM GSK101 compared with WT-CTD-expressing cells or 5K-CTD-expressing cells”Results, blocking ubiquitination in the N-terminal IDR increases TRPV4 channel activity

    • Adding the enzyme NEDD4 increased TRPV4 tagging and reduced channel activity, but did not reduce the amount of TRPV4 at the cell surface.

      “Conversely, enhancing TRPV4 ubiquitination via expression of an E3 Ub ligase reduced TRPV4 channel activity but did not decrease plasma membrane abundance.”Abstract; Results, Figures 5 and 6

    • The neuropathy mutant R269C carried fewer ubiquitin tags than normal TRPV4, both in cultured cells (about half at several sites) and in flies.

      “gain-of-function neuropathy-causing mutations in the TRPV4 gene led to reduced channel ubiquitination in both cellular and Drosophila models of TRPV4 neuropathy”Abstract; Results, Figure 7

    • The reduced tagging seems to be a result of the mutant channel's overactivity rather than its cause: blocking the channel with a drug in cells, or with a pore-blocking change in flies, restored normal tagging.

      “These data suggest that the reduced ubiquitination of neuropathy-causing TRPV4 mutants is downstream of their increased channel activity and not because of a structural inability of the protein to be ubiquitinated.”Results, neuropathy-causing mutations exhibit reduced ubiquitination

    • Extra NEDD4 increased tagging of R269C and partly reduced its overactivity in cells; it also reduced responses of another neuropathy mutant, R237L, in MN-1 cells.

      “NEDD4 expression both enhanced R269C TRPV4 ubiquitination and inhibited mutant channel activity upon antagonist withdrawal”Results, enhancing TRPV4 ubiquitination can partially rescue the gain-of-channel function

    What the authors concluded

    Ubiquitin tags on TRPV4's flexible N-terminal region act as a brake on channel activity without removing the channel from the cell surface. This is a new way of controlling an ion channel, and it may work partly through the channel's binding of the membrane lipid PIP2. Neuropathy mutations reduce this tagging as a knock-on effect of their overactivity, which may add to the problem. Boosting tagging with NEDD4 partly calmed the mutant channels. The authors note this may be relevant to emerging treatment approaches that change ubiquitination.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • ModerateLab models far from patients

      Most experiments used human kidney cells and other cell lines made to overproduce TRPV4 and NEDD4, plus flies. No patient cells or human nerves were studied. Adding extra NEDD4 to cells is a lab tool, and the paper does not show any way to do this in people.

      “Restoring ubiquitination of neuropathy mutant TRPV4 through expression of NEDD4 was able to ameliorate increased channel activity.”Discussion

    • MinorUbiquitin explains only part of the problem

      The paper shows that reduced tagging follows from overactivity, so at most it is a feedback loop that adds to the problem, not the root cause of CMT2C overactivity. The rescue with NEDD4 was partial. The authors themselves say the main cause of overactivity is still unknown.

      “In fact, it is still unknown how neuropathy-causing mutations result in increased channel activity.”Results, neuropathy-causing mutations exhibit reduced ubiquitination

    • MinorExclusion of cells with high starting calcium

      In one experiment, cells with high calcium before stimulation were left out, and more cells were excluded from the NEDD4 group (13.6%) than from the TRPV4-alone group (5.5%). Leaving out high-calcium cells could slightly favor finding lower responses with NEDD4. The reason given (to study healthy cells) is reasonable, but the effect on results is not reported.

      “To ensure assessment of a healthy population of cells, we analyzed only those cells that did not exceed two standard deviations above WT TRPV4 baseline intracellular calcium levels.”Results, enhancing TRPV4 IDR ubiquitination dampens stimulated channel responses

    • MinorSome supporting experiments done only once or twice

      Several supporting results, such as NEDD4 still binding the lysine mutants, come from one or two repeats without statistics. The main calcium results have larger numbers, but these particular supporting points are less certain.

      “Fig. S4 : ( D ) one transfection and IP.”Experimental procedures, statistics and reproducibility

    • MinorSmall counting and labeling inconsistencies

      The text says 15 tagged lysines but its list by region adds up to one fewer; Table 1 also lists lysine 407. The methods are headed 'TMT 8-plex' but describe 10-plex reagents. These do not change the conclusions.

      “five localized to the N-terminal IDR (Lys 70, 77, 101, 130, and 136), six to the ARD (Lys 192, 197, 340, 344, 352, and 382), and three to the CTD (Lys 766, 801, and 834)”Results; Table 1; Experimental procedures

    Three ways to explain it

    Scientific

    TRPV4 is multiubiquitinated predominantly within its N- and C-terminal intrinsically disordered regions, and N-terminal IDR ubiquitination negatively regulates channel activity without altering plasma membrane abundance, partly via PIP2 binding. The neuropathy mutant R269C showed reduced ubiquitination in HEK293T cells and Drosophila; ubiquitination was restored by an antagonist or a pore-inactivating mutation, indicating that the reduction is secondary to gain of function. NEDD4 overexpression partially suppressed R269C and R237L overactivity in cell lines.

    Easier

    Cells can stick tiny tags called ubiquitin onto proteins to change how they work. This study found that these tags on one end of TRPV4 act like a brake, reducing how much calcium the channel lets in, without removing the channel from the cell surface. A CMT2C-causing change (R269C) had fewer tags, seemingly because the channel was already overactive. Adding more tags with an enzyme called NEDD4 partly calmed the faulty channel in lab-grown cells.

    Simple

    TRPV4 is a door that lets calcium into cells, and the cell can stick small tags on it that work like a brake. In CMT2C the door is stuck too far open and has fewer brake tags. Adding more tags helped close the door a little in lab cells, but nobody has tried this in people.

    Why these ratings

    Relevance 4/5: Studies a natural control system for TRPV4 and how it is altered by CMT2C-causing changes such as R269C.

    Evidence 3/5: Thorough lab work using several methods, but in cell lines and flies with overproduced proteins, far from people.

    Funding and conflicts of interest

    Supported by NIH grants F31 NS105404 and R35 NS122306, the Deutsche Forschungsgemeinschaft (EXC 2051, Project ID 390713860), fellowships to individual authors, and NIH award S10OD023548 for the microscope facility. The authors declare no conflicts of interest.

    PubMed 35300980 · DOI

39. #39Full paperStructural biology study (cryo-electron microscopy) with calcium imaging in a cell line

    Structure of human TRPV4 in complex with GTPase RhoA

    Nadezhdin KD, Talyzina IA, Parthasarathy A et al. · 2023 · Nature communications

    Relevance 4/5Evidence 3/51 moderate · 4 minor problems

    This study shows in near-atomic detail that the protein RhoA sits on the part of TRPV4 where many neuropathy-causing changes are found, which supports the idea that these changes upset a natural brake on the channel. It also shows where an experimental blocking drug binds, but this is basic science and a long way from a treatment.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    Structural biology study (cryo-electron microscopy) with calcium imaging in a cell line

    Who or what

    Full-length normal human TRPV4 made in HEK 293S cells and purified in detergent, imaged on its own, with the activating compound 4α-PDD and with the blocking compound HC-067047. The cells' own RhoA protein came along bound to the channel. A few altered TRPV4 versions were tested by calcium imaging in transfected HEK 293 cells. No patients or animals were studied.

    What they did

    The team purified human TRPV4, froze it and used cryo-electron microscopy to build 3D structures at 3.0 to 3.5 Å resolution. They found the protein RhoA attached to the channel's ankyrin repeat domains and mapped which TRPV4 building blocks touch it. They also solved structures with an opening drug and a blocking drug and tested a few changed versions of the channel in cells.

    What they found

    • RhoA, a protein known to damp TRPV4 activity, sits on the membrane-facing side of the channel's ankyrin repeat domains. The closest contacts involve TRPV4 residues R232, R237, D263, R269, R315 and R316.

      “The strongest contacts are established between polar residues of TRPV4, including R232, R237, D263, R269, R315, and R316, and side chains and backbone carbonyls of RhoA residues R5, E40, E54, and D76”Results, Architecture of hTRPV4-RhoA complex

    • Most of the TRPV4 residues in this contact area are places where disease-causing changes have been reported, including in peripheral neuropathy.

      “Remarkably, most residues involved in polar TRPV4-RhoA interactions are subjects of disease-causing mutations in TRPV4-linked channelopathies, including peripheral neuropathy and skeletal dysplasia”Results, Architecture of hTRPV4-RhoA complex

    • The RhoA came from the cells themselves rather than being added by the researchers, and each channel had between zero and four RhoA molecules bound to it.

      “RhoA binds to hTRPV4 at various stoichiometries, ranging from none to four RhoA molecules bound to a single hTRPV4 tetramer.”Results, Functional and structural characterization of human TRPV4

    • Changing one contact residue (R316 to alanine) raised resting calcium entry through the channel, which the authors take as support for RhoA acting as a brake.

      “we introduced the R316A mutation that resulted in increased baseline calcium uptake, consistent with previous studies of neuropathy-causing mutations at the TRPV4-RhoA interface”Results, Architecture of hTRPV4-RhoA complex

    • The opening drug 4α-PDD and the blocker HC-067047 both bind near the base of the channel's S1-S4 region; the opener widens the pore, while the blocker locks the gate by refolding part of helix S6. In cells, HC-067047 blocked TRPV4 with an IC50 of 25.0 nM.

      “show that agonist binding leads to pore opening, while channel inhibition involves a π-to-α transition in the pore-forming helix S6.”Abstract; Results

    What the authors concluded

    The authors conclude that RhoA binds the membrane-facing surface of TRPV4's ankyrin repeat domains at residues often mutated in disease, which gives insight into how neuropathy-causing changes may work. They add that the drug-bound structures show how the channel opens and is blocked and could serve as a template for designing future treatments for TRPV4-related diseases.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • ModerateLink to neuropathy is inferred, not tested here

      No patient variant was imaged or tested in this paper. The connection to neuropathy comes from mapping known disease positions onto the structure and from earlier published work; the one interface change tested (R316A) is an alanine substitution, which the paper does not report as a patient variant.

      “Consistent with our structure, previous studies showed that neuropathy-related mutants R232C, R237L, R269C, and R315W disrupt protein-protein interactions between TRPV4 and RhoA”Results, Architecture of hTRPV4-RhoA complex

    • MinorArtificial conditions far from nerve cells

      The channel was made in a cell line, extracted into detergent and frozen, and drugs were added at 200 μM for the structures, far above the 25 nM blocking strength measured in cells. These conditions are standard for structural work, but the structures are snapshots and may not show how the channel behaves in human nerves.

      “we expressed full-length wild-type human TRPV4 in HEK 293S cells and purified it in glyco-diosgenin (GDN) detergent.”Results, Functional and structural characterization of human TRPV4

    • MinorHow RhoA's switch state affects binding is unresolved

      RhoA was seen in its inactive (GDP-bound) form, and an attempt to load an active-form mimic was inconclusive. So the structure does not show how RhoA's on/off state changes its grip on TRPV4.

      “making it unclear whether GTPγS did bind RhoA. Additional experiments are required to study the effects of nucleotides on RhoA regulation of hTRPV4.”Results, TRPV4 structure in complex with agonist 4α-PDD

    • MinorLimited functional data

      The calcium imaging that supports the structure is in the supplement, with potency measurements from 4 experiments and traces from at least 3 independent experiments of 20 to 40 cells each. This is enough to support the structural model but is a small functional data set.

      “The F340/380 ratios were averaged and plotted from at least 3 independent experiments.”Methods, Fura-2 calcium imaging

    • MinorSmall inconsistency in binding-site description

      D546A is described as a change in the 4α-PDD binding pocket, but D546 is not among the listed 4α-PDD contact residues; it is listed for the HC-067047 site. This does not affect the RhoA findings.

      “we introduced the N474A, D546A and Y591A mutations in the 4α-PDD binding pocket”Results, TRPV4 structure in complex with agonist 4α-PDD

    Three ways to explain it

    Scientific

    Cryo-EM structures of full-length human TRPV4 (about 3.0 Å) revealed endogenous GDP-bound RhoA attached to the membrane-facing surface of the ankyrin repeat domain, with interface residues (R232, R237, D263, R269, R315, R316) that largely coincide with disease-causing mutation sites. An interface substitution, R316A, increased baseline calcium uptake, consistent with RhoA acting as an inhibitor. The agonist 4α-PDD and antagonist HC-067047 both bind at the base of the S1-S4 bundle, with agonist binding opening the pore and inhibition involving a π-to-α transition in S6; the neuropathy link is inferred from mapping rather than tested with patient variants here.

    Easier

    Using a powerful electron microscope, researchers made a detailed 3D picture of the human TRPV4 channel. They found a protein called RhoA, which can reduce TRPV4 activity, stuck to the channel's ankyrin region. The exact spots where RhoA touches TRPV4 are mostly places where disease-causing changes have been found, including changes linked to neuropathy. They also showed where an opening drug and a blocking drug attach, which may help design medicines in the future.

    Simple

    TRPV4 is like a door that lets calcium into cells, and RhoA is like a hand that helps keep the door from opening too much. Scientists took a super-detailed picture and saw that the hand grips the door right where many disease changes are. That suggests these changes might loosen the grip, but this still needs testing.

    Why these ratings

    Relevance 4/5: Shows in structural detail how RhoA binds the part of TRPV4 where neuropathy-causing changes such as R269 and R315 lie, which bears directly on the mechanism behind CMT2C.

    Evidence 3/5: High-quality structural work with supporting mutation tests, but the link to neuropathy is inferred from mapping and earlier studies rather than tested with patient variants here.

    Funding and conflicts of interest

    Funded by US National Institutes of Health grants (R01 AR078814, R01 CA206573, R01 NS083660, R01 NS107253, R01 HL096647; facility support U24GM129547) and a German Research Foundation (DFG) Walter Benjamin Fellowship. The authors declare no competing interests.

    PubMed 37353478 · DOI

40. #40Full paperMouse model study (knock-in mice, cell-type-specific gene removal and drug treatment) with cultured cells from the mice; author manuscript

    Gain-of-function mutations of TRPV4 acting in endothelial cells drive blood-CNS barrier breakdown and motor neuron degeneration in mice

    Sullivan JM, Bagnell AM, Alevy J et al. · 2024 · Science Translational Medicine

    Relevance 4/5Evidence 3/51 major · 7 moderate · 1 minor problems

    In young mice with a severe, early-fatal form of TRPV4 disease, the damage appeared to start in the lining of the spinal cord's blood vessels, which became leaky, rather than in the nerve cells themselves. A TRPV4-blocking drug, started after symptoms appeared, closed the leaks and rescued the mice. This has not been shown in people with CMT2C: no patients or human tissue were studied, the authors say the mice match only the severe end of the human range, and the drug given to the mice (GSK2193874) is not the compound named in a cited human trial (GSK2798745).

    Full analysis: findings, problems and plain explanations

    The study

    Type

    Mouse model study (knock-in mice, cell-type-specific gene removal and drug treatment) with cultured cells from the mice; author manuscript

    Who or what

    Laboratory mice only; no people and no human tissue. Two knock-in lines in which the human disease-causing changes R269C and R232C were put into the mouse's own Trpv4 gene (not an added human gene). The main disease models were R269C mice with two mutant copies (homozygous, Trpv4R269C/R269C) and R232C mice with one mutant copy (heterozygous, Trpv4R232C/+). Mice with one copy of R269C appear in the survival and hang-test comparisons, but the text does not describe their results. Group sizes: 50 to 101 mice per genotype for survival, 22 to 43 for the forelimb hang test, 7 to 25 per group for the other movement tests, and usually 3 mice per group (up to 7) for tissue counts. The R269C line was crossed with 13 Cre mouse strains to remove the mutation from particular cell types. Blood vessel lining cells (neural vascular endothelial cells, NVECs) from the brain and spinal cord of the mice were grown in dishes. Male and female mice were used.

    What they did

    The team made two lines of mice, each carrying one of two TRPV4 changes that the paper says cause both distal spinal muscular atrophy (dSMA) and CMT2. They then tracked the mice's movement and survival and examined their spinal cords and muscles. They removed the mutant gene from one cell type at a time to find which cells drive the disease, and tested the mice's blood vessel lining cells in a dish. Finally, they gave a TRPV4-blocking drug (GSK219) by injection to R269C mice that already had symptoms.

    What they found

    • Mice with two copies of R269C developed movement problems in their third week of life and died by weaning age. Weakness mainly affected the neck and front legs, with labored breathing in the final hours. Motor neurons (the nerve cells that control muscles) were lost in a patch at the very top of the spinal cord (level C1). Neck muscles lost their nerve supply but front-leg muscles did not. Mice with one copy of R232C showed a comparable disease.

      “exhibited motor behavioral deficits with onset in the third postnatal week (Fig. 1, A and B, fig. S2, A to C) and lethality by weaning age”Results, 'Mutant TRPV4 mice develop marked neurological phenotypes' and 'Spinal motor neuron degeneration in TRPV4 mutant mice'

    • The R269C mice were crossed with 13 strains that remove the mutant gene from one cell type at a time (neurons, glia, muscle or blood vessel lining cells). Only removal from blood vessel lining (endothelial) cells, done with two independent strains, rescued survival and movement and prevented motor neuron loss. A reporter mouse showed the Trpv4 gene switched on in vessel lining cells of the spinal cord and brain, and the authors report it was not switched on in spinal motor neurons.

      “marked rescue of mutant mice from lethality was only observed following endothelial cell-specific deletion of the mutant allele by crossing to two independent Cre-expressing mouse strains”Results, 'Genetic deletion of the mutant allele from endothelial cells rescues TRPV4 mutant mice'; Discussion

    • Vessel lining cells grown from mutant mice reacted more strongly and faster than normal cells when a chemical that switches TRPV4 on (GSK101) was added, which the authors call a gain of function. Resting calcium levels were not different. In a dish test of barrier tightness, layers of mutant cells lost tightness faster at a low dose of the activator (50 nM) but not at a higher dose (500 nM), and both recovered by 24 hours.

      “resulted in elevations in cytosolic free calcium in NVECs from Trpv4R269C/R269C mice that were of greater amplitude and shorter latency than those observed in NVECs from WT littermates”Results, 'Mutant TRPV4 channels are overactive in NVECs'

    • Mice with symptoms had patchy, often one-sided leaks in the barrier between blood and spinal cord, mainly at the top of the spinal cord and in the brainstem, close to motor neurons. Leaks were not seen before symptoms, nor in mice whose vessel lining cells lacked the mutant gene, and were reduced or absent lower in the spinal cord. At leak sites, a tight junction protein (ZO-1) that helps seal the gaps between vessel cells was lost.

      “Symptomatic Trpv4R269C/R269C mice at Stage B exhibited marked, often asymmetric, leakage of all three molecules, most prominently within the upper cervical spinal cord”Results, 'Mutant TRPV4 expression in endothelial cells causes focal BSCB breakdown'

    • A TRPV4-blocking drug, GSK2193874 (GSK219), was injected into the belly (intraperitoneal) twice a day at 0.5 or 2 mg/kg, starting once R269C mice already had clear symptoms (Stage B, aged P17 to 21, that is, 17 to 21 days after birth). Treated mice lived longer and moved better on balance and rotarod tests than mice given the drug-free liquid (vehicle), with near complete rescue at 2 mg/kg. The drug was tested only in the R269C line.

      “Symptomatic Trpv4R269C/R269C mice (Stage B) treated systemically with GSK219 (0.5-2 mg/kg; twice daily intraperitoneal injections) exhibited marked improvements in both survival (Fig. 6A) and motor behavior”Results, 'TRPV4 antagonist administration rescues symptomatic mutant mice'

    • After an average of 37.6 ± 1.2 days of treatment (examined at P50 to 60, 3 mice per group), treated mice showed no tracer leak at the top of the spinal cord and no motor neuron loss or axon damage at level C1. ZO-1 returned after 10 days at 2 mg/kg. Treatment began at a stage the authors describe as having only limited motor neuron loss.

      “As in Cre-rescued mutant mice, EZ-Biotin extravasation was not detected in the upper cervical spinal cord of GSK219-treated mice (Fig. 6, D and E), demonstrating rescue of BSCB function.”Results, 'TRPV4 antagonist administration rescues symptomatic mutant mice'; Fig. 6 legend

    What the authors concluded

    The authors conclude that, in these mice, overactive mutant TRPV4 in the cells lining blood vessels makes the barrier between blood and spinal cord leak in patches, and that this leak leads to motor neuron damage, rather than the mutation acting inside the motor neurons themselves. Because a TRPV4-blocking drug given after symptoms began closed the leak and rescued the mice, they suggest TRPV4-blocking drugs are a potential treatment for people with TRPV4 mutations. They state that studies in humans are still needed, including on how early treatment would have to start.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • MajorMouse disease is far from typical human CMT2C

      The R269C mice that became ill carried two mutant copies, while the paper describes human TRPV4 disease as usually dominant, meaning one faulty copy is enough. Mice with one copy of R269C are included in the survival and hang-test figure, but the text does not say how they fared. The sick mice became weak in their third week of life and died by weaning age, and damage centered on the top of the spinal cord and brainstem. The authors themselves say the mice mirror only the severe end of the human spectrum. Results in these mice may not carry over to people, especially those with milder or later-onset disease.

      “As mutant mice exhibit either full (Trpv4R269C) or near-full lethality (Trpv4R232C) by weaning age, these lines mirror the severe end of the clinical spectrum of TRPV4-mediated motor neuron disease.”Discussion

    • ModeratePeripheral nerves, sensory nerves and vocal cords not examined

      The study looked at the spinal cord, brainstem, and neck and front-leg muscles. The main text does not report examining the nerves running out to the limbs, sensory nerve cells or the vocal cords, although the introduction lists vocal cord, limb and diaphragm weakness as part of the human disease. So it is unknown whether this blood vessel mechanism explains sensory or limb nerve problems.

      “Given the pronounced neck and forelimb motor behavioral deficits, we performed histopathological assessments of the cervical spinal cord and associated musculature.”Results, 'Spinal motor neuron degeneration in TRPV4 mutant mice'

    • ModerateNo human tissue or patient data

      No patient tissue, scans or clinical data were studied, so the barrier leak has not been shown in people. The link to humans rests on other groups' single-cell studies showing TRPV4 in human vessel lining cells. The statement that human motor neurons lack TRPV4 cites one reference (47), whose title in the reference list is about membrane cholesterol and TRPV4 evolution rather than human spinal cord cells; this may be a citation error, so the support for that point is unclear.

      “Future studies will be required to characterize BSCB impairments in humans.”Discussion; reference 47

    • ModerateHuman safety statement goes beyond the evidence, and involves a different drug

      The mice received GSK2193874 by injection. The human trials the paper cites involved an oral TRPV4 blocker, which one cited reference title names as GSK2798745, a different compound. The reference titles describe trials in heart failure, healthy volunteers and chronic cough, not TRPV4 neuropathy. Being well tolerated in those trials does not show there are no side effects. The main text does not report side-effect checks in the treated mice; it relies on earlier rodent work.

      “These findings suggest that TRPV4 inhibition within endothelial cells can be achieved in human patients without accompanying side effects or adverse events.”Discussion; references 80 to 82

    • ModerateDrug experiment is narrow

      The drug was tested in one line (R269C, two copies), at one starting point, by twice-daily injection. Treatment began at a stage the authors describe as having only limited motor neuron loss, so the result is best read as protection of remaining neurons and repair of the barrier, not regrowth of lost neurons. Tissue results come from 3 treated mice per group, and the vehicle mice were examined at P20 to 22 while treated mice were examined at P50 to 60. The text does not report what happens if the drug is stopped or over longer periods. The authors say it is unknown how early treatment would need to start in patients.

      “a stage characterized by leakage of serum products, motor behavioral deficits and motor neuronal dysfunction, but limited motor neuron soma loss”Results, 'TRPV4 antagonist administration rescues symptomatic mutant mice'; Fig. 6 legend; Discussion

    • ModerateNo randomization and unblinded movement tests

      Mice were not randomly assigned to groups, and the people scoring movement tests knew which mice were mutant; the text does not say whether they knew which mice got the drug. Scoring can be swayed by expectations. Average starting age differed a little between drug groups (P18.7 for vehicle, P19.9 for the higher dose). Sample sizes were chosen from pilot studies and earlier work. Results are not split by sex in the main text. In fairness, counts of motor neurons, nerve-muscle junctions and barrier leak were done blind to genotype and treatment, and survival is an objective measure.

      “No randomization was employed. Experimenters were not blinded to genotype for motor behavioral assessments.”Materials and Methods, 'Study design'

    • ModerateLeak causing nerve damage is inferred, not directly tested

      The evidence that the leak causes motor neuron damage is that both happen in the same places and at about the same time, and that removing or blocking mutant TRPV4 prevents both. No experiment closed the leak by another route to show the leak itself is the cause. The gene-removal strains act on vessel lining cells throughout the body, and the drug acts on TRPV4 everywhere. The authors also say they cannot completely exclude other organs contributing to death, and they acknowledge these limits.

      “Although the regionality and asymmetry of BSCB breakdown in symptomatic mutant mice strongly suggest that the neurological phenotypes arise due to direct effects of mutant TRPV4 in NVECs it will be important to verify this experimentally.”Discussion, limitations paragraph

    • ModerateSome wording goes beyond the mouse data

      The mutation is human, but the barrier breakdown was shown only in mice, so 'a human genetic mutation causing' barrier breakdown is a statement about mice. The discussion also says the data are consistent with TRPV4 blockade letting vessel cells 'fully' re-establish the barrier, which rests on a tracer test in 3 treated mice per group.

      “To our knowledge, the current findings represent the first example of a human genetic mutation causing acquired blood-CNS barrier breakdown as the primary driver of neurodegenerative pathology.”Discussion

    • MinorSmall tissue groups and reporting gaps

      Motor neuron counts and most barrier and tight junction measures come from 3 mice per group (up to 7). Some graphs count each spinal cord section as a separate data point (for example 30 hemisections from 3 mice), which can make results look more certain than they are, although per-mouse results are also shown. The legends for the survival curves of the gene-removal and drug experiments give no group sizes and name no statistical test; these may be in the figures or supplement, which were not available here. The main text confirms gene removal only for the two endothelial strains, so how completely the other strains removed the gene is not shown. Small groups are usual for this kind of tissue work.

      “Mean number of MNs per C1 spinal cord hemisection, based on assessments of the mean number per mouse (E; n=3 mice/genotype)”Fig. 1 legend; Fig. 2 and Fig. 6 legends

    Three ways to explain it

    Scientific

    In knock-in mice carrying the CMT2/dSMA-causing mutations R269C (homozygous) or R232C (heterozygous) in the endogenous Trpv4 gene, TRPV4 gain of function in neural vascular endothelial cells was associated with focal blood-spinal cord barrier breakdown in the upper cervical cord, loss of the tight junction protein ZO-1, regional motor neuron degeneration and early lethality. Deleting the mutant allele from endothelial cells (Cdh5Cre, Tie2Cre), but not from neurons, glia or muscle, rescued the phenotype, indicating a non-cell-autonomous mechanism. Systemic GSK2193874 started at symptom onset restored barrier integrity, protected C1 motor neurons and rescued survival in a dose-dependent manner in R269C mice. No human tissue was studied, and the mouse disease reflects only the severe end of the human spectrum.

    Easier

    TRPV4 is a channel, a kind of gate on the cell surface that lets calcium in. The researchers put two disease-causing TRPV4 changes into mice, which became weak, lost nerve cells at the top of the spinal cord and died young. The faulty channel worked too strongly in the cells lining small blood vessels, and this made the protective barrier between the blood and the spinal cord leak in patches next to motor neurons (the nerve cells that control muscles). Removing the faulty gene from those vessel cells, or injecting a TRPV4-blocking drug once symptoms began, stopped the leak and rescued the mice, but none of this has been tested in people.

    Simple

    TRPV4 is like a door that lets calcium into cells. In these mice, the door was stuck too far open in the walls of tiny blood vessels, so the vessels in the spinal cord leaked like a cracked pipe and nearby nerve cells got hurt. A medicine that pushes the door shut fixed the leak and helped the mice. But they were very sick young mice, and nobody knows yet whether the same thing happens in people or whether the medicine would help them.

    Why these ratings

    Relevance 4/5: A mouse study of the disease mechanism behind TRPV4 neuropathy, using two mutations that cause CMT2 in people, with a test of a TRPV4-blocking drug, but with no patient data.

    Evidence 3/5: Careful mouse work using many methods with built-in checks (two mutations, two endothelial gene-removal strains, blinded tissue counts), but with no randomization, unblinded movement tests, small tissue groups, no human data, and a severe early-fatal mouse disease that the authors say mirrors only the severe end of the human range.

    Funding and conflicts of interest

    Funded by the Muscular Dystrophy Association (grants 629305 and 2777551), several National Institutes of Health and National Institute of Neurological Disorders and Stroke grants, a New Discovery Award from Advance-CTR (supported by IDeA-CTR grant U54GM115677) and a Pamela Mars Wright Innovator Award. Competing interests: senior author C.J.S. has been a consultant to many companies, including Actio Biosciences, Novartis, Biogen and Roche; previously received research grant support from Ionis Pharmaceuticals and Argenx; and currently receives grant support from Roche, Biogen and Actio Biosciences. The paper does not say what Actio Biosciences works on or whether that support relates to this study. C.J.S. is an unpaid advisor to patient groups including the CMT Research Foundation and CMT Association, and co-holds two pending patent applications with Ionis Pharmaceuticals on antisense oligonucleotides targeting SMN-AS1; as described, these do not concern TRPV4. All other authors declare no competing interests. No GSK authors or GSK funding are listed, and the main text does not say where the GSK-named compounds (GSK1016790A, GSK2193874) came from.

    PubMed 38776392 · DOI

41. #41Detailed summary onlyFamily study of hearing, with repeated hearing tests in some members

    Intrafamilial variable hearing loss in TRPV4 induced spinal muscular atrophy

    Oonk AM, Ekker MS, Huygen PL et al. · 2014 · The Annals of otology, rhinology, and laryngology

    Relevance 4/5Evidence 3/54 moderate · 2 minor problems

    In this one family with the R269H change, everyone with the mutation developed hearing loss from the inner ear or hearing nerve, usually noticed in mid-life and slowly worsening. How severe it was did not match how weak their muscles were.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    Family study of hearing, with repeated hearing tests in some members

    Who or what

    One Dutch family with the TRPV4 change c.806G>A (R269H), first described in 1985 as having a dominantly inherited spinal muscular atrophy limited to the lower body. Eight relatives took part, four of them mutation carriers, with hearing tests at ages between 35 and 69.

    What they did

    Hearing specialists in Nijmegen invited the family. Each participant filled in a hearing and balance questionnaire and had an ear, nose and throat examination. Hearing was measured from 0.25 to 8 kHz by air and bone conduction, speech recognition was tested, and earlier hearing tests were used to compare each person's change over time with normal age-related hearing loss. A neurological examination was repeated. This analysis is based on a detailed secondary summary of the paper written by another reader, not on the paper itself.

    What they found

    • All four carriers had sensorineural hearing loss (hearing loss from the inner ear or hearing nerve). No other cause was found, and nobody reported balance problems.

      “All four carriers had sensorineural hearing loss. No other cause was found, and nobody reported balance problems.”Secondary summary, 'Hearing findings'

    • Hearing loss was first noticed between ages 35 and 62, and all four said it was getting worse. Severity was mild to moderate, with average thresholds from about 35 to about 69 dB.

      “The age at which each person noticed it ranged from 35 to 62. All four said it was getting worse.”Secondary summary, 'Hearing findings'

    • The pattern of hearing loss differed between relatives (sloping, U-shaped, rising, or a mild dip in the middle pitches). In the three people with earlier tests, real worsening beyond normal aging was confirmed, at different pitches in each.

      “Following each person over time confirmed real progression in three of them, at different pitches in each.”Secondary summary, 'Hearing findings' and table 'The four carriers'

    • How severe a person's nerve disease was did not match how severe their hearing loss was.

      “Severity of nerve disease and severity of hearing loss did not go together.”Secondary summary, 'Hearing findings'

    • Speech recognition was slightly below what age alone would predict, but as good as or better than expected for the measured hearing loss.

      “Speech recognition was slightly below what age alone would predict”Secondary summary, 'Hearing findings'

    • The authors recommend screening people with TRPV4 mutations for sensorineural hearing loss and suggest considering advice to be careful with noise, the latter based on findings in mice lacking TRPV4.

      “Screen people with TRPV4 mutations for sensorineural hearing loss, since early hearing support works better than late.”Secondary summary, 'Authors' recommendations'

    What the authors concluded

    Sensorineural hearing loss is part of the condition in this R269H family. It tends to start in mid-life and worsen, but its pattern and rate differ between relatives and do not follow the severity of the nerve disease. The authors recommend hearing screening for people with TRPV4 mutations and suggest considering advice about noise exposure.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • ModerateVery small group

      Only four people with the mutation were studied, all in one family, so the findings cannot show how common or how typical this hearing pattern is.

      “Four affected people in one family. The authors say the group is too small to test their own ideas.”Secondary summary, 'Limitations'

    • ModerateFamily diagnosis is spinal muscular atrophy, not CMT2C

      Whether people whose R269H change shows up as CMT2C have the same hearing pattern is assumed rather than shown.

      “This family's diagnosis is congenital spinal muscular atrophy.”Secondary summary, 'Limitations'

    • ModerateProgression based on few tests

      Earlier hearing tests existed for only three people, so the conclusion that hearing worsens over time rests on a handful of measurements per person.

      “Earlier hearing tests existed for only three people, so progression rests on a handful of data points each.”Secondary summary, 'Limitations'

    • ModerateNoise advice comes from mouse work

      The suggestion about noise exposure is based on a mouse study, and this family neither supports nor rules out the idea, so it goes beyond the human data.

      “The noise advice comes from one mouse study, with no human evidence.”Secondary summary, 'Limitations'

    • MinorNoise exposure by questionnaire

      Past noise exposure, a possible other cause of hearing loss, was assessed only by questionnaire.

      “Noise exposure was assessed by questionnaire.”Secondary summary, 'Limitations'

    • MinorRelatives without the mutation not used as a comparison

      The four relatives without the mutation are not described in detail, so there is no within-family comparison group.

      “The four relatives without the mutation are not described in detail as a comparison group.”Secondary summary, 'Limitations'

    How “unaffected” relatives were checked

    All four carriers had hearing loss, so there were no unaffected carriers. According to the summary, each participating relative completed a questionnaire and had an ear, nose and throat examination, but the four relatives without the mutation are not described in detail. Quote: “Eight relatives took part, four of them mutation carriers.”

    Three ways to explain it

    Scientific

    In one family carrying the TRPV4 R269H variant in the fourth ankyrin repeat, all four heterozygous carriers had mild to moderate sensorineural hearing loss with differing audiometric configurations, and three with serial audiograms showed progression beyond age-related norms at different frequencies. Hearing severity did not correlate with the severity of the neuromuscular phenotype. With four carriers and a spinal muscular atrophy phenotype, the findings describe this family and cannot establish frequency or mechanism in CMT2C.

    Easier

    Researchers tested the hearing of a Dutch family with a TRPV4 change, R269H, which is also found in some people with CMT2C. All four relatives with the change had hearing loss coming from the inner ear or hearing nerve, first noticed between ages 35 and 62. In three of them, tests confirmed it got worse over time. The pattern differed between relatives, and how bad someone's hearing was did not match how bad their muscle weakness was. The family is small, so these are observations rather than rules.

    Simple

    Four people in one family had the same change in their TRPV4 gene, and all of them found their hearing slowly getting worse as grown-ups. It was like four radios with the same fault, each losing different sounds. How weak their legs were did not tell you how bad their hearing would be.

    Why these ratings

    Relevance 4/5: It is a detailed hearing study in a TRPV4 neuromuscular family carrying R269H, which the summary notes is one of the common CMT2C mutations, though the family's own diagnosis is spinal muscular atrophy rather than CMT2C.

    Evidence 3/5: Standardized hearing tests, some repeated over years, but only four carriers in one family, and this analysis rests on a secondary summary.

    Funding and conflicts of interest

    The summary says funding came from the Heinsius Houbolt Foundation, the Oticon Foundation and ZonMw. Competing interests are not mentioned in the summary.

    PubMed 24963089 · DOI

42. #42Full paperLetter to the editor (correspondence)

    TRPV4 neuropathy-causing mutations localize to the convex face of the ankyrin repeat domain (reply in the Neurology correspondence “Phenotypic spectrum and incidence of TRPV4 mutations in patients with inherited axonal neuropathy”)

    Sullivan JM, Landouré G, Gaudet R et al. · 2014 · Neurology

    Relevance 4/5Evidence 2/51 moderate · 3 minor problems

    This letter is part of a scientific debate about the shape of the TRPV4 protein and where CMT2C mutations sit on it. It does not report anything new about symptoms or treatment.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    Letter to the editor (correspondence)

    Who or what

    No patients and no new experiments. A reply to Echaniz-Laguna et al. (Neurology 2014) about the TRPV4 Arg186Gln mutation and about where the mutated arginines (Arg186, Arg232, Arg269, Arg315 and Arg316) sit on the ankyrin repeat domain (ARD) of TRPV4.

    What they did

    Researchers who had earlier published TRPV4 neuropathy mutations and crystal structures of the TRPV4 ankyrin repeat domain wrote to the journal about another team's paper. They raised two concerns: a mutation described as new had already been reported, and the other paper placed the mutated arginines on the wrong face of the domain.

    What they found

    • The letter says the Arg186Gln mutation, described in the other paper as new, had already been reported by these authors, together with its location in the ankyrin repeat domain and its effects in cell studies.

      “In fact, we reported this mutation in Neurology [2] along with descriptions of its location in the TRPV4 ankyrin repeat domain (ARD) and its functional effects in cell-based studies.”Letter, paragraph 1

    • The original paper had stated that the side chains of the mutated arginines point toward the concave (inward-curving) part of the domain.

      “the side chain of Arg186 is directed toward the concave part of the domain, as is the case for Arg232, Arg269, Arg315, and Arg316.”Letter, paragraph 1 (quoting Echaniz-Laguna et al.)

    • The letter's authors say their own published structures show the mutated arginines sit on the convex (outward-bulging) face of the domain, so the original paper's statement contradicts their findings.

      “these conclusions appear to be in contradiction to our findings indicating that the mutated arginine residues reside on the convex face of the ARD [2-5].”Letter, paragraph 1

    • They argue that where the mutations sit matters for understanding how they cause disease, and ask the original authors to correct or fully justify their statement.

      “Given that the location of the mutations has very significant implications regarding their pathogenicity, we do not believe this is a minor point”Letter, paragraph 1

    What the authors concluded

    The authors say the Arg186Gln mutation was not new and that the mutated arginines lie on the convex face of the ankyrin repeat domain, not the concave face. Because they think location matters for how the mutations cause disease, they ask the original authors to either correct their statement or make a complete case for it.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • ModerateLetter with no new data, and only one side of a dispute

      The letter makes its case by pointing to earlier papers rather than showing evidence. The document does not include any reply from Echaniz-Laguna and colleagues, so a reader sees only one side of the disagreement and cannot judge it from this text alone.

      “feel it is important that the authors either correct or make a complete case for their conclusions.”Letter, paragraph 1

    • MinorSupport comes from the letter writers' own earlier work

      The authors are well placed to comment because they produced the structures in question. Still, the evidence for the convex-face position is their own published work (references 2 to 4), which is cited rather than shown, and one supporting reference is from another group (reference 5).

      “Despite being based on our published crystal structures of the TRPV4-ARD (although without appropriate citation)”Letter, paragraph 1

    • MinorWhy location matters is not explained

      The letter says the position of the mutations has very significant implications for pathogenicity but does not say what those implications are. A reader needs the cited papers to understand the point.

      “Given that the location of the mutations has very significant implications regarding their pathogenicity”Letter, paragraph 1

    • MinorTitle in the record does not match the letter

      The repository record cites this item under the title of the paper it responds to. The letter's own title is 'TRPV4 neuropathy-causing mutations localize to the convex face of the ankyrin repeat domain'. The version analyzed here is the author version from the Harvard DASH repository, so its wording may differ slightly from the published letter. Care is needed when citing it.

      “Phenotypic spectrum and incidence of TRPV4 mutations in patients with Inherited axonal neuropathy. Neurology (2014)”Repository cover page, Citation

    Three ways to explain it

    Scientific

    In correspondence on Echaniz-Laguna et al. (2014), Sullivan and colleagues state that TRPV4 Arg186Gln had already been reported, with its ankyrin repeat domain (ARD) location and cell-based functional effects. They also state that the mutated arginines (Arg186, Arg232, Arg269, Arg315 and Arg316) lie on the convex face of the ARD rather than the concave face, and argue that residue location bears on pathogenicity. The letter cites earlier crystal structures and reports but presents no new data.

    Easier

    This is a short letter from one research team to a journal about another team's paper on TRPV4 nerve disease. They say a mutation the other paper called new had already been described. They also disagree about where several CMT2C-linked mutations sit on one part of the TRPV4 protein, the ankyrin repeat domain: they say the mutations sit on the side that bulges outward, not the side that curves inward. They think this matters for understanding how the mutations cause disease.

    Simple

    Two teams of scientists disagreed about where the CMT2C changes sit on one part of the TRPV4 'door'. It is a bit like arguing whether a sticker is on the outside or the inside of a curved shell. This letter gives one team's side.

    Why these ratings

    Relevance 4/5: It concerns where CMT2C-linked TRPV4 mutations (including Arg269, Arg315 and Arg316) sit on the protein, which is part of the mechanism behind TRPV4 neuropathy.

    Evidence 2/5: A short letter with no new data that relies on the authors' earlier published structures and reports.

    Funding and conflicts of interest

    Not stated.

    PubMed 25404646 · DOI · Our copy is the authors' reply letter, not the original letter it answers.

43. #43Full paperCase report (two unrelated patients)

    Phenotypic variability of TRPV4 related neuropathies

    Evangelista T, Bansagi B, Pyle A et al. · 2015 · Neuromuscular disorders: NMD

    Relevance 4/5Evidence 2/54 moderate · 2 minor problems

    This paper adds two more examples of how differently TRPV4 changes can show up, from weakness and skeletal features in early childhood to leg weakness starting around age 40. It also shows that some people with TRPV4 nerve disease have no voice or hearing problems.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    Case report (two unrelated patients)

    Who or what

    Two unrelated patients described by a neuromuscular team in Newcastle, UK: an 8-year-old boy (Patient 1) with a de novo TRPV4 c.805C>T (p.Arg269Cys) variant, and a 48-year-old woman (Patient 2) with a new TRPV4 c.184G>A (p.Asp62Asn) variant. Parents were tested genetically, and for Patient 2 a 29-year-old brother was also tested.

    What they did

    The authors describe each patient's history, examination, nerve tests, muscle biopsy and (for Patient 2) muscle MRI. They used whole exome sequencing to find the genetic cause and checked the variants in relatives. They then compare the two cases with earlier reports.

    What they found

    • Patient 1 had weakness from infancy, scapular winging, foot drop and normal nerve conduction speeds, plus short stature, short legs and short fingers that the authors felt suggested a skeletal dysplasia.

      “There was a slight lumbar lordosis, short stature, short lower limbs, brachydactyly, flat feet and genus valgus, suggestive of skeletal dysplasia.”2.1.1. Patient 1

    • Patient 1's R269C variant arose for the first time in him (both parents tested negative). It has been reported before in CMT2C, SPSMA and congenital distal SMA, but the authors say it had not previously been reported with skeletal dysplasia.

      “To our knowledge this mutation has never been associated with skeletal dysplasia; however milder skeletal abnormalities may have been missed on routine neurological examination.”4. Discussion

    • Patient 2 had normal motor function until about age 40, then developed ankle twisting, foot drop and leg weakness (distal strength grade 2/5). Nerve conduction speeds were normal but motor responses were small, and she had minor sensory loss in the toes, a picture the authors call compatible with CMT2C.

      “Motor and sensory nerve conduction velocities of the lower limbs were normal, but reduced motor amplitudes in the right peroneal (1.8 mV) and right tibial (5 mV) and absent F-waves were observed.”2.1.2. Patient 2

    • Patient 2's new D62N variant was not found in her mother, was found in her brother who was born with bilateral talipes (clubfoot in both feet), and could not be tested in her father. Computer predictions and its rarity (seen once in 119,558 alleles) were used to support it being disease-causing.

      “This novel variant was absent in the healthy mother and was present in her brother, the father was deceased and DNA was not available for testing.”3.2. Genetic analysis

    • Patient 2's muscle biopsy showed nerve-related (neurogenic) changes plus unusual clumps of basophilic material in occasional fibers, which led the authors to speculate about a protein-clumping mechanism like that in heat shock protein neuropathies.

      “Large, mainly central accumulation of basophilic material was observed in occasional fibres”3.1. Muscle histology

    • Neither patient had vocal cord paralysis or hearing loss, features often described in TRPV4 neuropathy.

      “Vocal cord paralysis and, to a lesser extent, neurosensory deafness are described as clinical clues to diagnose TRPV4 associated neuropathies; however none of these were present in our patients.”5. Conclusions

    What the authors concluded

    TRPV4 changes cause a wide range of conditions with very different severity. Skeletal signs such as short stature and short fingers in someone with an axonal neuropathy should raise the possibility of TRPV4 changes. Basophilic inclusions in the muscle biopsy of someone with an axonal neuropathy should raise the possibility of TRPV4 or heat shock protein gene changes. The authors suggest TRPV4 disease may share a protein-clumping mechanism with heat shock protein neuropathies, and that muscle MRI is of limited use for diagnosis.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • ModerateRelatives described as healthy or not examined

      Patient 2's mother is called 'healthy' and 'asymptomatic', but no examination or nerve tests are reported for her. The brother who carries the same variant was not examined at all, so his status is known only from being born with talipes. The father had died and was not tested. As a result, the family evidence that the variant tracks with the disease is weak, and it says nothing reliable about how variable the condition is within this family.

      “He was unable to attend our clinic for neurological and electrophysiological examinations.”2.1.2. Patient 2

    • ModerateNew variant called pathogenic mainly on predictions

      The D62N variant was not tested in the lab. The case for it causing disease rests on computer prediction tools, its rarity, and its presence in an unexamined brother. That is suggestive but not proof, yet the paper describes both patients as carrying 'pathogenic mutations'.

      “The in silico studies and mutation prediction programmes indicate that this variant is probably pathogenic by changing the conformation of the protein.”4. Discussion

    • ModerateSkeletal dysplasia type named without reported X-rays

      Patient 1's skeletal findings are described from physical examination and a photograph. No skeletal X-rays are reported, yet the discussion names a specific dysplasia (metatropic dysplasia). Readers cannot check how firm this label is.

      “Patient 1 has a phenotype combining scapuloperoneal spinal muscular atrophy (SPSMA) and metatropic dysplasia.”4. Discussion

    • ModerateMechanism speculation from one biopsy

      The proposed link to heat shock protein neuropathies rests on inclusions seen in occasional fibers of a single biopsy, whose make-up was not identified. Patient 1's biopsy did not show them. The conclusion that such inclusions should point doctors toward TRPV4 goes further than one case can support.

      “In support of this we emphasize the fact that the muscle biopsy from patient 2 showed intracellular aggregates, but the exact protein composition of these aggregates has not been elucidated yet.”4. Discussion

    • MinorErrors and inconsistencies in the text

      The highlights and abstract describe two 'novel' mutations, but the results say R269C 'has been repeatedly reported'. The results also describe D62N as a change from aspartic acid to arginine, whereas its name (p.Asp62Asn) denotes a change to asparagine. These slips do not change the main message but show the paper needs careful reading.

      “The sequence change is predicted to cause the substitution of a moderately conserved aspartic acid for an arginine at codon 62”3.2. Genetic analysis

    • MinorGeneral statements drawn from very few patients

      The statement that muscle MRI is of limited diagnostic use is based on one patient's scan compared with earlier reports. This is an opinion, not a tested finding.

      “Therefore muscle MRI is of limited benefit as a diagnostic tool in TRPV4-related diseases.”4. Discussion

    How “unaffected” relatives were checked

    This is not a penetrance study, but relatives matter for judging the new variant. Patient 2's mother, who did not carry it, is described only as 'healthy' and 'asymptomatic', with no examination or nerve tests reported. Her brother, who does carry it and was born with bilateral talipes, was not examined: 'He was unable to attend our clinic for neurological and electrophysiological examinations.' Patient 1's mother is described as healthy and his father as having ankylosing spondylitis, again with no neurological examination reported.

    Three ways to explain it

    Scientific

    Two unrelated patients with heterozygous TRPV4 missense variants had divergent phenotypes: a de novo p.Arg269Cys variant with an SPSMA-like neurogenic phenotype plus clinical features suggestive of skeletal dysplasia, and a novel N-terminal cytoplasmic domain variant, p.Asp62Asn, with a late-onset axonal neuropathy compatible with CMT2C. Pathogenicity of p.Asp62Asn rests on in silico prediction, population rarity and incomplete segregation data. Basophilic inclusions in one muscle biopsy led the authors to propose a protein-aggregation mechanism shared with heat shock protein neuropathies, which remains untested.

    Easier

    The authors describe two people with changes in the TRPV4 gene. A boy had muscle weakness from babyhood plus short stature and short fingers, while a woman developed foot and leg weakness at about 40, like CMT2C. The woman's gene change had not been described before, and the evidence that it causes her illness comes mainly from computer predictions and how rare it is. Clumps seen in her muscle sample led the authors to suggest how the damage might happen, but this is an idea, not a proven finding.

    Simple

    The TRPV4 gene is like the instructions for a door that lets calcium into cells. This paper describes two people with mistakes in those instructions who got quite different illnesses, one as a small child and one as a grown-up. The doctors have ideas about why, but they have not proven them yet.

    Why these ratings

    Relevance 4/5: Describes two people with TRPV4-related nerve disease, one with an adult-onset picture compatible with CMT2C, and so speaks directly to how varied TRPV4 disease can be.

    Evidence 2/5: Two case descriptions, and the new variant is supported mainly by computer predictions, rarity and limited family testing.

    Funding and conflicts of interest

    Funding listed: Medical Research Council (UK) (G1000848), European Research Council (309548), Medical Research Council UK grant G1002274, European Union Seventh Framework Programme grants 305444 (RD-Connect) and 305121 (Neuromics), and Wellcome Trust, NIHR and MRC support for one author. No competing interests statement appears in this text.

    PubMed 25900305 · DOI

44. #44Full paperNews and views commentary (no new data)

    Channelopathies converge on TRPV4

    Nilius B, Owsianik G · 2010 · Nature genetics

    Relevance 4/5Evidence 2/53 moderate · 3 minor problems

    This piece marks the moment in 2010 when TRPV4 was linked to CMT2C. Its account of the gene discovery is more reliable than its symptom figures, some of which come from very few families or have no source.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    News and views commentary (no new data)

    Who or what

    No new patients or experiments. A short commentary on three 2010 genetic studies: Deng et al. (two unrelated families), Auer-Grumbach et al. (five unrelated families with SPSMA or CMT2C) and Landouré et al. (two families with CMT2C). Between them they found the TRPV4 changes R269C, R269H, R315W and R316C.

    What they did

    The authors explain what the TRPV4 channel is, how it is built and where in the body it is found. They summarize the three studies that linked TRPV4 to scapuloperoneal spinal muscular atrophy (SPSMA) and CMT2C. They then discuss how the studies disagreed about what the mutations do to the channel, and list other open puzzles.

    What they found

    • Three separate studies showed that SPSMA and CMT2C are allelic disorders, meaning different conditions caused by changes in the same gene, TRPV4.

      “Three new studies demonstrate that they are allelic disorders caused by mutations in the vanilloid transient receptor potential cation-channel gene TRPV4.”Page 98, standfirst

    • All the mutations found affect residues in the ankyrin (ANK) repeat region at the start of the protein, a region thought to interact with regulatory proteins.

      “All the mutations identified affect residues located in the ANK region of TRPV4.”Page 99, 'TRPV4-related channelopathies'

    • The studies disagreed on what the mutations do. One found that mutant channels in HeLa cells did not respond to any stimulus and stayed inside the cell, while the other two found that the mutant channels were overactive (gain of function).

      “Surprisingly, these data do not match the findings of the two other groups, who convincingly showed gain-of-function activity of the TRPV4 mutants in either HEK293 cells or Xenopus laevis oocytes”Pages 99 to 100, 'Functional dilemmas'

    • Citing the Landouré study, the commentary reports bladder overactivity, hearing loss and worse hand weakness in the cold as common in CMT2C.

      “Given the expression of TRPV4 in the inner ear, it is also not surprising that 59% of individuals with CMT2C have bilateral sensorineural hearing loss”Page 100, 'Functional dilemmas'

    • The authors say the links between where TRPV4 mutations sit and which organs are affected are unclear. For example, some skeletal dysplasia mutations lie close to R315W and R316C, yet people with that bone condition do not show obvious motor neuron defects.

      “In considering all known disease-related mutations in TRPV4 (Fig. 1), links between disease symptoms and affected organs are not obvious.”Page 100, 'Functional dilemmas'

    • The authors suggest TRPV4 could become a treatment target, while noting the functional questions were unresolved.

      “Although these functional dilemmas remain unresolved, the new studies suggest that TRPV4 could constitute a promising target for treatment of neurodegenerative diseases.”Page 100, final paragraph

    What the authors concluded

    SPSMA and CMT2C are caused by mutations in the same gene, TRPV4, all in the ankyrin repeat region. How these mutations damage motor and sensory nerves is unclear, because the studies disagreed on whether the channel is overactive or not working. TRPV4 might become a treatment target, and future research should explain its role in these diseases.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • ModerateCommentary, not original research

      This piece reports and interprets other papers. Its statements are only as reliable as the three studies it summarizes, and the methods of those studies cannot be judged from this text.

      “Three new studies demonstrate that they are allelic disorders”Page 98, standfirst

    • ModerateUnsourced claim that CMT2C is the most common inherited neurological disease

      No source is given for this statement and nothing else in the piece supports it. The commentary itself describes the CMT2C mutations being found in only a handful of families. The sentence may have been meant to refer to CMT as a whole, but it does not say so. It should not be relied on.

      “CMT2C, the most common inherited neurological disease, leads to progressive weakness of distal limbs, vocal cords, diaphragm, and intercostal and laryngeal muscles.”Page 98, opening paragraph

    • ModeratePercentages from a few families presented as true for everyone with CMT2C

      The bladder and hearing figures are cited to the Landouré study, which the commentary describes as finding mutations in two families with CMT2C. Figures from so few families may not apply to people with CMT2C in general, but the wording presents them as if they do.

      “about 90% of individuals with CMT2C suffer from overactive bladders”Page 100, 'Functional dilemmas'

    • MinorInconsistent mutation labels

      The same DNA position, 946, is given for two different protein changes at neighboring residues: R316C in one sentence and R315W in the next. One DNA position cannot sit in two different codons, so one label is probably a typing error. It is unclear which.

      “as well as a third heterozygous mutation (C946T) causing a R315W substitution”Page 99, 'TRPV4-related channelopathies'

    • MinorSome clinical statements made without evidence

      The piece states that altered pain perception is 'clearly not the case' in CMT2C, and earlier that sensory deficits often impair vision, without citing any data for either. These read as the authors' impressions rather than findings.

      “one would also expect to find hyperalgesia or altered nociception in individuals with SMD or CMT2C, which is clearly not the case.”Page 100, 'Functional dilemmas'

    • MinorEarly snapshot

      This piece was written in February 2010, right after the gene discovery, when the functional results conflicted. It reflects what was known then and cannot be used to describe the current state of the science.

      “Although these functional dilemmas remain unresolved”Page 100, final paragraph

    Three ways to explain it

    Scientific

    This News and Views piece summarizes three 2010 linkage and sequencing studies showing that SPSMA and CMT2C are allelic disorders caused by heterozygous TRPV4 missense mutations (R269C, R269H, R315W, R316C), all in the ankyrin repeat region. It highlights conflicting functional data: no response and cytoplasmic retention in HeLa cells in one study, versus gain of function in HEK293 cells and Xenopus oocytes in the other two. The authors note that genotype-phenotype links are unclear and suggest TRPV4 as a possible therapeutic target, without presenting new data.

    Easier

    In 2010 three research teams found that changes in the TRPV4 gene cause CMT2C and a related condition called scapuloperoneal spinal muscular atrophy (SPSMA). This short commentary explains those findings. All the changes were in one part of the TRPV4 protein, the ankyrin repeat region, which is thought to help it connect with other proteins. The teams disagreed on whether the changes made the channel work too much or not at all, and the authors list this and other puzzles as open questions.

    Simple

    Three teams of scientists found that CMT2C is caused by small changes in a gene called TRPV4, which makes a door that lets calcium into cells. They did not yet agree on whether the changes leave the door stuck too far open or stuck shut.

    Why these ratings

    Relevance 4/5: A commentary on the discovery that TRPV4 mutations cause CMT2C and SPSMA, covering TRPV4 neuropathy generally and its possible mechanism.

    Evidence 2/5: A short commentary with no new data; its main point rests on three independent genetic studies, but several side statements are unsourced or generalized from a few families.

    Funding and conflicts of interest

    Not stated.

    PubMed 20104247 · DOI

45. #45Full paperFamily study (case report of one kindred)

    Essential Tremor in a Charcot-Marie-Tooth Type 2C Kindred Does Not Segregate with the TRPV4 R269H Mutation

    Louis ED, Hernandez N, Ottman R et al. · 2014 · Case reports in neurology

    Relevance 4/5Evidence 2/52 moderate · 3 minor problems

    In this one CMT2C family, tremor also appeared in relatives without the TRPV4 mutation, so the authors think it was a separate, common condition (essential tremor) rather than part of CMT2C. One family cannot show how often this applies to other people with CMT2C and tremor.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    Family study (case report of one kindred)

    Who or what

    One family with CMT2C caused by the TRPV4 mutation R269H and self-reported tremor, enrolled through a US family study of essential tremor. Seven relatives were enrolled and genotyped; four with tremor (aged 61, 72, 81 and 87) are described in detail. Members of this family had been reported before by other researchers.

    What they did

    Relatives filled in questionnaires and had a videotaped neurological examination, and one author rated tremor severity on a standard 0 to 36 score. Essential tremor was diagnosed using published research criteria. Seven relatives were tested for R269H to see whether tremor and the mutation are passed on together.

    What they found

    • All four relatives with tremor met research criteria for essential tremor, a common inherited shaking condition.

      “The clinical diagnosis of ET was confirmed in all 4 tremor cases based on stringent published research criteria.”Abstract, Results

    • Two relatives with essential tremor but no CMT did not carry R269H, and neither did two relatives described as normal.

      “Neither family members II.5 and III.4 (both with a diagnosis of ET) nor III.8 and III.11 (normal) carried the TRPV4 R269H mutation.”Results, Identification of TRPV4 mutations

    • Relatives with both CMT2C and tremor carried R269H, including a third person (III.10) who is shown in the pedigree but not described clinically.

      “Subject III.10 with CMT type 2C and ET carried the TRPV4 R269H mutation”Results, Identification of TRPV4 mutations

    • The proband's CMT2C began with leg weakness and voice changes, and nerve tests showed a mainly axonal neuropathy in the arms and legs.

      “The subject had been diagnosed with CMT at age 37 but had been symptomatic for 1-2 years with leg weakness and changes in the quality of her voice”Results, Phenotypes

    • The authors interpret the tremor in this family as a separate condition rather than part of CMT2C.

      “This suggests that tremor in this family is unlikely to be an expression of CMT and unlikely to represent incomplete penetrance or variable expressivity of the TRPV4 R269H mutation.”Discussion

    What the authors concluded

    In this family the tremor appears to be a separate condition, essential tremor, rather than part of CMT2C, because relatives without the TRPV4 mutation and without CMT had the same kind of tremor. The authors say this shows that in some CMT families tremor is a second disorder.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • ModerateOne family; conclusion worded too strongly

      The findings rest on seven genotyped relatives in one family. Saying this 'establishes' that tremor is a second disorder in some CMT families is stronger than a single family can support, although the finding in this family is clear.

      “This establishes that in some CMT families the tremor diathesis likely represents a second disorder, namely ET.”Abstract, Conclusions

    • ModerateCannot tell what causes the tremor in people who have both conditions

      The diagnostic criteria require tremor with no 'other known cause', yet two of the people diagnosed with essential tremor also had CMT, and the introduction notes tremor is reported to be common in CMT. Finding tremor in non-carriers shows essential tremor runs in the family. It does not show whether the tremor in the mutation carriers comes from essential tremor, the neuropathy, or both.

      “moderate or greater amplitude kinetic tremor during ≥3 activities or a head tremor in the absence of Parkinson's disease or another known cause”Methods, Subjects/Evaluation

    • MinorSummary does not match the results

      The abstract and discussion say the mutation was found in '2 family members with ET and CMT', but the results report a third carrier, III.10, with CMT2C and essential tremor, who is not described clinically.

      “We confirmed the presence of the TRPV4 R269H mutation in the 2 family members with ET and CMT”Abstract, Results; compare Results, Identification of TRPV4 mutations

    • MinorHow 'no CMT' and 'normal' were decided is not fully described

      Two relatives are said not to have CMT and two are labeled 'normal', but no nerve conduction studies are reported for them. Their status seems to rest on history and a videotaped examination. Because none of them carried the mutation, this matters less here than in a penetrance study.

      “She did not have CMT.”Results, Phenotypes (subject III.4)

    • MinorRecruited through a tremor study, with a single rater

      The family came to the researchers through a genetic study of essential tremor, and tremor ratings were made by one author from videotapes. The paper does not say whether the rater knew who carried the mutation.

      “E.D.L. reviewed these examinations and rated the severity of postural and kinetic arm tremors”Methods, Subjects/Evaluation

    How “unaffected” relatives were checked

    Enrolled relatives had an in-person evaluation with questionnaires and a videotaped neurological examination, and Table 1 records limb weakness and areflexia (both absent in II.5 and III.4). Their 'no CMT' status appears to rest on this clinical examination and history; no nerve conduction studies are reported for them. Two relatives are labeled 'normal' without details of how this was decided: “Neither family members II.5 and III.4 (both with a diagnosis of ET) nor III.8 and III.11 (normal) carried the TRPV4 R269H mutation.” Only the proband's nerve conduction study is reported.

    Three ways to explain it

    Scientific

    In a CMT2C kindred carrying TRPV4 R269H, four relatives met research criteria for essential tremor, but R269H was found only in relatives with both CMT2C and ET and was absent from two relatives with ET alone. The authors conclude that tremor in this family is unlikely to reflect incomplete penetrance or variable expressivity of R269H and more likely represents co-occurring ET. The evidence comes from a single kindred with seven genotyped members, and the tremor in mutation carriers cannot be firmly attributed to one cause.

    Easier

    This family has CMT2C caused by a TRPV4 change called R269H, and several relatives also have a shaking of the hands called essential tremor. Two relatives with the tremor had neither the TRPV4 change nor CMT, so the tremor seems to run in the family separately from CMT2C. This study covers only one family, so it cannot say how often tremor in CMT2C has the same explanation.

    Simple

    In one family, some people had CMT2C and some had shaky hands. A few people had shaky hands without the CMT2C gene change, so the shaking seems to be a separate family trait. It is a bit like freckles and curly hair: they are passed down separately, even though some people get both.

    Why these ratings

    Relevance 4/5: It concerns a CMT2C family with a known TRPV4 mutation, but its question is narrow: whether the family's tremor is part of CMT2C.

    Evidence 2/5: A single family with seven people genotyped; the tremor examination was careful, but one family cannot show how tremor and CMT2C relate in general.

    Funding and conflicts of interest

    Supported by National Institutes of Health grant R01 NS073872. The authors declare no conflicts of interest and no competing financial interests.

    PubMed 24575025 · DOI

46. #46Full paperCase report with literature summary

    Combined Phenotypes of Spondylometaphyseal Dysplasia-Kozlowski Type and Charcot-Marie-Tooth Disease Type 2C Secondary to a TRPV4 Pathogenic Variant

    Faye E, Modaff P, Pauli R et al. · 2019 · Molecular syndromology

    Relevance 4/5Evidence 2/51 moderate · 4 minor problems

    This case shows that one person can have both CMT2C and a TRPV4 skeletal dysplasia, and that for her the nerve problems affected daily life more than the bone problems. It is one person's story, so it cannot say how common this overlap is.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    Case report with literature summary

    Who or what

    One woman, whose history is described from 18 months to 27 years of age, cared for from age 5 by the Midwest Regional Bone Dysplasia Clinic and pediatric orthopedics at the University of Wisconsin (USA). She has a de novo TRPV4 c.943C>T (R315W) variant and a second TRPV4 variant of unknown significance (c.904A>T, N302Y) inherited from her father. The paper also tabulates earlier published patients and families with both skeletal and nerve features.

    What they did

    The authors describe one patient's history, X-rays, nerve tests, operations and genetic results over many years. They then compare her with earlier published patients who had both a TRPV4 skeletal dysplasia and a TRPV4 neuropathy.

    What they found

    • From early childhood she had a skeletal dysplasia, defined on X-rays as spondylometaphyseal dysplasia Kozlowski type (SMD-K), with flattened vertebrae, scoliosis and abnormal hips. She needed spinal fusion at 10 years of age.

      “Her diagnosis was further defined as SMD-K based on significant platyspondyly, anterior vertebral body abnormalities, abnormal odontoid development, and metaphyseal changes of the long bones”Clinical Report

    • From age 5 she had leg weakness, absent knee and ankle reflexes and high-arched feet, with normal sensation at first. Sensory loss appeared in adolescence, and later nerve tests showed a motor-more-than-sensory axonal neuropathy, diagnosed as CMT2C.

      “Repeat electromyography/nerve conduction studies showed evidence of a generalized, chronic, motor greater than sensory, axonal polyneuropathy.”Clinical Report

    • The nerve condition affected her daily function more than the skeletal condition. At 27 she walked without aids, had weak hands and feet, and had completed a Master's degree.

      “CMT2C findings affected her function more than her skeletal findings.”Clinical Report

    • Lung function was checked regularly because of her scoliosis and early spinal fusion and did not get worse. She reported no hearing problems.

      “Regular pulmonary function monitoring was performed due to her scoliosis and early fusion surgery. These have shown no deterioration.”Clinical Report

    • The R315W change arose for the first time in her (her mother had no TRPV4 variants). Her father carries the second variant, N302Y, which is of unknown significance.

      “Parental testing was performed and showed the patient's father has the same TRPV4 c.904A>T, N302Y variant of unknown significance.”Results, Molecular Findings

    • Combining this case with earlier reports, the authors argue that where a variant sits in the TRPV4 protein does not reliably predict whether it causes skeletal disease, nerve disease or both.

      “Based on this report in addition to previous reports, the domain location of TRPV4 pathogenic variants does not appear to exclusively predict the phenotype.”Discussion

    What the authors concluded

    TRPV4 skeletal dysplasias and TRPV4 neuropathies are not separate diseases caused by separate sets of variants, but two phenotypes on the same spectrum that may or may not overlap. The authors suggest that people with any TRPV4-related condition be assessed for both skeletal and neurological findings.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • ModerateFather's second variant and family status not examined

      The father carries the N302Y variant of unknown significance, but he and other relatives are described only by family history and reported height. No examination or nerve tests are reported for them, and the paper does not discuss whether N302Y might add to her features. The authors call the overlap 'presumably' due to R315W, which is reasonable but not proven.

      “Family history is negative for documented neuromuscular disease, peripheral neuropathy, or skeletal dysplasia.”Clinical Report

    • MinorLimited testing for other genetic causes

      Before TRPV4 testing she had tests for PMP22, MPZ and SMN only, and TRPV4 was then sequenced on its own. No wider gene panel or exome is reported, so other genetic contributors were not ruled out.

      “Prior molecular genetic evaluation of the patient's peripheral neuropathy included normal CMT1A testing of PMP22 , CMT1B testing of MPZ (CMT2 testing was not available at that time)”Results, Molecular Findings

    • MinorErrors in the text

      The discussion reverses the amino acid change (R315W replaces arginine with tryptophan, as the results correctly say), and 'scapuloperoneal' is spelled 'scapuloperitoneal'. The paper calls her the '11th' published individual, but its own table lists some whole families and several fetuses as single entries, so this count is unclear.

      “This patient's R315W pathogenic variant causes an arginine to be substituted for a tryptophan in the Ankyrin repeats of TRPV4 .”Discussion

    • MinorNerve findings reported without detail

      No nerve conduction numbers are given, and early on doctors considered distal spinal muscular atrophy as well as a motor neuropathy. Voice and diaphragm function, features often linked to CMT2C, are not mentioned. This makes it harder to compare her nerve disease with other reports.

      “Based on these findings and normal spinal cord anatomy on MRI, her clinical picture was thought suggestive of either distal spinal muscular atrophy or a predominately motor neuropathy.”Clinical Report

    • MinorWide conclusion from one case

      The idea that skeletal and nerve forms of TRPV4 disease overlap is supported by earlier reports as well as this case, so it is reasonable. But a single case cannot show how often the overlap happens or who is at risk of it.

      “This and prior reports illustrate that TRPV4 -related skeletal dysplasias and TRPV4 -related neuropathies are not fully distinct disorders secondary to unique sets of pathogenic variants as originally postulated”Abstract

    How “unaffected” relatives were checked

    Relatives were not examined in the report. The family history was 'negative for documented neuromuscular disease, peripheral neuropathy, or skeletal dysplasia', and the parents and sister are described only by reported height. The father carries the N302Y variant of unknown significance, but no neurological examination or nerve tests are described for him.

    Three ways to explain it

    Scientific

    A woman with a de novo heterozygous TRPV4 c.943C>T (p.R315W) variant had radiographically defined spondylometaphyseal dysplasia Kozlowski type from early childhood and a progressive motor-greater-than-sensory axonal polyneuropathy diagnosed as CMT2C. She also carried a paternally inherited N302Y variant of uncertain significance whose contribution was not assessed. Drawing on prior reports, the authors argue that TRPV4 skeletal and neuromuscular phenotypes form an overlapping spectrum that is not predicted by variant domain alone.

    Easier

    This report describes one woman whose TRPV4 gene change caused both a bone growth condition affecting her spine and hips (a skeletal dysplasia) and CMT2C nerve disease. Her gene change was new in her, not passed on from her parents. The authors say TRPV4 bone and nerve conditions are not completely separate and can happen together, and they suggest checking for both.

    Simple

    TRPV4 is like a door that lets calcium into cells in both bones and nerves. In this one woman, a mistake in the door affected both her bones and her nerves. Doctors used to think these were separate illnesses, and this case adds to the idea that they can happen together.

    Why these ratings

    Relevance 4/5: Describes a person with CMT2C caused by a known TRPV4 variant who also has a skeletal dysplasia, which bears directly on the range of features in TRPV4 disease.

    Evidence 2/5: A single, well-documented case followed over many years, with a literature summary.

    Funding and conflicts of interest

    No funding source is stated. The authors state they have no conflicts of interest to disclose.

    PubMed 31191204 · DOI

47. #47Detailed summary onlyShort report of two families, with a table of all published cases of the same variant

    The remarkable phenotypic variability of the p.Arg269HiS variant in the TRPV4 gene

    Jędrzejowska M, Dębek E, Kowalczyk B et al. · 2019 · Muscle & nerve

    Relevance 4/5Evidence 2/53 moderate · 3 minor problems

    In these Polish families the same TRPV4 change, R269H, caused disease of very different severity, even between a mother and her daughters, and one adult needed breathing support at night. The paper shows that the same change can be called CMT2C or a form of spinal muscular atrophy.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    Short report of two families, with a table of all published cases of the same variant

    Who or what

    Two Polish families with the TRPV4 change c.806G>A (R269H). Family 1 spans four generations: four affected relatives tested and found to carry it (a mother of 56, an uncle, a woman of 34 and her sister of 30) and three deceased affected relatives who were not tested. In family 2, the patient is a girl of 5 in whom the change was new. The paper also tabulates every published R269H family.

    What they did

    Family 1 was tested by Sanger sequencing of TRPV4, and the girl in family 2 by a broad gene panel confirmed by Sanger sequencing, with both her parents tested. The authors describe the clinical features, nerve tests, EMG and X-rays, and compare them with published R269H cases. This analysis is based on a detailed secondary summary of the paper written by another reader, not on the paper itself.

    What they found

    • Severity varied widely with the same R269H change, from an adult with mild shoulder blade winging to a child who walks only with braces and crutches.

      “Two Polish families with the same R269H mutation show disease ranging from mild shoulder blade winging to a child who cannot walk without braces”Secondary summary, opening summary

    • Within family 1, the 56-year-old mother had only mild, non-progressive shoulder blade winging, her daughters had intermediate and severe disease, and an uncle of intermediate severity uses non-invasive ventilation at night.

      “Waddling gait, winged shoulder blades, and non-invasive ventilation at night.”Secondary summary, table 'Family 1, across four generations', row 'Uncle'

    • The family 1 proband was born with clubfeet and hip dysplasia and was stable for many years. Her nerve conduction speeds were normal but leg motor responses were very small (0.2 mV in both peroneal nerves), and EMG showed long-standing nerve damage.

      “Course: stable for many years. Since 30 she has noticed more fatigue when walking.”Secondary summary, 'The family 1 proband in detail'

    • The two most severe patients had arm contractures from birth and cranial nerve signs, neither of which had been reported with R269H before. Skeletal changes (hip dysplasia, short stature, scoliosis) were also present.

      “The two most severe patients had arm contractures from birth and cranial nerve signs (facial asymmetry, drooping eyelid, squint).”Secondary summary, 'Authors' conclusions'

    • Across published families, R269H has been labeled CMT2C, scapuloperoneal spinal muscular atrophy and congenital distal spinal muscular atrophy.

      “The same mutation has therefore been given all three labels.”Secondary summary, table 'Every published R269H family'

    • The authors found no pattern of the disease starting earlier in each generation, and conclude that something beyond the mutation itself shapes severity.

      “Across the published families there is no pattern of the disease starting earlier in each generation.”Secondary summary, 'Authors' conclusions'

    What the authors concluded

    The same R269H change can cause anything from mild shoulder blade weakness to severe congenital disease with contractures, skeletal changes and, in an adult, night-time breathing support. The authors add arm contractures and cranial nerve signs to the features seen with R269H, find no sign of earlier onset in later generations, and conclude that a modifier or more complex mechanism shapes severity.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • ModerateOlder relatives described from family history

      Three older affected relatives, including two described as mild, had died, were not genetically tested, and are described from family history. Their status and severity therefore rest on family accounts, which weakens comparisons of severity across generations.

      “Three older affected relatives were not genetically tested and are described from family history.”Secondary summary, 'Limitations'

    • ModerateWeak test of anticipation

      The conclusion that the disease does not start earlier in later generations comes from reading published case tables, not from a formal analysis.

      ““No anticipation” comes from reading published case tables, which is a weak test.”Secondary summary, 'Limitations'

    • ModerateHearing and vocal cords not assessed

      No hearing tests or vocal cord examinations are reported, so these features of TRPV4 disease may have been missed in these families.

      “No hearing tests or vocal cord examinations are reported for either family.”Secondary summary, 'Limitations'

    • MinorShort report with details in a supplement

      Details for most relatives were in a supplement the summary writer did not see, so they could not be checked here.

      “A short report on two families. Details for most relatives sit in a supplement I did not see.”Secondary summary, 'Limitations'

    • MinorYoung girl's diagnosis rests on clinical picture and gene result

      The 5-year-old had normal nerve conduction and EMG at age 3, so nerve involvement in her was not confirmed by tests at that time.

      “The 5-year-old had normal nerve and muscle tests at age 3, so her diagnosis rests on the clinical picture and the gene result.”Secondary summary, 'Limitations'

    • MinorChannel effect taken from other studies

      The summary says the claim that R269H raises channel activity comes from cited work, with no new experiments in this paper.

      “The claim that R269H raises channel activity comes from cited studies, with no new experiments.”Secondary summary, 'Limitations'

    How “unaffected” relatives were checked

    No unaffected carriers are reported. The mildly affected 56-year-old mother was genetically tested and is described clinically; the summary does not say whether she had nerve conduction tests. Three deceased relatives in older generations, two of them described as mild, were not genetically tested and their features come from family history. In family 2, both parents are described as healthy and did not carry the change. Quote: “Three older affected relatives were not genetically tested and are described from family history.”

    Three ways to explain it

    Scientific

    In two Polish families, the TRPV4 R269H variant showed marked variability within and between families, from mild scapular winging to congenital contractures with skeletal involvement, and nocturnal non-invasive ventilation in one adult. Congenital arm contractures and cranial nerve signs were described with R269H for the first time. Across published R269H families, the same variant has been labeled CMT2C, scapuloperoneal SMA and congenital distal SMA. The authors find no anticipation, suggesting that modifiers influence severity, although the data are descriptive and partly based on family history.

    Easier

    Doctors in Poland describe two families with the same TRPV4 change, R269H. In one family a mother had only mild shoulder blade weakness into her fifties, while one daughter was born with stiff joints and has never walked without help, and an uncle uses a breathing machine at night. In earlier reports, the same change has been given three different diagnosis names, and the illness did not seem to start earlier in each new generation. The authors think other factors besides the gene change affect how severe it is.

    Simple

    In one Polish family, the same TRPV4 gene change affected each person differently: a mom was only a little affected, while her daughters were more affected. It is like the same recipe turning out differently each time it is baked. Doctors think something else, not just the gene change, also affects how strong the illness is.

    Why these ratings

    Relevance 4/5: It is about TRPV4 neuromuscular disease caused by R269H, under labels that include CMT2C, and the summary notes that it describes Polish families.

    Evidence 2/5: A short descriptive report of two families, partly relying on family history and a supplement, read here only through a secondary summary.

    Funding and conflicts of interest

    The summary says the work was funded by the Polish National Science Centre. Competing interests are not mentioned in the summary.

    PubMed 30230566 · DOI

48. #48Detailed summary onlyFamily study of mosaic mutations (two families), with exome sequencing and clinical, nerve and MRI assessment

    De novo somatic mosaicisms of INF2 and TRPV4 in patients with Charcot-Marie-Tooth disease

    Lee AJ, Kanwal S, Awasthi M et al. · 2025 · Genes & genomics

    Relevance 4/5Evidence 2/54 moderate · 2 minor problems

    A TRPV4 change can be present in only some of a person's cells (mosaicism) and still cause severe CMT2C, as in this one girl. The summary notes this matters for families because a mildly affected or apparently unaffected parent could be mosaic.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    Family study of mosaic mutations (two families), with exome sequencing and clinical, nerve and MRI assessment

    Who or what

    Two Korean families, 3 affected and 8 unaffected people in all. The TRPV4 case is one girl, aged 11 at examination with onset at age 1, who carries a new TRPV4 variant, c.2187C>A (S729R), in about 30% of her blood DNA. For comparison, the paper describes a father who carries an INF2 variant (C104Y) in 24% of his blood DNA, and his daughter who carries it in full.

    What they did

    A Korean CMT research group used whole exome sequencing filtered for rare variants in CMT genes, Sanger sequencing in available relatives, and DNA markers to confirm parentage. They measured the mosaic level by sequencing about 100 bacterial colonies per mutation, and assessed patients with nerve conduction studies, strength scoring, two disability scales and 3 tesla MRI of the hips, thighs and calves. This analysis is based on a detailed secondary summary of the paper written by another reader, not on the paper itself.

    What they found

    • The girl's TRPV4 variant was not found in either healthy parent, and about 30% of her blood DNA carried it. The summary explains that this means it arose after fertilization and is present in only some of her cells (mosaicism).

      “Neither healthy parent carries it. About 30% of the girl's blood DNA does.”Secondary summary, 'The TRPV4 mutation'

    • Despite being mosaic, the girl was severely affected: CMT neuropathy score 26, functional disability score 7 with wheelchair use, absent knee and ankle reflexes, vocal cord paresis, hearing loss, scoliosis and developmental delay.

      “A Korean girl with severe CMT2C carries a new TRPV4 mutation in only about 30% of her blood DNA”Secondary summary, opening summary and table 'The three people studied'

    • The variant, S729R, sits next to the TRP box, outside the ankyrin repeats where most CMT2C mutations lie. It is absent from four population databases, conserved from fish to humans, rated damaging by prediction programs and classed by the authors as likely pathogenic.

      “This is far from the ankyrin repeats where most CMT2C mutations lie.”Secondary summary, 'The TRPV4 mutation'

    • Her leg MRI showed severe fat replacement of muscle, worst in the calves with the thighs relatively spared, a pattern the summary says fits nerve damage that depends on nerve length. Sural sensory responses could not be recorded.

      “MRI showed severe fat replacement in the hips, thighs and calves, with the thighs relatively spared and the calves worst.”Secondary summary, 'More on the girl with the TRPV4 mutation'

    • In the comparison INF2 family, the mosaic father had such mild symptoms that he did not think he was affected, and was only tested after his daughter's diagnosis.

      “The father's symptoms were so mild that he did not think he had his daughter's condition.”Secondary summary, 'The INF2 family, for comparison'

    • The authors conclude that severity depends on which mutation is present and how much of it is in the relevant tissues, and that mosaicism may explain some differences in severity within and between CMT families.

      “The authors conclude that severity depends on which mutation it is and on how much of it is present in the tissues that matter.”Secondary summary, 'What the authors say about TRPV4'

    What the authors concluded

    A TRPV4 mutation present in only part of a person's cells can still cause severe CMT2C. The authors conclude that severity depends on the specific mutation and on how much of it is present in the tissues that matter, and that mosaicism may explain some of the variation in severity seen in CMT families.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • ModerateSingle patient with this variant

      Nobody else is known to carry S729R, so there is no one to compare her severity with and the variant's effect rests on one person.

      “One patient with this mutation. No other family carries it, so there is nothing to compare her severity with.”Secondary summary, 'Limitations'

    • ModerateNo laboratory test of the variant

      The case that S729R is harmful rests on it being rare, at a position unchanged across species and predicted damaging by computer programs, not on any test of how the channel behaves.

      “No laboratory test of channel function. The case for harm rests on rarity, conservation and computer prediction.”Secondary summary, 'Limitations'

    • ModerateMosaic level measured in blood only

      The proportion of nerve cells carrying the variant is unknown, so the link between mosaic level and severity is uncertain.

      “Mosaic level was measured in blood only. The level in nerve, the tissue that matters, is unknown and could be higher or lower.”Secondary summary, 'Limitations'

    • ModerateFeatures not typical of CMT2C, second cause not fully excluded

      The summary notes that developmental delay is not typical of CMT2C and that another genetic cause was not specifically ruled out beyond the exome filter, so not every feature can be safely put down to TRPV4.

      “A second genetic cause was not specifically excluded beyond the exome filter.”Secondary summary, 'Limitations'

    • MinorInconsistencies within the paper

      The summary writer found the girl described as a woman in the text but aged 11 in the table, and sex labels swapped between a table and a figure. This raises some doubt about other details.

      “The text calls her a woman while the table gives her age as 11, and the table's sex labels for the other family are swapped against the MRI figure.”Secondary summary, 'Limitations'

    • MinorSeverity comparison is an impression

      The statement that she is as severe as typical CMT2C patients was not backed by a formal comparison.

      “The claim that her severity resembles other CMT2C patients is an impression, with no formal comparison.”Secondary summary, 'Limitations'

    How “unaffected” relatives were checked

    For the TRPV4 girl, the summary calls her parents healthy and reports that neither carries the variant in blood, but does not say whether they were examined or had nerve tests. The 8 unaffected people across both families are not described in detail. In the comparison INF2 family, the father, who did not think he was affected, was examined and had nerve conduction tests only after his daughter's diagnosis, with a median nerve conduction speed of 51.8 m/s. Quote: “He was only tested after her diagnosis.”

    Three ways to explain it

    Scientific

    A girl with severe early-onset TRPV4 axonal neuropathy carried a de novo S729R variant, located near the TRP box outside the ankyrin repeat domain, at an estimated 30% somatic mosaic level in blood. Despite mosaicism, her phenotype was severe, including vocal cord paresis, hearing loss and a length-dependent pattern of fatty muscle replacement on MRI. The variant was classed as likely pathogenic on the basis of rarity, conservation and in silico prediction, without functional testing, and the mosaic level in nerve is unknown.

    Easier

    A Korean girl with severe CMT2C had a new change in the TRPV4 gene that was not inherited from either parent. Only about 30% of the DNA in her blood carried the change, which is called mosaicism, yet her disease was still severe. The researchers did not test the change in the lab and could only measure it in blood, not in nerves.

    Simple

    Our bodies are built from many tiny cells, like bricks in a wall. In this girl only some of the bricks had the TRPV4 change, but she was still very affected. She is only one person, so we cannot say this always happens.

    Why these ratings

    Relevance 4/5: One of the two cases is a girl with severe TRPV4 neuropathy, and the idea that a TRPV4 change can be mosaic is relevant to families, but half the paper concerns a different gene (INF2).

    Evidence 2/5: The TRPV4 part is a single patient with no laboratory test of channel function, read here only through a secondary summary.

    Funding and conflicts of interest

    Not stated in the summary.

    PubMed 40257654 · DOI

49. #49Abstract onlyGenetic linkage study in one family

    Genetic heterogeneity in Charcot-Marie-Tooth neuropathy type 2

    Yoshioka R, Dyck PJ, Chance PF · 1996 · Neurology

    Relevance 4/5Evidence 1/51 moderate problem

    This early study showed that CMT2C is not caused by the same gene region as CMT2A. It is part of the history of how CMT2C came to be recognized as its own condition.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    Genetic linkage study in one family

    Who or what

    One large family with CMT2C (diaphragm and vocal cord weakness). The abstract does not give the number of family members.

    What they did

    The authors tested DNA markers from the CMT2A gene region on chromosome 1 to see whether they traveled with the disease in a large CMT2C family. This analysis is based only on the abstract, so the methods cannot be judged.

    What they found

    • There was no evidence that the CMT2C gene lies in the CMT2A region on chromosome 1.

      “There was no evidence to support linkage of the CMT2C gene to the region of the CMT2A locus on chromosome 1.”Abstract

    • The authors conclude that CMT2C is not a variant form of CMT2A, adding evidence that axonal CMT has several genetic causes.

      “CMT2C is not an allelic variant of CMT2A.”Abstract

    What the authors concluded

    CMT2C is genetically separate from CMT2A, which supports the view that inherited axonal neuropathies have more than one genetic cause.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • ModerateMethods cannot be judged from the abstract

      Only the abstract was available. It does not give the family size, how relatives were classed as affected or unaffected, which markers were used or the linkage scores, so the strength of the exclusion cannot be checked.

    How “unaffected” relatives were checked

    Cannot be judged: the abstract does not say how family members were classed as affected or unaffected, or whether they were examined or had nerve tests.

    Three ways to explain it

    Scientific

    Linkage analysis in a large CMT2C pedigree using markers from the CMT2A locus on the short arm of chromosome 1 found no evidence of linkage, indicating that CMT2C is not allelic to CMT2A. The result adds to evidence of genetic heterogeneity among inherited axonal neuropathies. Pedigree size, phenotyping and LOD scores cannot be assessed from the abstract.

    Easier

    Researchers checked whether CMT2C in one large family came from the same area of chromosome 1 as another type, CMT2A. It did not. This showed CMT2C is a separate genetic condition.

    Simple

    Scientists checked whether CMT2C came from the same spot in our DNA as another kind of CMT, and found it did not. So CMT2C has its own cause.

    Why these ratings

    Relevance 4/5: It is directly about a CMT2C family, but its result is an early genetic exclusion that is mainly of historical interest.

    Evidence 1/5: Rated 1 because only the abstract was available, so the family data, markers and linkage scores could not be checked.

    Funding and conflicts of interest

    Not stated in the abstract.

    PubMed 8614538 · DOI

50. #50Abstract onlyFamily report with long-term follow-up

    Long-Term Observations in an Affected Family with Neurogenic Scapuloperoneal Syndrome Caused by Mutation R269C in the TRPV4 Gene

    Vill K, Kuhn M, Gläser D et al. · 2015 · Neuropediatrics

    Relevance 4/5Evidence 1/51 moderate problem

    In this one family with TRPV4 R269C, the father stayed stable over two decades and his son, who was very ill as a newborn, improved and walked with braces by age 5. One family cannot show how others will do.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    Family report with long-term follow-up

    Who or what

    One family with two affected people, a father and his son, carrying TRPV4 c.805C>T (p.R269C), followed for over two decades.

    What they did

    The authors used multi-gene panel sequencing to find the cause, then followed the father and son clinically, with muscle ultrasound and nerve conduction studies (neurography), over a long period. This analysis is based only on the abstract, so the methods cannot be judged.

    What they found

    • The father had a classical scapuloperoneal syndrome (weakness around the shoulder blades and lower legs), while the son had a severe start at birth with breathing difficulty, feeding problems and joint contractures (arthrogryposis multiplex).

      “The father clinically suffered from a classical scapuloperoneal syndrome, while the son presented with a severe neonatal onset with congenital respiratory distress, feeding problems and arthrogryposis multiplex.”Abstract

    • Over two decades the father showed no relevant progression, and the son improved markedly after a very difficult newborn period, walking with orthoses by age 5. Muscle ultrasound matched this course.

      “Long-term observation over two decades showed no relevant disease progression in the father and, after a dramatic neonatal period, a significant improvement in the son who became ambulant with orthoses at the age of 5 years”Abstract

    • Nerve conduction studies showed a late-onset sensory neuropathy in the father, which the authors say had not been described in TRPV4 neuropathies.

      “Neurography revealed a late-onset sensory neuropathy in the father, which was so far not described in TRPV4 neuropathies.”Abstract

    What the authors concluded

    TRPV4 R269C caused disease of very different severity in father and son, but the course was stable in the father and improved in the son, suggesting a reasonably good outlook even after severe onset at birth. The father developed a late-onset sensory neuropathy.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • ModerateMethods cannot be judged from the abstract

      Only the abstract was available, so the examination and test details cannot be checked, and it is not stated whether other causes of the father's sensory neuropathy were looked for. The suggestion of a reasonably good outlook after severe onset at birth rests on one child.

      “suggesting a reasonably good prognosis even in cases with severe neonatal onset.”Abstract

    How “unaffected” relatives were checked

    The abstract reports two affected people, father and son, but does not say whether other relatives were examined or tested.

    Three ways to explain it

    Scientific

    In a father and son heterozygous for TRPV4 R269C, phenotypes ranged from classical scapuloperoneal syndrome to severe neonatal onset with respiratory distress and arthrogryposis multiplex. Over two decades the father showed no relevant progression and the son improved to independent ambulation with orthoses by age 5, paralleled by stable or improved muscle ultrasound findings. Neurography showed a late-onset sensory neuropathy in the father. Prognostic conclusions rest on one family, and methods cannot be assessed from the abstract.

    Easier

    Doctors followed a father and son with the same TRPV4 change, R269C, for more than two decades. The father had weakness around the shoulder blades and lower legs that hardly changed, while the son was very ill at birth but improved a lot and was walking with leg braces by age 5. Later, nerve tests showed the father had also developed some damage to the nerves that carry feeling.

    Simple

    A dad and his son had the same TRPV4 change. The son was very sick as a baby but got much stronger and could walk with braces by age 5, and the dad stayed about the same for many years.

    Why these ratings

    Relevance 4/5: It gives rare long-term follow-up of TRPV4 neuromuscular disease with R269C, including late sensory nerve involvement, though the family's diagnosis is scapuloperoneal syndrome rather than CMT2C.

    Evidence 1/5: Rated 1 because only the abstract was available; it is also one family of two people.

    Funding and conflicts of interest

    Not stated in the abstract.

    PubMed 26110311 · DOI

51. #51Abstract onlyCase report

    A case of congenital spinal muscular atrophy with pain due to a mutation in TRPV4

    Fleming J, Quan D · 2016 · Neuromuscular disorders: NMD

    Relevance 4/5Evidence 1/51 moderate problem

    This single report describes a person with a TRPV4 change who had pain and a sense of reduced feeling as well as weakness. It also shows that a diagnosis in one family member does not always explain another relative's condition.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    Case report

    Who or what

    One patient with congenital spinal muscular atrophy, pain and subjective sensory loss, carrying TRPV4 c.806G>A (Arg269His). A sister and niece were reported to have CMT1A. The abstract does not give the patient's age or sex.

    What they did

    The authors describe the patient's symptoms, EMG findings and genetic result, and draw lessons for diagnosis. This analysis is based only on the abstract, so the methods cannot be judged.

    What they found

    • The patient, who carried the TRPV4 Arg269His change, had congenital spinal muscular atrophy with pain, a sense of reduced feeling, a clubfoot on the right, late walking and walking that worsened over time.

      “We present a patient with congenital spinal muscular atrophy associated with pain, subjective sensory loss, right talipes equinovarus, delayed walking, and progressive gait impairment.”Abstract

    • Relatives were reported to have CMT1A, but the patient's EMG showed an axonal motor neuropathy or neuronopathy, pointing to a different diagnosis.

      “A sister and niece reportedly had Charcot-Marie-Tooth 1A, but the patient's electromyogram showed an axonal motor neuropathy or neuronopathy.”Abstract

    • The authors suggest that skeletal X-rays can find bone causes of pain in people with TRPV4 mutations and help point to a TRPV4 diagnosis, and that nerve tests are worthwhile even when a family's genetic diagnosis is thought to be known.

      “Even when a patient's genetic diagnosis is presumed to be known, electrodiagnostic testing is warranted to verify the diagnosis.”Abstract

    What the authors concluded

    TRPV4 mutations cause variable conditions, and pain can be part of the picture. Skeletal X-rays may help explain pain and suggest a TRPV4 diagnosis, and nerve tests should be used to check a diagnosis even when the family's genetic cause seems known.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • ModerateMethods cannot be judged from the abstract

      Only the abstract was available, so the patient's age, examination, nerve test values and the cause of the pain cannot be checked. The relatives' CMT1A is described as reported, and the abstract does not say whether they were examined or tested for TRPV4. One case cannot show how often pain occurs with TRPV4 disease.

      “A sister and niece reportedly had Charcot-Marie-Tooth 1A”Abstract

    Three ways to explain it

    Scientific

    A patient with congenital spinal muscular atrophy, pain, subjective sensory loss, talipes equinovarus and progressive gait impairment carried TRPV4 c.806G>A (Arg269His), although relatives reportedly had CMT1A. EMG showed an axonal motor neuropathy or neuronopathy. The authors propose skeletal radiography to identify orthopedic causes of pain and support a TRPV4 diagnosis, and electrodiagnostic confirmation even when a familial genotype is presumed. Clinical detail cannot be assessed from the abstract.

    Easier

    Doctors describe a person born with weakness from damaged motor nerve cells who also had pain and felt that their sensation was reduced. Relatives were said to have a different type of CMT, CMT1A, but nerve tests pointed elsewhere, and genetic testing showed this person had a TRPV4 change, Arg269His. The authors say X-rays can help find bone causes of pain, and that nerve tests are useful even when a family's diagnosis seems known.

    Simple

    One person with a TRPV4 change had weak legs and pain. Their relatives were thought to have a different kind of CMT, so checking this person's own nerves helped find the right answer, like checking a map instead of guessing the way.

    Why these ratings

    Relevance 4/5: It describes pain and sensory symptoms in a person with the TRPV4 change Arg269His (c.806G>A), and makes points about diagnosis relevant to TRPV4 neuromuscular disease generally.

    Evidence 1/5: Rated 1 because only the abstract was available; it is also a single case.

    Funding and conflicts of interest

    Not stated in the abstract.

    PubMed 27751652 · DOI

52. #52Abstract onlyGenetic linkage (exclusion) study in two families

    Clinical and genetic heterogeneity in peroneal muscular atrophy associated with vocal cord weakness

    McEntagart M, Dunstan M, Bell C et al. · 2002 · Journal of Neurology, Neurosurgery, and Psychiatry

    Relevance 4/5Evidence 1/51 moderate problem

    This was one of the steps that narrowed down where the CMT2C gene could be, by showing it was not in one suspected region.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    Genetic linkage (exclusion) study in two families

    Who or what

    Two families: one with HMSN IIC (an older name for CMT2C), and one with vocal cord paralysis, hearing loss and distal muscle wasting.

    What they did

    The authors tested whether the condition in each family was linked to the chromosome 2q14 region, where the gene for a similar condition (distal hereditary motor neuropathy type VII) had been placed. This analysis is based only on the abstract, so the methods cannot be judged.

    What they found

    • Neither family showed linkage to the chromosome 2q14 region.

      “Two-point LOD scores at chromosome 2q14 markers encompassing the DHMNVP gene were negative in both families.”Abstract, Results

    • The authors concluded that HMSN IIC and distal hereditary motor neuropathy type VII are unlikely to share the same gene.

      “dHMN-VII and HMSN-IIC are unlikely to be allelic disorders”Abstract, Conclusions

    • The two conditions look alike, but HMSN IIC also involves the sensory nerves.

      “dHMN-VII and HMSN-IIC are clinically similar but are differentiated by sensory involvement in HMSN-IIC”Abstract, Background

    What the authors concluded

    At least one more gene causes distal weakness with vocal cord paralysis, and HMSN IIC is probably not caused by the dHMN-VII gene. More HMSN IIC families are needed to confirm this.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • ModerateMethods cannot be judged from the abstract

      Only the abstract was available, so family sizes, how affected status was decided and the LOD score values cannot be checked. The authors themselves say more families are needed.

      “Analyses of further HMSN-IIC families are required to confirm this.”Abstract, Conclusions

    How “unaffected” relatives were checked

    Cannot be judged from the abstract.

    Three ways to explain it

    Scientific

    Linkage analysis of chromosome 2q14 microsatellites spanning the DHMNVP locus gave negative two-point LOD scores in one HMSN IIC family and one family with distal weakness, vocal cord paralysis and sensorineural deafness. The authors conclude that dHMN-VII and HMSN IIC are unlikely to be allelic and that at least one further locus exists.

    Easier

    Before the CMT2C gene was found, researchers suspected it might be the same gene as a similar condition that also affects the vocal cords. They tested two families and found the condition did not travel with that part of chromosome 2, so the cause was probably a different gene.

    Simple

    Scientists were hunting for the CMT2C gene. They checked one hiding place on chromosome 2 and it was empty, which told them to look somewhere else.

    Why these ratings

    Relevance 4/5: It includes an HMSN IIC family and helped rule out one possible gene region before TRPV4 was found.

    Evidence 1/5: Rated 1 because only the abstract was available, and linkage exclusion in two families is a narrow finding.

    Funding and conflicts of interest

    Not stated in the abstract.

    PubMed 12438487 · DOI

53. #53Abstract onlyMulticenter genetic screening study with family follow-up

    Phenotypic spectrum and incidence of TRPV4 mutations in patients with inherited axonal neuropathy

    Echaniz-Laguna A, Dubourg O, Carlier P et al. · 2014 · Neurology

    Relevance 4/5Evidence 1/51 moderate · 1 minor problems

    In this French series, TRPV4 changes were found only in people whose neuropathy came with extra features such as vocal cord paralysis or skeletal problems, and in none of the 95 people with 'pure' CMT2. Many people with TRPV4 neuropathy had been given other labels, such as distal hereditary motor neuropathy.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    Multicenter genetic screening study with family follow-up

    Who or what

    169 unrelated French patients with inherited axonal neuropathy: 95 with dominant 'pure' CMT2, and 74 with extra features (vocal cord paralysis and/or skeletal dysplasia), including 39 with distal hereditary motor neuropathy, 14 with congenital SMA and arthrogryposis, 13 with CMT2 and 8 with scapuloperoneal SMA. 17 patients with TRPV4 mutations were studied in 12 families.

    What they did

    The authors tested all 169 patients for TRPV4 mutations, then studied affected relatives of those who had one, and described their features. This analysis is based only on the abstract, so the methods cannot be judged.

    What they found

    • No harmful TRPV4 mutation was found in any of the 95 patients with 'pure' CMT2.

      “No deleterious TRPV4 mutation was identified in the 95 patients with “pure” CMT2 (0/95).”Abstract, Results

    • 12 of the 74 patients with vocal cord paralysis and/or skeletal dysplasia (16%) had a disease-causing TRPV4 mutation. Most of them had been labeled distal hereditary motor neuropathy; only one had been labeled CMT2.

      “12 of 74 patients (16%) with neuropathy and vocal cord paralysis and/or skeletal dysplasia presented pathogenic TRPV4 mutations, including 7 patients with distal hereditary motor neuropathy, 2 with scapuloperoneal spinal muscular atrophy, 2 with congenital spinal muscular atrophy and arthrogryposis, and one with CMT2”Abstract, Results

    • All 17 patients with a TRPV4 mutation had a motor neuropathy starting in childhood. Foot deformities, curvature of the spine and raised creatine kinase were seen in all, and vocal cord paralysis in 94%.

      “All patients had childhood-onset motor neuropathy and showed a variety of associated findings, including foot deformities (100% of cases), kyphoscoliosis (100%), elevated serum creatine kinase levels (100%), vocal cord paralysis (94%)”Abstract, Results

    • Other features were less common: scapular winging 53%, breathing insufficiency 29%, hearing loss 24%, skeletal dysplasia 18% and arthrogryposis 12%.

      “scapular winging (53%), respiratory insufficiency (29%), hearing loss (24%), skeletal dysplasia (18%), and arthrogryposis (12%)”Abstract, Results

    • Six of the mutations were new in the patient rather than inherited (de novo), and two carriers had no symptoms.

      “Six mutations were de novo events, and 2 asymptomatic carriers were identified.”Abstract, Results

    What the authors concluded

    TRPV4 mutations are a major cause of inherited axonal neuropathy when it comes with extra features such as vocal cord paralysis or skeletal dysplasia, and these patients show a wide range of additional problems.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • ModerateMethods cannot be judged from the abstract

      Only the abstract was available. It does not say how the 'asymptomatic carriers' were assessed (examined, nerve-tested, or only asked), how features such as hearing loss were measured, or how families were chosen. The percentages come from 17 people and describe this selected group, not everyone with TRPV4 neuropathy.

      “2 asymptomatic carriers were identified”Abstract, Results

    • Minor16% applies to a group chosen for TRPV4-like features

      The 16% figure is among patients already selected because they had vocal cord paralysis or skeletal dysplasia, features known to go with TRPV4. It is not the share of all axonal neuropathy that TRPV4 explains, which in this study was 12 of 169.

    How “unaffected” relatives were checked

    Cannot be judged: the abstract reports 2 asymptomatic carriers but does not say whether they were examined or had nerve conduction tests.

    Three ways to explain it

    Scientific

    Screening of 169 French probands with axonal neuropathy found no pathogenic TRPV4 variants in 95 with pure dominant CMT2, but 12 of 74 (16%) with vocal cord paralysis and/or skeletal dysplasia carried one, most diagnosed as dHMN. Among 17 affected individuals, all had childhood-onset motor neuropathy with foot deformities, kyphoscoliosis and raised CK, and 94% had vocal cord paralysis. Six variants were de novo and 2 asymptomatic carriers were reported, with assessment methods not stated in the abstract.

    Easier

    French doctors tested 169 people with an axonal type of inherited neuropathy for TRPV4 changes. None of the 95 people with 'pure' CMT2 had one, but 12 of the 74 people who also had vocal cord paralysis or bone problems did. Almost all of the 17 people with a TRPV4 change had vocal cord paralysis, and all had weakness from childhood, foot shape changes and a curved spine. Two relatives carried the change without symptoms, but the abstract does not say how they were checked.

    Simple

    Doctors looked for the CMT2C gene change in 169 people. They only found it in people who also had a weak voice or bone problems. Those problems are like a clue that points detectives to the right suspect.

    Why these ratings

    Relevance 4/5: It is one of the larger screening studies of TRPV4 neuropathy and describes the features of 17 people with it, though most were labeled motor neuropathy or SMA rather than CMT2C.

    Evidence 1/5: Rated 1 because only the abstract was available, so how patients were chosen, examined and tested cannot be checked.

    Funding and conflicts of interest

    Not stated in the abstract.

    PubMed 24789864 · DOI

54. #54Full paperRetrospective multicenter imaging cohort

    Whole-Body Muscle MRI in Non-5q Spinal Muscular Atrophy: Patterns, Genotype Prediction, and Diagnostic Implications

    Berling E, Reyngoudt H, Le Guillou A et al. · 2026 · Neurology. Genetics

    Relevance 3/5Evidence 3/51 major · 3 moderate · 3 minor problems

    In this small French series, people with TRPV4-related SMA had very varied muscle MRI patterns, and 2 of 4 had a badly affected neck muscle not seen in anyone else. This might one day help doctors recognize TRPV4 disease on scans, but it rests on very few people and the authors say it needs checking in larger groups.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    Retrospective multicenter imaging cohort

    Who or what

    28 adults with non-5q spinal muscular atrophy (chronic, motor-predominant weakness usually starting in the upper arms or thighs) seen at four French neuromuscular centers. Median age at evaluation was 46 years (interquartile range 26 to 50). 23 (82.1%) had a genetic diagnosis across 13 genes, including 4 with TRPV4 variants. Quantitative fat-fraction MRI was done in 21 patients at one center.

    What they did

    They reviewed whole-body muscle MRI scans, grading fatty replacement in 42 paired muscles, 4 limb muscle groups and the tongue on the Mercuri scale, and measured fat fraction in thigh and leg muscles at one center. They looked for patterns specific to each gene and compared each patient's scan with patterns previously published for that gene.

    What they found

    • TRPV4 was the second most common genetic cause in this group of 28 adults, found in 4 patients.

      “Among 28 patients (median age 46 [26-50] years), 23 (82.1%) carried pathogenic variants in 13 genes, most commonly VWA1 (n = 5), TRPV4 (n = 4), and VRK1 and DYNC1H1 (n = 3 each).”Abstract, Results

    • The four TRPV4 patients had very different MRI patterns from one another, and none closely matched the lower-limb pattern previously published for TRPV4.

      “Similarly, the TRPV4 pattern showed poor concordance with the imaging findings in all 4 TRPV4 patients.”Discussion

    • A neck muscle, the sternocleidomastoid, was severely affected in 2 of the 4 TRPV4 patients and was unaffected in every other patient in the study.

      “Notably, the sternocleidomastoid muscle was severely affected in 2 patients (MS = 3-4) and spared in the other 2, while it was consistently unaffected in all other patients in the cohort.”Results, TRPV4-Related Cases

    • In the lower legs, 3 of the 4 TRPV4 patients had involvement, with the extensor digitorum and peroneus muscles more affected than the tibialis anterior, and the medial gastrocnemius relatively spared in 3 of 4.

      “leg involvement was observed in 3 of the 4 patients, with preferential involvement of the extensor digitorum and peroneus muscles (MS = 2-4) compared with the tibialis anterior (MS = 1-2).”Results, TRPV4-Related Cases; Figure 2D

    • Two of the TRPV4 patients needed non-invasive ventilation. One TRPV4 patient, a 34-year-old man, had mild sensory nerve conduction abnormalities.

      “Noninvasive ventilation was required in a BSCL2 -related case, 2 TRPV4 -related cases, and a genetically undiagnosed patient.”Results, Characteristics of the Cohort

    • Clearer, repeatable patterns were found for three other genes (VRK1, DYNC1H1 and VWA1), which the authors suggest can help guide genetic testing.

      “Distinct and reproducible MRI patterns were identified in VRK1 -related, DYNC1H1 -related, and VWA1 -related cases.”Abstract, Results

    What the authors concluded

    Whole-body muscle MRI appears useful for guiding genetic diagnosis in non-5q SMA, with gene-specific patterns for VRK1, DYNC1H1 and VWA1. Involvement of the sternocleidomastoid (a neck muscle) may be a supportive clue for TRPV4-related SMA, although it is not seen in every TRPV4 patient. The authors stress that numbers per gene were small and larger cohorts are needed.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • MajorVery small TRPV4 group

      The TRPV4 findings come from only 4 people, and the neck muscle clue from 2 of them. No statistical test supports it. Such a small number can easily produce patterns by chance, as the authors themselves acknowledge.

      “These findings should be interpreted with caution because sample sizes for each genetic subtype were small.”Discussion, conclusion

    • ModerateSelected, referral-center patients

      Patients were included only if they had whole-body MRI, which the authors say is often done when diagnosis is difficult. People with typical or milder disease may therefore be missing, so the patterns may not represent everyone with these conditions.

      “the inclusion criterion of whole-body MRI, which in clinical practice is often performed in diagnostically challenging cases”Discussion

    • ModerateMay not apply to people with CMT2C

      Patients had to have an SMA picture with only or mainly motor involvement, usually starting near the trunk, and the paper itself separates these motor conditions from Charcot-Marie-Tooth disease. People with the CMT2C form of TRPV4 disease would therefore mostly not be included, so the findings may not carry over to them. Which TRPV4 variants and clinical features each patient had is only in supplementary tables, not the main text.

      “The diagnosis of SMA was based on a chronic, progressive motor-predominant weakness, typically starting proximally in either upper or lower limbs”Methods, Study Population

    • ModerateRater blinding and agreement not reported

      The paper does not say whether the people grading the scans knew each patient's genetic result. If they did, this could influence which patterns they noticed. No measure of agreement between raters is reported.

      “Forty-two paired muscles, 4 limb muscle groups, and the tongue were evaluated by 2 experienced raters (Y.F. or R.Y.C. and E.B.).”Methods, MRI Acquisition and Analysis

    • MinorMixed ages and disease stages, single time point

      Each patient was scanned once, at very different ages and stages of disease. Muscle patterns can change as disease progresses, so differences between patients may partly reflect stage rather than gene. Two MRIs were done in childhood although the study was meant to include adults.

      “Although we focused our analysis on adult patients to limit this bias, 2 participants had MRIs performed during childhood, and 1 at age 64.”Discussion, limitations

    • MinorErrors and unclear figures in tables and text

      Table 1 gives an impossible count of 4.7 patients. Its sensory conduction counts (3 with a genetic diagnosis, 2 without) do not match the five patients listed in the text (four with a genetic diagnosis, one without), and bladder dysfunction is 7.1% in the table but 7.4% in the text. The sentence comparing the TRPV4 pattern scores with previously published patients is unclear about what is being compared. These errors do not change the main message but suggest the numbers were not carefully checked.

      “Respiratory involvement, n (%) 5 (17.9) 4.7 (17.4) 1 (20)”Table 1; Results

    • MinorClaims about automated scoring go beyond what was done

      The conclusion says fat-fraction mapping removes the burden of visual assessment, but in this study the muscle regions were drawn by hand, and no statistic is given for how well the two methods agreed.

      “By providing results comparable with manual scoring while removing the burden of visual assessment, qMRI-based FF mapping paves the way”Discussion, conclusion

    Three ways to explain it

    Scientific

    In a retrospective cohort of 28 adults with non-5q SMA, whole-body muscle MRI (Mercuri grading, plus Dixon fat fraction in 21) identified reproducible gene-specific patterns for VRK1, DYNC1H1 and VWA1. The four TRPV4 patients showed heterogeneous involvement and poor concordance with previously published TRPV4 lower-limb patterns, but severe sternocleidomastoid involvement occurred in 2 of 4 and in no other cohort member. The authors propose this as a supportive but inconsistent marker requiring validation in larger cohorts.

    Easier

    The researchers looked at whole-body MRI scans of the muscles of 28 adults with a group of rare motor nerve conditions; 4 of them had TRPV4 changes. The scans show which muscles have been replaced by fat. For some genes there was a clear, repeatable pattern, but the scans of the 4 TRPV4 patients all looked different from each other. Two of them had heavy damage in a neck muscle that nobody else in the study had, which could be a clue worth testing in more people.

    Simple

    Doctors took pictures of all the muscles of 28 adults with weak muscles. The pictures of the 4 people with TRPV4 changes all looked different. But 2 of them had a weak neck muscle that nobody else had. That might be a clue, like a footprint, though only a few people were checked.

    Why these ratings

    Relevance 3/5: Its TRPV4 patients had the SMA form of TRPV4 disease rather than classic CMT2C, and there were only four of them.

    Evidence 3/5: A small retrospective series; the TRPV4-specific neck muscle finding rests on 2 of 4 patients.

    Funding and conflicts of interest

    The authors report no targeted funding; the article processing charge was funded by the authors. The authors report no relevant disclosures.

    PubMed 42662709 · DOI

55. #55Full paperRetrospective single-center cohort study

    Hip Dysplasia in Charcot-Marie-Tooth Disease: Insights From a Large Cohort of Children and Adolescents

    Tan KA, Ryan MM, Kennedy RA et al. · 2025 · Journal of the peripheral nervous system: JPNS

    Relevance 3/5Evidence 3/53 moderate · 3 minor problems

    In this Australian children's clinic, all 4 children with TRPV4-related CMT had hip dysplasia on X-ray and 2 needed hip surgery. This suggests hips can be a concern in childhood TRPV4 neuropathy, but with only 4 children involved, it cannot say how common hip dysplasia is.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    Retrospective single-center cohort study

    Who or what

    178 children with CMT or related inherited neuropathies seen at the Royal Children's Hospital, Melbourne, Australia, who had at least one pelvic X-ray between 2000 and 2020 (256 identified; 77 excluded for having no hip X-rays and one for a complex neurodevelopmental disorder). The group included 4 children with TRPV4-related CMT, 100 with CMT1A, 10 with CMT2A and 7 with Dejerine-Sottas disease.

    What they did

    The team looked back through hospital records and had one senior pediatric orthopedic surgeon measure every pelvic X-ray using three standard hip measurements. They counted how many children had hip dysplasia on their first and last X-ray, how many needed surgery, and how this differed between types of CMT.

    What they found

    • Hip dysplasia was common: 64 of 178 children (36.0%) had it on their first X-ray, 50 (28.1%) at last follow-up, and 17 (9.6%) needed hip surgery.

      “At the time of last follow up, 50/178 children (28.1%) had hip dysplasia and 17/178 children (9.6%) required surgical intervention.”Abstract, Results

    • All 4 children with TRPV4-related CMT had hip dysplasia, the highest rate of any group, although the group was very small.

      “3/10 with CMT2A and 4/4 with TRPV4 -related phenotypes”Results, final outcomes

    • Two of the 4 children with TRPV4-related CMT needed hip surgery at the time hip dysplasia was diagnosed. One child with TRPV4-related CMT never walked independently.

      “Three children required surgery (two had TPRV4 -related CMT and one had no genetic diagnosis).”Results; Figure 3

    • Hip dysplasia was often silent: most children found to have it had no hip symptoms at the time.

      “Therefore, only 11/42 (26.2%) children with available data regarding the indication for their initial pelvic radiograph were symptomatic at the time of diagnosis of hip dysplasia.”Results

    • Hip findings changed with growth: of 52 children with dysplasia who were followed with repeat X-rays, 20 had hips that returned to normal, while 6 children with a normal first X-ray developed dysplasia later.

      “Of these, 12 (18.8%) required immediate surgical intervention. Based on their first pelvic radiograph, serial pelvic radiography was performed in the remaining 52 children, of which 20 normalized with age.”Results

    • The authors suggest the bone effects of TRPV4 changes may add to the risk of hip dysplasia in TRPV4-related CMT, and suggest more frequent hip monitoring for children with TRPV4 changes or Dejerine-Sottas disease and for children who cannot walk.

      “The association of bone dysplasia with TRPV4 mutations may also be a factor contributing to hip dysplasia in this specific CMT subtype”Discussion

    What the authors concluded

    Hip dysplasia is common in children with CMT and often causes no symptoms. The abstract estimates its prevalence at between 9.6% and 28.1%, while the discussion gives between 9.6% and 22.9%. Because early findings can normalize and new ones can appear, the authors support regular hip X-ray surveillance until near skeletal maturity. Children with Dejerine-Sottas disease or TRPV4-related CMT appeared especially affected, and the authors suggest closer hip monitoring in these groups.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • ModerateVery small TRPV4 group

      The '100%' rate for TRPV4-related CMT comes from 4 children. With so few, the true rate in children with TRPV4 neuropathy could be much lower. The authors say findings in rarer subtypes need repeating in larger groups.

      “The small numbers of the less common CMT subtypes in this genetically heterogeneous cohort mean this finding needs to be replicated in larger cohorts.”Discussion

    • ModerateWho had X-rays was not systematic

      X-rays were ordered at each doctor's discretion, the reason was unclear for many children, and 77 children with no hip X-rays were excluded. Children with symptoms or more severe disease may have been more likely to have X-rays, which can distort the rates.

      “As such, the acquisition of pelvic radiographs was based on individual clinician discretion and awareness of hip dysplasia risk.”Discussion, limitations

    • Moderate'Independent risk factor' claimed without the analysis to test it

      The authors say not walking is likely an independent risk factor, but they chose not to run the regression analyses needed to separate one risk factor from another. For the TRPV4 children, the paper cannot separate the effect of not walking from the effect of TRPV4 on bone.

      “Many of these children were also non ambulant, indicating that ambulation status is likely an independent risk factor of hip dysplasia in children with CMT.”Discussion

    • MinorInconsistent figures

      The abstract gives the prevalence range as 9.6% to 28.1% but the discussion gives 9.6% to 22.9%. The abstract says 35.8% at first X-ray while the results say 36.0%, and Table 3 is headed n = 179 rather than 178. The differences are small but show the figures need careful reading.

      “we estimate that the true prevalence of hip dysplasia in children with CMT and related disorders may be between 9.6% and 22.9%.”Discussion

    • MinorSingle reader and a broad definition

      One surgeon measured all X-rays, and the paper does not report a second reader or blinding to CMT type. The definition used is broader than an earlier study's, and 12 children were counted as having dysplasia from a single X-ray that radiologists had reported as normal, with no follow-up imaging.

      “This included 12 children who were retrospectively diagnosed with hip dysplasia with our radiographic criteria after a single hip radiograph but had no further imaging as the hip radiograph was reported to be normal by the radiologist.”Results

    • MinorTRPV4 children not described in detail

      The paper does not describe the TRPV4 children's specific variants or whether they had other skeletal features, such as a known skeletal dysplasia. This information would help readers judge whether their hip problems came from nerve weakness, from bone development, or both.

    Three ways to explain it

    Scientific

    In a retrospective single-center cohort of 178 children with CMT and related neuropathies, radiographic hip dysplasia (migration percentage, acetabular index, lateral center edge angle) was present in 36.0% at first radiograph and 28.1% at last follow-up, with 9.6% requiring surgery. All 4 children with TRPV4-related CMT had hip dysplasia and 2 required surgery, but this subgroup is very small and selection for radiography was not systematic. The authors propose that TRPV4-related skeletal effects and non-ambulation may contribute, without multivariable analysis to test this.

    Easier

    This study looked back at hip X-rays of 178 children with CMT at one Australian hospital. 64 of them had a shallow or poorly fitting hip socket (hip dysplasia) on their first X-ray, often without any hip symptoms, and 17 needed an operation. All 4 children with the TRPV4 form of CMT had hip dysplasia, but 4 is too few to know how common this is in TRPV4 nerve disease.

    Simple

    The hip is a ball that sits in a cup. In some children with CMT the cup is too shallow, so the ball does not sit snugly. All 4 children in this study with the TRPV4 type had this, but that is only a few children, so we cannot be sure how often it happens.

    Why these ratings

    Relevance 3/5: A general childhood CMT study with a small but striking TRPV4 result: all 4 children with TRPV4-related CMT had hip dysplasia and 2 needed surgery.

    Evidence 3/5: A fairly large retrospective cohort for a rare condition, but X-rays were taken inconsistently and the TRPV4 group has only 4 children.

    Funding and conflicts of interest

    The authors received no specific funding for this work. Disclosures: E.M.Y. leads the Australian Neuromuscular Diseases Registry, which is supported by grants from TREAT-NMD, MD-NSW, two foundations, the Western Australian government and the companies Biogen, Roche, Novartis, PTC Therapeutics and Pfizer; E.M.Y. has also received advisory board honoraria from Biogen and Roche and speaker fees (paid to the institution) from Biogen and PTC Therapeutics; R.K. has received speaker honoraria from Biogen; K.C. has received research support from Pfizer. All are stated to be unrelated to this manuscript.

    PubMed 39887493 · DOI

56. #56Full paperCase series with follow-up (text analyzed is the Research Square preprint, version 1)

    Comparison of the natural course of clinical and radiologic features in 13 patients with TRPV4-related skeletal dysplasias

    Güneş N, Alkaya DU, Kurugoğlu S et al. · 2025 · Pediatric radiology

    Relevance 3/5Evidence 3/54 moderate · 3 minor problems

    This paper is mainly about TRPV4 bone and growth conditions rather than CMT2C. It shows that TRPV4 skeletal problems can change with age and vary within families, and it includes one child with congenital distal SMA who was walking normally by about 9 years.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    Case series with follow-up (text analyzed is the Research Square preprint, version 1)

    Who or what

    13 patients (11 children and 2 affected parents; 8 male, 5 female) from 10 families with heterozygous TRPV4 variants: 5 with spondylometaphyseal dysplasia Kozlowski type (SMDK), 3 with classic metatropic dysplasia (MD), 3 with brachyolmia type 3 (BCYM3), 1 with a mild form of spondyloepiphyseal dysplasia Maroteaux type (SEDM) and 1 with congenital distal spinal muscular atrophy (CDSMA). Median age at first examination was 3 years (range 1 to 44), and median follow-up was 6.5 years (range 3.5 to 16.5). There were eight different TRPV4 variants, one of them new.

    What they did

    The team examined patients yearly and took skeletal X-rays, recording height, pain, walking, and spine and limb deformities. They grouped patients into skeletal dysplasia types by clinical and X-ray features and described how findings changed with age in each type. Genetic testing used a skeletal dysplasia gene panel, or whole-exome sequencing in the CDSMA patient.

    What they found

    • In SMDK and MD, short stature and pain when running, walking far or climbing stairs appeared after about age 5 and worsened with age.

      “Short stature, and bone pain during running, walking, and stair climbing occurred in SMDK and MD patients after the age of 5 years and became more pronounced with increasing age.”Abstract (Results); Results sections on SMDK and MD

    • Forward curvature of the spine (kyphosis) was more marked in SMDK and MD, while sideways curvature (scoliosis) was more marked in BCYM3 and the CDSMA patient.

      “Kyphosis was more pronounced in SMDK and MD patients, and scoliosis in BCYM3 and CDSMA.”Abstract (Results)

    • Hip and pelvis X-rays best told SMDK and MD apart: irregular, widened areas near the top end of the thigh bone (proximal femoral metaphyses) in SMDK, and marked widening of the femoral neck in MD.

      “Radiologic differentiators between SMDK and MD phenotypes were primarily radiographs of the pelvis”Discussion (conclusion paragraph)

    • The one child labeled CDSMA had the p.Arg269His variant. At 12 months he had hip dislocation, torticollis (a twisted neck), laryngomalacia (floppy tissue in the voice box) and contractures, with a normal EMG; by about 9 years he walked normally and had only mild remaining problems.

      “At around 9 years of age, he was almost normal height and had no other problems apart from mild laryngomalacia, scoliosis, and mild knee limitation.”Results, 'A patient whose phenotype is compatible with CDSMA'; Discussion

    • The same variant could look different within one family: in the SMDK and BCYM3 families, relatives sharing a variant differed in severity or features. The recurrent variant p.Arg594His was found in five SMDK patients from three families.

      “Intrafamilial variability was observed in the SMDK and BCYM3 families.”Discussion (conclusion paragraph); Results, 'Molecular findings'

    What the authors concluded

    SMDK and classic MD lead to a short trunk, severe short stature, kyphosis and activity-related bone pain from about age 5, while the milder types allow normal walking. Pelvic X-rays help distinguish SMDK from MD, X-ray features change with age, and there is variability within families. The authors believe these findings will help diagnosis and treatment of TRPV4 skeletal dysplasias.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • ModeratePreprint version analyzed

      The text supplied is the Research Square preprint posted before peer review. A version was later published in Pediatric Radiology, and the published version may differ.

      “This is a preprint; it has not been peer reviewed by a journal.”Preprint header

    • ModerateVery small groups, descriptive only

      Thirteen patients are split across five types, two of them with a single patient, and comparisons are descriptive without statistics. The detailed patient tables were not included in the text supplied. Statements comparing types are therefore observations in a few people.

      “one each with the mild form of Maroteaux-type spondyloepiphyseal dysplasia (SEDM), and congenital distal spinal muscular atrophy (CDSMA)”Abstract (Material and methods)

    • ModerateConclusion contradicts case details

      The conclusion says all BCYM3 and CDSMA patients had severe scoliosis and were of normal height, but the results describe mild scoliosis in the CDSMA child and a BCYM3 mother with short stature (-3.9 SDS). The summary statements overstate the uniformity of the findings.

      “All BCYM3 and CDSMA patients had severe scoliosis. However, at the follow-up, the BCYM3, SEDM, and CDSMA patients were able to walk normally and were of normal height.”Discussion (conclusion paragraph); compare Results on P11 and the CDSMA patient

    • MinorUnclear how many were followed up

      The abstract says 11 of the 13 were followed, but the methods say all 13 were followed for a median of 6.5 years. It is unclear which patients had follow-up.

      “Thirteen patients with a mutation in TRPV4 from 10 families were included in the study, and 11 were followed for a median of 6.5 years.”Abstract (compare Material and methods, 'Patients')

    • ModerateNerve and muscle involvement not assessed

      Patients with TRPV4 nerve or muscle disease but no skeletal findings were excluded, and no nerve conduction studies or detailed strength testing are reported. The one child labeled CDSMA had a normal EMG, so the basis for the neuromuscular label is not clear from the paper, and nerve involvement in the other patients is unknown.

      “His EMG was normal.”Results, 'A patient whose phenotype is compatible with CDSMA'

    • MinorMechanism claim without supporting data

      The authors state that finding an in-frame deletion rules out gain of function as the explanation for all cases, but the paper contains no functional testing of this or any other variant, so the statement is not supported by its own data.

      “the observation of a heterozygous in-frame deletion mutation rules out the possibility that they are all explained by gain-of-function”Discussion

    • MinorBackground statement contradicts later text

      The introduction says the channel is impermeable to calcium, but the next paragraph says TRPV4 increases calcium inside cells. This inconsistency suggests the background section should be read with caution.

      “a cation channel protein that is selectively impermeable to calcium”Introduction

    How “unaffected” relatives were checked

    This is not a penetrance study. The paper says 'While the disease was de novo in eight families, it was familial in two families' (Results), but does not describe how parents or other relatives in the de novo families were examined or tested to decide this.

    Three ways to explain it

    Scientific

    In 13 patients from 10 families with heterozygous TRPV4 variants, followed for a median of 6.5 years, SMDK and classic metatropic dysplasia showed short stature, kyphosis and activity-related pain emerging after about age 5 and progressing with age, whereas brachyolmia type 3 and the single CDSMA patient (p.Arg269His) showed scoliosis with preserved ambulation. Pelvic radiographs best distinguished SMDK (irregular proximal femoral metaphyses) from MD (femoral neck widening), and intrafamilial variability was observed. The series is small and descriptive and is analyzed here in its preprint form.

    Easier

    This study followed 13 people, mostly children, with bone and growth conditions caused by TRPV4 changes, for about six and a half years on average. In the two more severe types, children grew much shorter than average and developed a forward-curving back and pain on walking or climbing stairs, starting around age 5 and worsening with age. Hip X-rays helped tell these two types apart. One child had a nerve-related form called congenital distal SMA and was walking normally at about 9 years.

    Simple

    Doctors took X-rays of 13 people with TRPV4 bone conditions for several years. In some types, the back curved and walking got painful as children grew. This study is mostly about bones, not CMT2C.

    Why these ratings

    Relevance 3/5: Mainly about TRPV4 bone growth conditions (skeletal dysplasias), with one congenital distal SMA patient and little direct CMT2C content.

    Evidence 3/5: A small descriptive series with useful long-term X-ray follow-up, but with very small groups, no statistics, internal inconsistencies, and analyzed here in its non-peer-reviewed preprint form.

    Funding and conflicts of interest

    Funding is not stated in the preprint text. 'No competing interests reported.'

    PubMed 39825918 · DOI · Our copy is the Research Square preprint (version 1), not the published Pediatric Radiology article.

57. #57Detailed summary onlySmall case series (three patients) with genetic testing and nerve studies

    TRPV4-pathy manifesting both skeletal dysplasia and peripheral neuropathy: a report of three patients

    Cho TJ, Matsumoto K, Fano V et al. · 2012 · American journal of medical genetics. Part A

    Relevance 3/5Evidence 3/54 moderate · 2 minor problems

    This paper describes three children who had both a TRPV4 bone condition and nerve involvement, which supports the idea that TRPV4 bone and nerve conditions belong to one spectrum. It suggests bone changes may go unnoticed in people known to have only TRPV4 neuropathy.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    Small case series (three patients) with genetic testing and nerve studies

    Who or what

    Three unrelated girls referred for TRPV4 skeletal dysplasia who also had nerve involvement: a Korean girl followed from age 1 to 6 (E278K, c.832G>A), a Japanese girl aged 14 (P799R, c.2396C>G) and an Argentine girl aged 5 (A217S, c.649G>T, new in her). The Korean girl's mother, who carries E278K, is also described.

    What they did

    Bone specialists in Seoul, Saitama and Buenos Aires, working with a genetics laboratory in Japan, sequenced TRPV4 and carried out nerve conduction studies and EMG, plus one muscle biopsy. They built a computer model of where the new A217S mutation sits in the protein, based on the known structure of the chicken channel. This analysis is based on a detailed secondary summary of the paper written by another reader, not on the paper itself.

    What they found

    • All three girls had a TRPV4 skeletal dysplasia (two Kozlowski type, one Maroteaux type) together with signs of nerve involvement, showing the bone and nerve forms of TRPV4 disease can occur in the same person.

      “Three children had both a TRPV4 bone disorder and a TRPV4 nerve disorder, which shows the two disease families are not separate.”Secondary summary, opening summary

    • The nerve findings differed between the girls: slow motor conduction and absent peroneal responses with normal sensory nerves in patient 1, a motor and sensory axonal neuropathy in the legs in patient 2, and normal nerve conduction but active denervation on EMG in patient 3.

      “Motor and sensory axonal neuropathy in the legs, arms normal”Secondary summary, table 'The three patients', row 'Nerve tests'

    • Patient 1's mother carries the same E278K mutation and has short stature (145 cm) and spine changes, but is described as having no neuropathy. The bone disorder passed from mother to daughter while the nerve disorder appeared only in the daughter.

      “The bone disorder passed from mother to daughter. The nerve disorder appeared only in the daughter.”Secondary summary, 'The mother of patient 1'

    • The authors found no strict rule linking where a mutation sits, or what type it is, to bone versus nerve disease. None of the three mutations replaces an arginine, and P799R lies outside the ankyrin region.

      “There is no strict rule linking a mutation's position or type to bone versus nerve disease.”Secondary summary, 'Authors' conclusions'

    • All three girls had heights in the normal range. The authors suggest bone changes are probably missed in people known to have only neuropathy, and recommend X-rays in TRPV4 neuropathy and careful neurological examination in TRPV4 skeletal dysplasia.

      “X-rays are worth doing in people with TRPV4 neuropathy, and a careful neurological examination in people with TRPV4 skeletal dysplasia.”Secondary summary, 'Authors' conclusions'

    • The new A217S mutation was absent from both parents and from 121 controls, lies at a position unchanged from fish to humans, and is predicted by the model to disturb a short helix.

      “A217S was absent from both parents and from 121 controls of mixed ancestry.”Secondary summary, 'Other details'

    What the authors concluded

    TRPV4 bone disorders and TRPV4 nerve disorders are not separate: the same person can have both, and no simple rule links mutation position or type to one or the other. The authors think bone involvement is nearly always expressed while nerve involvement is not, and suggest looking for bone changes in TRPV4 neuropathy and for nerve problems in TRPV4 skeletal dysplasia.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • ModerateThree patients assessed differently

      Each child came from a different center and was examined with different tests, so the cases are hard to compare and general rules cannot be drawn from them.

      “Three patients, each from a different centre, examined with different tests.”Secondary summary, 'Limitations'

    • ModerateUnclear how the mother was judged free of neuropathy

      The authors' point that nerve involvement is not always expressed leans on patient 1's mother. The summary does not say whether she had a neurological examination or nerve conduction tests, so her 'no neuropathy' status cannot be checked.

      “Her daily life is not limited and she has no neuropathy.”Secondary summary, 'The mother of patient 1'

    • ModerateEarlier P799R patients judged from records only

      Two earlier children with P799R were judged free of neuropathy by reviewing their records, not by examining them, which is a weak way to rule out nerve involvement.

      “Records of other P799R patients were reviewed on paper for neuropathy”Secondary summary, 'Limitations'

    • ModerateNo laboratory test of the mutations

      There is no test of how the mutations change the channel. Evidence that A217S is harmful rests on its absence from parents and controls, conservation and a structural model.

      “No laboratory test of how the mutations change the channel. The A217S evidence is a structural model only.”Secondary summary, 'Limitations'

    • MinorShort follow-up in young children

      The children were young, so how their nerve and bone problems develop over time is unknown.

      “The children were young and follow-up short, so progression is unknown.”Secondary summary, 'Limitations'

    • MinorPossible second genetic condition in patient 3

      Patient 3's parents are related to each other, which raises the chance of another genetic condition, although the TRPV4 change was new in her.

      “Patient 3's parents were related to each other, which raises the chance of a second unrelated genetic condition”Secondary summary, 'Limitations'

    How “unaffected” relatives were checked

    Patient 1's mother carries E278K and is described as having no neuropathy, but the summary does not say whether this was based on a neurological examination or nerve conduction tests. Two earlier children with P799R were judged free of neuropathy from a review of their records, not by re-examination. Quote: “Her daily life is not limited and she has no neuropathy.”

    Three ways to explain it

    Scientific

    Three unrelated girls with TRPV4 skeletal dysplasia (spondylometaphyseal dysplasia Kozlowski type or spondyloepimetaphyseal dysplasia Maroteaux type) also had neurogenic involvement, ranging from motor or sensorimotor axonal neuropathy to denervation with normal nerve conduction, and carried E278K, P799R or de novo A217S. The authors find no consistent genotype-phenotype rule separating skeletal from neuropathic expression, and suggest skeletal features are highly penetrant while neuropathy shows reduced penetrance, based partly on an E278K-carrying mother without reported neuropathy. Evidence is clinical and genetic only, with no functional studies.

    Easier

    This paper describes three girls who each had a TRPV4 bone growth condition and also had damage to the nerves that control muscles. The changes in their TRPV4 genes were in different places, and the authors found no simple rule for which changes cause bone problems, nerve problems or both. One girl's mother had the same change and the bone condition, but was described as having no nerve problems. The authors suggest checking for bone changes in people with TRPV4 nerve disease, and the other way round.

    Simple

    The TRPV4 gene can cause bone problems or nerve problems, and these three girls had both. It is like one faulty part in a machine that can make it break in more than one way. Doctors still cannot predict which way it will go for each person.

    Why these ratings

    Relevance 3/5: It describes TRPV4 skeletal dysplasia (a bone condition caused by the same gene as CMT2C) overlapping with neuropathy, which supports seeing CMT2C as part of a wider TRPV4 spectrum.

    Evidence 3/5: A small series of three well-described cases supports the main point that bone and nerve disease can co-occur, but there are no lab tests, and this analysis rests on a secondary summary.

    Funding and conflicts of interest

    The summary says funding came from the Korean and Japanese health ministries. Competing interests are not mentioned in the summary.

    PubMed 22419508 · DOI

58. #58Detailed summary onlySmall case series with gene screening, tissue staining and cell experiments

    TRPV4 mutations in children with congenital distal spinal muscular atrophy

    Fiorillo C, Moro F, Brisca G et al. · 2012 · Neurogenetics

    Relevance 3/5Evidence 3/54 moderate · 2 minor problems

    In this small group of children born with leg weakness from motor nerve damage, the two with TRPV4 changes were the ones who also had vocal cord or bone problems. One of the changes, R232C, has also been reported in people with CMT2C, which shows the same change can be given different diagnosis labels.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    Small case series with gene screening, tissue staining and cell experiments

    Who or what

    Six patients aged 2.5 to 21 with genetically unexplained congenital distal spinal muscular atrophy, chosen from the databases of pediatric neuromuscular centers in Pisa and Genoa. Two had new TRPV4 mutations: a girl of 12 with P97R (c.290C>G) and a boy of 2.5 with R232C (c.694C>T). P97R was tested in HeLa cells.

    What they did

    The team sequenced the TRPV4 coding exons, nearby intron sequence, both untranslated ends and the predicted promoter, and checked new changes with prediction programs and comparisons across species. They expressed normal and P97R channels in HeLa cells and measured calcium inside the cells and channel current, at rest and after swelling the cells. They also stained TRPV4 protein in the two children's muscle, skin and cultured skin cells. This analysis is based on a detailed secondary summary of the paper written by another reader, not on the paper itself.

    What they found

    • Two of the six patients had new TRPV4 mutations, and these were the only two with voice, vocal cord or bone involvement. None of the four without a mutation had these features.

      “Two of six children with unexplained congenital distal spinal muscular atrophy had new TRPV4 mutations, and they were the two with a hoarse voice or bone changes.”Secondary summary, opening summary and 'Shared findings'

    • The girl with P97R has never stood or walked without support, developed a hoarse voice at 7, and her vocal cords fail to open when she breathes in. Her lung function was close to normal (forced vital capacity 92% of predicted).

      “Endoscopy showed both vocal cords failing to open on breathing in, while closing normally for speech.”Secondary summary, 'Patient 1, a girl of 12 with P97R'

    • The boy with R232C had both nerve disease and bone changes (flattened vertebrae, short finger bones, misshapen hip joints). R232C had previously been reported under several labels, including CMT2C.

      “R232C was new in him. It had been reported in three families, nine patients in all, variously labelled distal SMA with vocal cord paralysis, distal and scapuloperoneal SMA, and CMT2C.”Secondary summary, 'Patient 2, a boy of 2.5 with R232C'

    • In HeLa cells, P97R gave a smaller calcium rise after cell swelling and about 60% of normal channel current, which the authors read as loss of function. Resting calcium was the same as with the normal channel.

      “Channel current after swelling was about 60% of normal (p below 0.01, at least four cells per group).”Secondary summary, 'Laboratory findings for P97R'

    • The authors found no clean link between the type of mutation, its position, its measured effect on the channel and the clinical picture, and suggest other genetic or epigenetic factors contribute. They recommend TRPV4 testing in congenital distal SMA with bone abnormalities or vocal cord paralysis.

      “TRPV4 testing is warranted in congenital distal SMA with bone abnormalities or vocal cord paralysis.”Secondary summary, 'What the authors argue'

    What the authors concluded

    TRPV4 mutations are a cause of congenital distal spinal muscular atrophy, especially when it comes with bone abnormalities or vocal cord paralysis, and TRPV4 testing is warranted in those children. The P97R mutation reduced channel function in their lab tests, while R232C had been reported elsewhere to increase it, so the authors see no simple link between mutation, channel effect and illness, and call for experiments in patients' own cells.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • ModerateSmall, selected group

      Six patients were picked from two centers' files, so two out of six cannot be read as how often TRPV4 causes this condition.

      “Six patients chosen from two centres' files. The two-in-six figure cannot be read as a frequency.”Secondary summary, 'Limitations'

    • ModerateLab test in an overexpressing cancer cell line

      The channel tests used HeLa cells made to produce large amounts of TRPV4, with small numbers of recordings. This is a long way from human nerve cells, and the authors themselves urge caution.

      “The functional tests used a cancer cell line forced to make large amounts of the channel, with small numbers of recordings.”Secondary summary, 'Limitations'

    • ModerateLoss versus gain comparison not like for like

      Only P97R was tested here. The contrast with R232C relies on another laboratory's results using different methods, so the two cannot be fairly compared.

      “Only P97R was tested. R232C was not run in the same system”Secondary summary, 'Limitations'

    • ModerateResult conflicts with most other work

      The summary notes the loss-of-function finding goes against most other studies of neuropathy mutations, and the authors list method differences or unrepresentative cells as possible reasons.

      “The loss-of-function result conflicts with most other work on neuropathy mutations.”Secondary summary, 'Limitations'

    • MinorTissue staining judged by eye

      The finding that TRPV4 protein looked normal in the children's tissues was not measured with numbers.

      “Protein staining in tissue was judged by eye.”Secondary summary, 'Limitations'

    • MinorVery young second patient

      The boy was only 2.5 years old, so how his condition develops is unknown.

      “Patient 2 was only 2.5 years old, so his later course is unknown.”Secondary summary, 'Limitations'

    Three ways to explain it

    Scientific

    Screening six patients with genetically unexplained congenital distal SMA identified new TRPV4 variants in two: P97R, located before the ankyrin repeats and the first distal SMA mutation found outside them, and R232C, previously reported with CMT2C; only these two had laryngeal or skeletal involvement. In HeLa cells, P97R reduced swelling-evoked calcium entry and reduced current to about 60% of normal, which the authors interpret as loss of function, in contrast to gain of function reported elsewhere for R232C. The authors find no consistent genotype-phenotype or genotype-function correlation and propose additional modifying factors.

    Easier

    Doctors tested the TRPV4 gene in six young patients who were born with leg weakness caused by damage to motor nerve cells, but had no genetic diagnosis. Two had a new TRPV4 change, and these were the only two who also had a hoarse voice or bone changes. In lab cells, one of the changes (P97R) made the channel work less than normal, while another lab had reported that the other change (R232C) makes it work more. The authors say the link between the type of change and the illness is not yet clear.

    Simple

    Two of six children born with weak legs had a change in their TRPV4 gene, and they were the ones with a croaky voice or bone problems. In a lab test, one child's TRPV4 door opened less than it should, not more. Scientists are still working out why different changes in the same door can cause similar illness.

    Why these ratings

    Relevance 3/5: It concerns congenital distal spinal muscular atrophy, another condition caused by TRPV4 changes, and includes R232C, a mutation also reported in CMT2C.

    Evidence 3/5: A small, selected clinical series with some laboratory work in overexpressing cells, read here only through a secondary summary.

    Funding and conflicts of interest

    Not stated in the summary.

    PubMed 22526352 · DOI

59. #59Full paperCase report with 5-year follow-up of an exercise program

    The case of a highly trained TRPV4 related scapuloperoneal spinal muscular atrophy patient: a 5-year follow-up

    Crisafulli O, Fortunati M, Quintiero V et al. · 2025 · Acta myologica: myopathies and cardiomyopathies: official journal of the Mediterranean Society of Myology

    Relevance 3/5Evidence 2/51 major · 3 moderate · 2 minor problems

    One young man with TRPV4-related scapuloperoneal SMA did regular, supervised strength and endurance training for about five years without reported harm. His measured strength was maintained or improved, and the authors put a dip in his fitness at the last test down to a two-month break in training. One person cannot show that exercise slows the condition, and his experience may not apply to others.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    Case report with 5-year follow-up of an exercise program

    Who or what

    One man, aged 26 at the time of writing, with TRPV4-related scapuloperoneal spinal muscular atrophy (SPSMA) and a heterozygous TRPV4 change c.281C>G (p.S94W), followed at a sports medicine center in Italy from age 21. He had three laboratory assessments, at about ages 21, 23 and 26.

    What they did

    The patient followed a supervised program of resistance training three times a week and endurance cycling twice a week, plus exercises for the shoulder-blade and lower-leg muscles. The team measured fitness (VO2max on a cycling test), grip strength, arm and leg strength on the MRC scale, body composition (phase angle from bioimpedance) and a questionnaire on well-being and fatigue (FACIT-F). His results were compared with published values for healthy people, athletes and other neuromuscular patients.

    What they found

    • Yearly clinical check-ups found no new weakness over the follow-up.

      “Annual clinical evaluations did not reveal any additional deficits compared to the initial assessment, indicating a stable clinical condition.”Results

    • His summed MRC strength score for six muscle groups on each side (maximum 60) was 45, then 52, then 52, so it did not fall and in fact rose after the first assessment.

      “Total score (point) 45 52 52”Table I

    • His fitness (VO2max) was 50.0, 51.9 and 43.8 mL/min/kg, and at its peak was similar to that of athletic groups. The authors link the last, lower value to a two-month break from training before that test.

      “His relative V̇O 2 max, which peaked above 50.0ml/min/kg on two occasions, aligns with a large cohort of athletic populations”Discussion; Table I

    • Grip strength (57.3, 58.8 and 55.7 kg in the dominant hand) stayed in the upper range for healthy people of his age and sex.

      “between the 75 th and the 90 th percentile of reference values for age and sex-matched healthy subjects”Discussion

    • Self-reported well-being improved and fatigue scores moved toward those of healthy people, and he reported no new limitations or side effects from training.

      “Functional well-being 16 19 24 Fatigue subscale 43 45 49”Table I; Discussion

    What the authors concluded

    The authors conclude that long-term regular exercise was feasible and, in their words, effective for this patient, who reached fitness, strength and body composition comparable to healthy or athletic people. They describe the results as preliminary and call for studies in more patients.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • MajorOne person, no comparison: cannot show exercise was effective

      Stable strength over five years in one person could reflect a slow natural course rather than the training. The authors admit the natural history of SPSMA is poorly known, which makes any effect impossible to separate from what would have happened anyway. The word 'effectiveness' in the conclusion goes further than one uncontrolled case can support; 'feasibility' is better supported.

      “This study has shown the long-term feasibility and effectiveness of regular PE in a patient affected by TRPV4 related SPSMA”Conclusion

    • ModerateAn unusually motivated, already active patient

      He was already training on his own before joining the program and was described as highly trained. His fitness and experience may not reflect what other people with TRPV4 conditions, especially those with more weakness, could achieve or tolerate.

      “Since age 20, the patient has been independently exercising at a local gym”Case report, Patient

    • ModerateCoarse or indirect measures, assessed without blinding

      The MRC score is a rough strength scale and here covers six muscle groups per side. Phase angle is used as a stand-in for muscle mass and quality, not a direct measure. The paper does not say the assessors were blinded, and they were the same team that prescribed the exercise. The rise in MRC score from 45 to 52 is not explained.

      “PhA is proposed as a proxy indicator of skeletal muscle mass and quality, with higher values reflecting better muscle condition”Materials and methods, Body composition evaluation

    • ModerateGenetic diagnosis and nerve testing thinly described

      The variant is named but the paper does not say how it was classified, whether family members were tested, or whether other causes were excluded. EMG and nerve conduction studies were normal at 3 months of age and no later nerve tests are reported, so the diagnosis rests on the clinical pattern plus the variant.

      “harbouring a heterozygous missense mutation in TRPV4 gene: c.281C > G/p.S94W”Case report, Patient

    • MinorFollow-up timing does not quite add up

      Two gaps of 25 months make about 4 years and 2 months between the first and last laboratory tests, not five years, and the stated ages (21, 23, 26) do not match 25-month steps. The overall clinical follow-up may be longer, but the exact timing is unclear.

      “at the ages of 21, 23, and 26 years (rounded to the nearest year), he underwent three laboratory assessments (25 months apart)”Results

    • MinorExplanation for the fall in fitness is speculative

      VO2max fell from 51.9 to 43.8 mL/min/kg at the last test. The authors attribute this to a two-month break in training rather than disease progression, and say themselves this is speculative. Disease progression cannot be ruled out from these data.

      “although speculative in nature, would suggest that such decrease is physiological and does not appear to be mediated by disease progression”Discussion

    Three ways to explain it

    Scientific

    This single-patient longitudinal report describes TRPV4 (p.S94W) scapuloperoneal SMA in a man followed from about 21 to 26 years of age during supervised combined resistance and endurance training. MRC sum score (45, 52, 52), handgrip strength, VO2max (50.0, 51.9, 43.8 mL/min/kg) and phase angle were maintained or improved, with no reported adverse effects. Without a control or established natural history, the data support feasibility in this individual but cannot establish a disease-modifying effect.

    Easier

    This report follows one young man with a TRPV4-related condition that weakens the muscles around the shoulder blades and lower legs. He trained at a gym with professional guidance for about five years. His strength and sense of well-being stayed the same or got better, his fitness dipped only after a two-month break in training, and he reported no harmful effects. Because only one person was studied and there was no comparison group, it is not possible to say whether the exercise slowed the condition.

    Simple

    One young man with a TRPV4 condition went to the gym regularly for five years and stayed strong. That is good news for him, but from just one person we cannot tell whether exercise would help everyone.

    Why these ratings

    Relevance 3/5: About scapuloperoneal SMA, another condition caused by TRPV4 changes, and about exercise, which is of practical interest to people with CMT2C, but it describes only one patient.

    Evidence 2/5: A single case without a comparison group, measured by the team that designed the program.

    Funding and conflicts of interest

    No external funding. The authors declare no conflicts of interest.

    PubMed 41199732 · DOI

60. #60Full paperNarrative review

    Roles for TRPV4 in disease: A discussion of possible mechanisms

    Hernández-Vega AM, García-Villegas R, Rosenbaum T · 2024 · Cell calcium

    Relevance 3/5Evidence 2/53 moderate · 2 minor problems

    This review explains the main laboratory ideas about how TRPV4 changes may harm nerves, chiefly too much calcium entering cells and disturbed links with a protein called RhoA. Most of this comes from cells and mice, so it describes possible mechanisms rather than proven effects in people with CMT2C.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    Narrative review

    Who or what

    Published research on the TRPV4 channel: its structure, how it is controlled, where in the body it is found, and reported human mutations. Three tables list mutations linked to skeletal disorders (Table 1), nerve and muscle disorders including CMT2C (Table 2) and, possibly, polycystic kidney disease (Table 3). No new patients or experiments.

    What they did

    The authors summarized earlier studies of TRPV4 structure, regulation and calcium handling. They listed reported mutations with what is known about how each changes channel activity, and discussed possible disease mechanisms in bone and cartilage, peripheral nerve and kidney. They ended by presenting their own group's hypothesis that TRPV4 changes may influence polycystic kidney disease.

    What they found

    • The review reports that more than eighty TRPV4 mutations have been described in people, and that most disease-linked mutations make the channel more active (gain of function), mainly causing skeletal and nerve or muscle conditions.

      “mutations in the TRPV4 channel have been associated to a broad spectrum of congenital diseases, with most of these mutations mainly resulting in gain-of-function”Abstract; Introduction ('To date, over eighty mutations have been described')

    • Summarizing lab studies, the review says neuropathy-causing mutant channels damage nerve cells and their fibers, an effect linked to raised calcium inside cells and to disrupted transport of mitochondria along axons.

      “The expression of neuropathy-causing GOF mutant TRPV4 channels results in neurotoxicity and axonal and dendritic degeneration”Section 4, 'TRPV4 and neuromuscular disease'

    • Mutations in the ankyrin repeat domain (the part of TRPV4 where most CMT2C mutations sit) are described as interfering with TRPV4's binding to RhoA, a protein that controls the cell skeleton.

      “Mutations in the ARD of TRPV4 lead to neuropathies [35,192] through the interruption of binding of RhoA to TRPV4 [60]”Section 4, 'TRPV4 and neuromuscular disease'

    • The review cites a mouse study in which mice carrying R232C or R269C mutations developed motor neuron loss linked to a leaky blood-spinal cord barrier, and in which TRPV4-blocking molecules prevented early death in R269C mice.

      “treatment of mice expressing the R269C mutation with small TRPV4 antagonist molecules [198] resulted in rescue from lethality due to protection of BSCB integrity [118]”Section 4, 'TRPV4 and neuromuscular disease'

    • The authors stress that too much calcium alone does not fully explain how TRPV4 mutations cause nerve disease, and that more precise mechanisms are only starting to be understood.

      “the neurotoxicity caused by excess [Ca2+]i levels does not fully explain the pathophysiological characteristics of these neuropathies”Section 4, 'TRPV4 and neuromuscular disease'

    What the authors concluded

    TRPV4 is found throughout the body and helps control calcium inside cells, and its mutations cause a widening range of conditions, mostly of the skeleton and peripheral nerves. Cell and animal models support gain of function as a main mechanism, but there are gaps between what is seen in isolated cells and in the whole body, and the links between specific mutations and the conditions they cause are not yet understood. The authors also propose, from their own small study, that gain-of-function TRPV4 changes may affect polycystic kidney disease.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • ModerateReview, not new data, with no search method

      This article summarizes other studies. It does not say how studies were found or chosen, so it may not cover the evidence evenly, and readers should go to the original studies for firm conclusions.

      “No data was used for the research described in the article.”Data availability

    • ModerateMechanisms come mainly from cells and mice

      Most mechanism evidence summarized comes from cell lines made to produce mutant channels, often switched on with synthetic chemicals, and from mice. The authors acknowledge this may not reflect what happens in a whole human body. The review cites no evidence that the drug rescue seen in mice works in people.

      “there are still experimental limitations regarding what is measured in isolated cell cultures and what could be happening in a complete physiological system”Section 6, 'Conclusions and future perspectives'

    • MinorMutation tables do not grade evidence

      Tables 1 and 2 label each mutation as gain of function, loss of function, normal or undefined, but some labels rest on single experiments in non-nerve cell lines (for example HeLa cells), and many are 'Undefined'. The tables do not say how strong the evidence is that each variant causes disease, so they should not be read as a definitive variant list.

      “In transiently transfected HeLa cells [288].”Table 2, footnote

    • ModerateKidney hypothesis stated too strongly

      The polycystic kidney disease section relies on one small study by the authors' own group, yet says the data 'strongly suggest' a role for TRPV4. One small association study cannot establish this; the authors themselves call for larger studies and animal models. This does not affect the CMT2C content.

      “Although this report is the first and only association of TRPV4 mutations to PKD severity, the data strongly suggest the participation of TRPV4 in the development of this sickness”Section 5.3, 'TRPV4 and ADPKD'

    • MinorConfusing section heading

      Section 3 is titled 'skeletal muscle disorders' but discusses bone and cartilage conditions (skeletal dysplasias), not muscle. Readers could wrongly think it is about muscle disease.

      “3. TRPV4 and skeletal muscle disorders”Section 3 heading

    Three ways to explain it

    Scientific

    This narrative review summarizes TRPV4 structure, regulation and tissue expression and catalogs reported skeletal, neuromuscular and putative PKD-associated variants, most characterized as gain of function. For neuropathy it cites evidence that mutant channels raise intracellular Ca2+, disrupt axonal mitochondrial transport and RhoA binding at the ankyrin repeat domain, and, in R232C and R269C knock-in mice, impair blood-spinal cord barrier integrity, with antagonist rescue of lethality in R269C mice. The authors note that Ca2+ excess does not fully explain the phenotypes and that most data come from heterologous expression systems.

    Easier

    This article reviews earlier research rather than reporting new experiments. TRPV4 is a channel, a tiny gate in the cell surface that lets calcium in, and most disease-causing changes seem to make this gate too active. In cells grown in the lab and in mice, this was linked to damaged nerve fibers, and in one mouse study a TRPV4-blocking drug prevented early death. The authors say extra calcium does not explain everything and that results from lab cells may not match the whole human body.

    Simple

    TRPV4 is like a door that lets calcium into cells. Many TRPV4 changes seem to leave the door open too much, which harmed nerve cells in lab dishes and in mice. Scientists still do not know exactly how this works in people.

    Why these ratings

    Relevance 3/5: A broad TRPV4 mechanism review in which nerve disease, including CMT2C, is one section among several, with much of the space given to bone and kidney.

    Evidence 2/5: A narrative review with no stated method for choosing studies, summarizing mostly cell and mouse work, plus a kidney hypothesis resting on one small study by the same group.

    Funding and conflicts of interest

    T.R. was supported by a grant from the Secretaría de Educación, Ciencia, Tecnología e Innovación (SECTEI) of the Government of Mexico City (SECTEI/208/2019); the first author held a CONAHCYT doctoral scholarship and wrote the review as part of a doctoral program. The authors declare no competing financial interests.

    PubMed 39609180 · DOI

61. #61Full paperLaboratory study in a rat cell line, rat nerve cells and mouse tissue

    Axonal neuropathy-associated TRPV4 regulates neurotrophic factor-derived axonal growth

    Jang Y, Jung J, Kim H et al. · 2012 · The Journal of biological chemistry

    Relevance 3/5Evidence 2/51 major · 3 moderate · 2 minor problems

    This lab study suggests TRPV4 normally helps nerve fibers grow, which helps explain why the channel matters for nerves. It did not test the gene changes that cause CMT2C, so it says little directly about what happens in people with the condition.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    Laboratory study in a rat cell line, rat nerve cells and mouse tissue

    Who or what

    PC12 cells (a rat cell line used as a model of nerve cells), sensory nerve cells (dorsal root ganglion neurons) taken from newborn rats, and spinal cord, nerve ganglion and muscle tissue from newborn and adult mice. Normal human TRPV4 and one loss-of-function form (P19S) were tested. No CMT2C-causing TRPV4 variants and no human patients were studied.

    What they did

    They looked at where TRPV4 is found in mouse sensory and motor nerve cells. They then increased, reduced, or chemically switched TRPV4 on or off in rat cells grown in dishes and measured how long the cells' nerve fibers (neurites) grew. They also tested which signals (nerve growth factor, cAMP and the enzyme phospholipase A2) act upstream of TRPV4, and whether TRPV4 activation changes the cell skeleton (actin) and how well cells stick to a surface.

    What they found

    • TRPV4 was found in sensory nerve cells and large motor nerve cells of the spinal cord in mice, and at the junction between nerve and muscle.

      “These results suggest that TRPV4 is highly expressed in sensory and motor neurons in neonatal as well as adult mice.”Results, TRPV4 Was Highly Expressed in Developing Peripheral Neurons; Figure 1

    • Reducing TRPV4 with siRNA shortened nerve fibers and reduced the share of cells growing them. Adding extra normal TRPV4 increased fiber growth, while the loss-of-function form did not.

      “TRPV4 siRNA significantly reduced ( p < 0.001, n = 222-318) neurite length and the percentage of PC12 cells with neurites”Results, Effects of Knockdown and Overexpression of TRPV4 on Neuritogenesis; Figure 3

    • Chemicals that switch TRPV4 on increased nerve fiber growth in rat cells, while TRPV4 blockers reduced it.

      “Treatment with 10 μ m 4α-PDD or AA significantly increased neurite length and the percentages of cells bearing neurites in NGF-treated PC12 cells and DRG neurons”Results, Pharmacological Perturbation of TRPV4 Alters Neuritogenesis; Figure 4

    • Nerve growth factor and cAMP raised the levels of TRPV4 and the enzyme phospholipase A2, and blocking this enzyme reduced fiber growth, suggesting a pathway that runs from the growth factor to phospholipase A2 to TRPV4.

      “when PC12 cells were treated with NGF, the PLA 2 and TRPV4 transcript levels were markedly increased”Results, NGF-induced Neuritogenesis Required TRPV4 Activity via PLA2 Pathway; Figures 5 and 6

    • Switching TRPV4 on increased actin (part of the cell skeleton) and made cells stick better to the dish.

      “When PC12 cells were treated 10 μ m 4α-PDD or 300 n m 5,6-EET for 24 h, the number of attached cells after shaking for 1 h was significantly increased ( p < 0.01, n = 15)”Results, TRPV4 Increased Actin Polymerization and Cell Adhesion; Figure 7

    What the authors concluded

    TRPV4 helps nerve growth factor and cAMP drive the growth of nerve fibers in developing sensory and motor nerve cells, acting through a phospholipase A2 pathway. Because growth factors are needed to keep peripheral nerves healthy, the authors suggest that abnormal TRPV4 activity could lead to some types of inherited sensory and motor nerve disease.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • MajorNo disease-causing variants tested

      The paper studies normal TRPV4 and one loss-of-function form, but none of the variants that cause CMT2C. It therefore shows what TRPV4 normally does in growing nerve cells in a dish, not what goes wrong in the disease, despite a title that names axonal neuropathy.

      “Twenty-four hours after transfection with human TRPV4 wild type or mutant TRPV4 P19S”Experimental Procedures, Calcium Imaging

    • ModerateFindings not reconciled with the gain-of-function idea

      The paper's own background says disease variants make TRPV4 overactive and cause calcium overload. Its results show that more TRPV4 activity helps nerve fibers grow. The paper does not explain how an overactive channel would damage nerves under its model, so the link to disease remains a suggestion.

      “Neurodegeneration caused by Ca 2+ overload due to the gain-of-function mutation of TRPV4 was suggested as the molecular mechanism for the neuropathies.”Abstract

    • ModerateRodent cells in dishes, far from human nerves

      Most experiments used PC12 cells, a rat cell line, and nerve cells from newborn rats grown for days in culture. Fiber growth in a dish is a model of early nerve development and may not reflect how adult human nerves behave over many years.

      “Dorsal root ganglion (DRG) neurons were dissected in neonatal rats”Experimental Procedures, Cell Culture

    • ModerateConclusion goes beyond the experiments

      In adult mouse tissue, the study only looked at whether TRPV4 is present; no experiment tested whether TRPV4 keeps adult nerves healthy. The paper also says the adhesion result 'confirmed' that TRPV4 acts downstream of nerve growth factor, although that effect was seen without nerve growth factor.

      “The present study shows that TRPV4 is essential for axonal growth of sensory/motor nerves in early development as well as for their maintenance in the adult.”Discussion

    • MinorInconsistencies in labeling and methods

      The Figure 1 legend calls tissue both adult and P1 (newborn); the results describe newborn spinal cord while the legend says adult; the MEK inhibitor is spelled three different ways; and cortical neuron measurements are described in the methods but no cortical results are shown. These suggest limited checking, though they do not overturn the main results.

      “B, immunostaining of adult mouse ( P1 ) DRG neurons with anti-TRPV4”Figure 1 legend

    • MinorNumber of independent experiments not stated

      The n values are counts of individual cells. The paper does not say how many separate cultures or repeat experiments these came from, so it is hard to judge how reproducible the differences are.

      “Percentages of cells bearing neurites were determined by counting more than 100 cells in randomly chosen fields.”Experimental Procedures, Measurement of Neurite Lengths

    Three ways to explain it

    Scientific

    In rat PC12 cells and neonatal rat DRG neurons, TRPV4 overexpression or agonists (4α-PDD, 5,6-EET, arachidonic acid, GSK1016790A) enhanced NGF-driven neuritogenesis, whereas siRNA knockdown or antagonists (HC-067047, ruthenium red) reduced it. NGF and cAMP upregulated PLA2 and TRPV4 through a MEK-dependent pathway, and TRPV4 activation increased F-actin and cell adhesion. No neuropathy-causing TRPV4 variants were examined, so the relevance to CMT2C pathogenesis remains inferential.

    Easier

    In mice, TRPV4 is found in the nerve cells that control movement and feeling. In rat cells grown in dishes, turning TRPV4 up or switching it on with chemicals made nerve fibers grow longer, and turning it down made them shorter. The study did not test the gene changes that cause CMT2C, so it shows what TRPV4 normally does rather than what goes wrong in the disease.

    Simple

    TRPV4 is like a door that lets calcium into nerve cells. In rat cells in a dish, opening the door more helped the nerve's long arms grow, and closing it slowed them down. But the scientists did not test the faulty door that causes CMT2C.

    Why these ratings

    Relevance 3/5: It studies the normal role of TRPV4 in nerve cells, motivated by CMT2C, but tests no disease-causing variants.

    Evidence 2/5: Cell and rodent work far from people, with no disease variants tested and several reporting inconsistencies.

    Funding and conflicts of interest

    Supported by Grant R31-2008-000-10103-0 from the World Class University program and Creative Research Initiatives Program Grant 20110018358 of the Ministry of Education Science and Technology and the National Research Foundation of Korea. Competing interests: not stated.

    PubMed 22187434 · DOI

62. #62Full paperCase report of two unrelated patients, with genetic testing and computer-based structural modeling

    De novo TRPV4 Leu619Pro variant causes a new channelopathy characterised by giant cell lesions of the jaws and skull, skeletal abnormalities and polyneuropathy

    Ragamin A, Gomes CC, Bindels-de Heus K et al. · 2022 · Journal of medical genetics

    Relevance 3/5Evidence 2/55 moderate · 2 minor problems

    This paper describes two women with a rare TRPV4 change near the channel's pore. Like some people with CMT2C, they had nerve damage, vocal cord weakness and hearing loss, but they also had aggressive bone lesions in the jaw and skull. It widens the range of known TRPV4 conditions; the bone lesions were linked to this specific change and are not described here as part of typical CMT2C.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    Case report of two unrelated patients, with genetic testing and computer-based structural modeling

    Who or what

    Two unrelated women, both aged 20, one Dutch and one Brazilian, each with a new (de novo) TRPV4 change that alters amino acid 619 from leucine to proline (p.Leu619Pro). They were linked through GeneMatcher. Both had exome sequencing with their parents; the Brazilian woman also had sequencing of jaw lesion tissue.

    What they did

    The authors describe both women's medical histories from birth to age 20. They report the genetic tests that found the TRPV4 change in each woman and not in her parents, and the proportion of DNA reads carrying it in blood and in jaw lesions. They used a published 3D structure of frog TRPV4 to predict how the change might affect the channel; no laboratory tests of the channel were done.

    What they found

    • Both women had a combination of nerve problems and repeated, aggressive giant cell bone lesions of the jaws and skull, along with other skeletal changes.

      “From an early age, both patients had several lesions of the nervous system including progressive polyneuropathy, and multiple aggressive giant cell-rich lesions of the jaws and craniofacial/skull bones, and other skeletal lesions.”Abstract, Results

    • Features shared with TRPV4 neuromuscular disease included vocal cord paresis (needing a tracheostomy at 8 months in the Dutch woman), hearing loss and pes cavus (high-arched feet) in both women. In the Brazilian woman, nerve tests showed motor nerve fiber damage in the arms and legs, and no sensory abnormality was found on examination.

      “Nerve conduction studies demonstrated motor axonal damage in the upper and lower limbs.”Results, Subject 2

    • In both women the change was absent from both parents. In the Brazilian woman it was classed as likely pathogenic under American College of Medical Genetics rules and was not found in population databases.

      “According to American College of Medical Genetics recommendations, this variant was classified as likely pathogenic.”Results, Subject 2

    • In the milder case the change was found in only 21.6% of blood DNA reads, and in 42% and 10% of reads from two jaw lesions, which the authors interpret as the change being present in only some of her cells (mosaicism).

      “The low VAF for this novel variant is suggestive of postzygotic somatic mosaicism”Results, Subject 2

    • The jaw and skull lesions came back repeatedly after surgery and seemed to become worse each time. Pamidronate eased pain and slowed the process in one woman but did not help the other.

      “the giant cell-rich lesions affecting the jaws and skull of both patients recurred several times and, unfortunately, they seem to worsen with each new surgical intervention.”Discussion

    • Computer modeling placed Leu619 in the S5 helix next to the channel's pore and predicted that the change would increase ion flow through the channel.

      “Based on this analysis, the Leu619Pro mutation is predicted to trigger S6 movement, allowing for increased ion permeability.”Results, TRPV4 variant modelling

    What the authors concluded

    The authors conclude that the de novo TRPV4 p.Leu619Pro change causes a newly recognized syndrome combining aggressive giant cell lesions of the jaws and skull, skeletal changes and polyneuropathy. They also conclude that mosaicism may explain the milder case, and that MEK inhibitors or TRPV4 blockers might be worth exploring because surgery seemed to make the lesions worse.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • ModerateA new syndrome defined from two people

      Finding the same new change, absent in parents, in two unrelated women with similar features is good evidence that it matters. But describing a whole syndrome, and attributing every feature to TRPV4, from two cases is provisional; some features may be chance or have other causes.

      “Our findings define a novel polysystemic syndrome due to germline TRPV4 p.Leu619Pro”Abstract, Conclusion

    • ModerateGain of function predicted, not tested

      The idea that the change makes the channel leak comes from modeling on a frog (Xenopus tropicalis) TRPV4 structure and from an earlier study of the rat channel in frog eggs. The human channel carrying this change was not tested in this paper.

      “structure of TRPV4 from Xenopus tropicalis , which shares a 78% sequence identity with the human protein”Results, TRPV4 variant modelling

    • ModerateNeuropathy in the first patient not confirmed by nerve tests

      No nerve conduction results are reported for the Dutch woman. Her 'polyneuropathy' rests on examination findings at age 15, and tongue and hand wasting were attributed to motor neuron disease. She also had a spinal cord malformation (meningomyelocele with syrinx and tethered cord), which can itself cause leg and bladder signs.

      “impaired sensibility, minimal gnostic impairment and low reflexes of the lower extremities, interpreted as signs of polyneuropathy”Results, Subject 1

    • ModerateOther genetic causes not fully excluded in the first patient

      Her exome analysis was limited to a panel of 2753 genes linked to multiple congenital anomalies (plus earlier tests of SH3BP2, KRAS, BRAF and PTPN11). A second genetic cause for features such as the spinal malformation cannot be ruled out from what is reported.

      “in a panel of 2753 genes believed to be involved in multiple congenital anomalies”Methods, Genetic analyses, Subject 1

    • MinorMosaicism interpretation rests on small read counts

      The first patient's variant was seen in 19 of 26 reads (73%), above the 50% expected for a simple heterozygous change, which is not discussed. The second patient's 21.6% came from 11 of 51 reads. With such small numbers the percentages are imprecise, so mosaicism, and its link to the milder illness, is plausible but not proven.

      “The total number of reads for this codon was 26, with seven wild-type reads and 19 times delinsCT (VAF=73%).”Results, Subject 1

    • ModerateTreatment statements go beyond the evidence

      The abstract states that surgery worsens the lesions, based on the course of two patients without any comparison. MEK inhibitors and TRPV4 antagonists are suggested, but neither was tried in these patients and no evidence of benefit for this condition is presented.

      “we assume that this targeted treatment could be potentially useful in our subjects, reducing the need for additional surgical treatments.”Discussion

    • MinorSmall reporting errors

      The second patient's neck vertebra findings are referred to figure 1H, but figure 1 shows the first patient and the matching image is figure 3H. The ethics section says patient consent for publication was not required, while the methods say written consent for images was obtained. These do not affect the conclusions.

      “including C1 ( figure 1H ).”Results, Subject 2; Ethics statements

    Three ways to explain it

    Scientific

    Two unrelated women carried de novo TRPV4 variants producing p.Leu619Pro in the S5 transmembrane helix and shared a multisystem phenotype: recurrent aggressive giant cell-rich lesions of the jaws and skull, skeletal anomalies, vocal cord paresis, hearing loss and polyneuropathy (motor axonal on nerve conduction in one). A low variant allele fraction in the milder case (21.6% in blood) was interpreted as postzygotic mosaicism. Gain of function was inferred from structural modeling and earlier work rather than tested, so the genotype-phenotype link rests on two cases.

    Easier

    Two young women in different countries had the same new change in the TRPV4 gene, which neither set of parents had. Both had nerve damage, weak vocal cords, hearing loss and skeletal problems, plus bone growths in the jaw and skull that kept coming back after surgery. One woman may carry the change in only some of her cells, which could explain why she was less severely affected. The researchers predicted from a computer model that the change makes the channel leak, but did not test this in the lab.

    Simple

    TRPV4 is like a door that lets calcium into cells. Two women had a change near the opening of the door that might make it leak, and they had problems with their nerves, hearing and face bones. Doctors know of only two people with this change, so there is still a lot to learn.

    Why these ratings

    Relevance 3/5: Describes a different TRPV4 condition that combines neuropathy, vocal cord paresis and hearing loss with skeletal and jaw lesions, widening the TRPV4 spectrum rather than describing typical CMT2C.

    Evidence 2/5: Two well-documented cases, each sequenced along with both parents, but no lab test of how the variant affects the channel, and a new syndrome defined from two people.

    Funding and conflicts of interest

    No specific grant was declared for this research. Competing interests: two authors (RG and CCG) are research fellows at the National Council for Scientific and Technological Development, Brazil.

    PubMed 33685999 · DOI

63. #63Full paperSingle-family case report with exome sequencing and cell experiments

    Homozygous TRPV4 mutation causes congenital distal spinal muscular atrophy and arthrogryposis

    Velilla J, Marchetti MM, Toth-Petroczy A et al. · 2019 · Neurology. Genetics

    Relevance 3/5Evidence 2/51 major · 3 moderate · 2 minor problems

    This report shows that TRPV4 disease can, at least in one child, come from inheriting the same change from both parents, giving a severe form present from birth. Whether people with one copy of this particular change have any signs was not tested by examination.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    Single-family case report with exome sequencing and cell experiments

    Who or what

    One family of Puerto Rican descent: a 2-year-old boy (the only affected person), his parents and his sister. HEK293 cells made to produce normal TRPV4 or the variants S94L, R269C, R315W and T701I (n = 3 experiments for cell death and protein levels).

    What they did

    The team sequenced the exomes of the boy and his parents, filtered the results for rare changes fitting the family pattern, and confirmed the leading candidate by Sanger sequencing in all four family members. They modeled where the change sits in the TRPV4 structure. In HEK293 cells they measured protein levels, cell death (with and without the TRPV4 blocker HC-067047) and where the channel sat in the cell.

    What they found

    • The boy was born with multiple joint contractures, hip and foot deformities and torticollis (a twisted neck), and his vocal cords did not move on either side.

      “Vocal cords were clearly visible and appeared to be immobile bilaterally in the paramedian position.”Results, clinical presentation

    • Nerve tests showed a motor nerve problem with normal sensory responses, so he was diagnosed with congenital spinal muscular atrophy and arthrogryposis (CSMAA).

      “The electrophysiologic findings are suggestive of generalized motor axonopathy with coexisting denervation and reinnervation changes.”Results, clinical presentation

    • Exome sequencing found the TRPV4 change p.S94L on both copies of the gene in the boy. Each parent carried one copy, and his unaffected sister carried none. The other 14 candidate genes were ruled out.

      “Sanger sequencing confirmed the homozygosity of this variant in the proband (BGM0049-1) and heterozygous inheritance from both parents”Results, whole-exome sequencing; Figure 1B

    • The change is rare in the general population, with no one recorded as carrying two copies.

      “only 6 heterozygous and no homozygous individuals are reported in a control population of 246210 people”Discussion

    • In HEK293 cells, S94L increased cell death and lowered protein levels compared with normal TRPV4, and the TRPV4 blocker HC-067047 reversed both. The effect was smaller than with the known neuropathy changes R269C and R315W.

      “Quantification showed significantly higher levels of cell death in the p.S94L transfected cells in comparison to WT TRPV4-transfected cells.”Results, functional modelling; Figure 3

    • A different reported neuropathy change, T701I, did not increase cell death but was held near the nucleus instead of reaching the cell surface, suggesting different TRPV4 changes may act in different ways.

      “Interestingly, the p.T701I mutant protein showed a perinuclear localization pattern, and in contrast to WT protein, no protein was detected on the plasma membrane”Results, functional modelling; Figure 4

    What the authors concluded

    Two copies of the TRPV4 change p.S94L cause a recessive form of congenital spinal muscular atrophy with arthrogryposis. The authors interpret the cell results as gain of function, suggest that one copy has reduced penetrance, and note that different TRPV4 changes may disturb the channel in different ways.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • MajorCarrier parents called healthy without described examination

      The parents each carry one copy and are described as healthy with no phenotype, which the authors use to suggest reduced penetrance of one copy. No neurological examination or nerve conduction tests of the parents are described. Mild signs could be missed without examination, so the penetrance suggestion is not well supported. The authors do advise neurology follow-up for the parents.

      “the proband's parents who are heterozygous for the TRPV4 p.S94L variant did not exhibit any phenotype”Discussion

    • ModerateGain of function claimed without measuring channel activity

      No calcium measurements or electrical recordings were made. The gain-of-function conclusion is inferred from cell death and protein levels that change with a TRPV4 blocker. This fits overactivity but does not show it directly.

      “Functional studies by Western blot and immunohistochemical analysis show that p.S94L increased TRPV4 activity-based cytotoxicity and resultant decreased TRPV4 expression levels, therefore involves a gain-of-function mechanism.”Abstract

    • ModerateCausal claim from one family

      The title and conclusion state that this change causes the disease. The genetic fit is good, but the evidence comes from one affected child; confirmation in other unrelated families would be needed for a firm causal claim.

      “This study identifies a novel homozygous mutation in TRPV4 as a cause of the recessive form of CSMAA.”Abstract, conclusions

    • ModerateKidney-derived cell line and few repeats

      All cell work used HEK293 cells given extra TRPV4, with three repeats per test. The authors acknowledge these are not nerve-like cells, so how well the results reflect nerve cells is uncertain.

      “A limitation of this work is that these studies were performed in HEK293 cells rather than neuronal-like cells.”Discussion

    • MinorSkeletal versus nerve origin unclear

      The boy had both major skeletal problems and nerve findings. The authors note it is hard to know which drives the contractures, which makes the classification of his condition less certain.

      “it is hard to predict which of these contributed to primary disease pathology”Discussion

    • MinorSmall inconsistencies

      The clinical section reports moderate conductive hearing loss with middle ear fluid, but the next paragraph says he has normal hearing. The structure file is given as 6bdj in Methods and 6bbj in the Figure 2 legend, a variant is written 'p.R314W' once, and the abstract says 'immunohistochemical' for what Methods describe as immunofluorescence in cells.

      “Visual reinforced audiometry showed a moderate conductive hearing loss for at least 1 ear at 500 and 2000 Hz.”Results, clinical presentation; Methods; Figure 2 legend

    How “unaffected” relatives were checked

    The parents are described as 'unrelated healthy parents' who 'did not exhibit any phenotype'; no neurological examination or nerve conduction tests of either parent are described, yet their status is used to suggest reduced penetrance of one copy. The sister is described as unaffected and does not carry the change. Quote: 'The male proband was born to unrelated healthy parents of Puerto Rican descent after 37 weeks of gestation' (Results, clinical presentation).

    Three ways to explain it

    Scientific

    Exome sequencing in a boy with congenital distal SMA with arthrogryposis, bilateral vocal cord immobility and normal sensory responses identified homozygous TRPV4 p.S94L in the N-terminal disordered region, with both parents heterozygous and an unaffected sister homozygous for the reference allele. In HEK293 cells S94L increased cytotoxicity and lowered protein levels, both reversed by the TRPV4 antagonist HC-067047, which the authors interpret as gain of function, although channel activity was not measured directly. They propose a recessive form of the disease and reduced penetrance in heterozygotes, but the parents' status is not described as based on examination.

    Easier

    Most TRPV4 conditions happen when one copy of the gene is changed. This boy had the same change in both copies, one from each parent. He was born with stiff, bent joints, could not move his vocal cords and had weak legs. In lab cells the changed channel harmed the cells, and a TRPV4 blocker stopped this. The parents, who each carry one copy, were described as healthy, but the paper does not say they were examined.

    Simple

    Everyone has two copies of the TRPV4 instructions. This boy got the same small mistake from both his mom and his dad, and he was born with stiff joints and weak legs. In a dish, the faulty calcium door harmed cells, and a door-blocking chemical stopped it. His parents each have one faulty copy and seem well, but the paper does not say anyone checked their nerves.

    Why these ratings

    Relevance 3/5: It concerns congenital distal SMA, another condition caused by TRPV4 changes, and is unusual for showing a two-copy (recessive) form, with some lab comparison against CMT2C-linked changes.

    Evidence 2/5: A single affected child with good genetic work-up, but the causal claim rests on one family and limited cell tests that did not measure channel activity directly.

    Funding and conflicts of interest

    Funded by K01 AR062601, the Eleanor and Miles Shore Fellowship and a Brigham and Women's Hospital Career Development Award (V.A.G.), a Brigham Biomedical Research Institute Director's Transformative Award (Brigham Genomic Medicine) and the American Heart Association 16GRNT27250119 (R.G.). Disclosures: B.T. Darras reports advisory board roles, honoraria and research support from several drug companies (including Hoffmann-La Roche, Biogen, Sarepta, AveXis, PTC Therapeutics and Cytokinetics); N.Y. Frank holds patents licensed to companies and advises Veritas Genetics; R. Gaudet's spouse is employed by and holds stock in Bristol-Myers Squibb. None of these is described as related to TRPV4.

    PubMed 31041394 · DOI

64. #64Full paperCase report

    Homozygous TRPV4 Mutation Broadens the Phenotypic Spectrum of Congenital Spinal Muscular Atrophy and Arthrogryposis: A Case Report

    Lugo E, Graulau E, Ramos Cortes E et al. · 2023 · Cureus

    Relevance 3/5Evidence 2/51 major · 2 moderate · 3 minor problems

    This report describes one child with two copies of a TRPV4 change who had a motor-only condition present from birth, which is different from typical CMT2C. It cannot tell us whether people with one copy of this change are truly unaffected, because the parents' assessments are not described.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    Case report

    Who or what

    One boy born to unrelated, healthy parents of Puerto Rican descent, first seen at six days old and described up to about two years of age, with two copies (homozygous) of the TRPV4 variant c.281C>T; p.S94L. Both parents carry one copy (heterozygous).

    What they did

    The authors describe the boy's clubfeet and stiff knees at birth, their treatment with casting and Achilles tendon surgery, and his development. He had a bone survey, a 358-gene panel for skeletal disorders, nerve conduction studies and needle electromyography (EMG). They compare him with one earlier reported child with the same variant.

    What they found

    • The boy was born with stiff knees (bilateral knee arthrogryposis) and clubfeet, which needed casting and surgery and came back once. At two years he could sit and walk on his knees but could not stand or walk on his own, while his arms, speech, hearing and vision were normal.

      “At two years of age, the patient could sit independently and walk on his knees. However, he was unable to stand or walk independently.”Case presentation

    • Gene panel testing found two copies of the TRPV4 change c.281C>T (p.S94L). Both parents carry one copy and are reported to have no signs of disease.

      “The parents are heterozygous for the same mutation, without any clinical manifestations.”Case presentation

    • Sensory nerve tests (right median and right sural) were normal, the right peroneal motor nerve had a low response size (0.625 mV at the ankle) with normal speed, and needle EMG of two leg muscles showed signs of denervation. The authors read this as a pure motor neuron disease rather than CMT.

      “Electromyography studies were performed, revealing evidence of a pure motor neuron disease, ruling out the possibility of a neuropathy (CMT)”Case presentation; Tables 1 to 3

    • Unlike the earlier child with the same variant, this boy had no vocal cord paralysis, torticollis, hearing loss or skeletal dysplasia; a bone survey showed no bone dysplasia.

      “In contrast to Velilla's patient, our subject did not exhibit vocal cord paralysis, torticollis, sensorineural hearing loss, or skeletal dysplasia.”Discussion

    • Both known children with this homozygous variant were born to unrelated healthy parents of Puerto Rican descent, and the authors raise the possibility of a founder effect (a change inherited from a shared ancestor in that population).

      “This suggests the possibility of a founder effect.”Discussion

    What the authors concluded

    The boy has congenital spinal muscular atrophy and arthrogryposis caused by a homozygous TRPV4 variant. His condition differs from the one previous case, which widens the known range of TRPV4 conditions. The authors say links between TRPV4 mutations and symptoms are not uniform, and that genetic testing is needed for a definite diagnosis.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • MajorParents called unaffected without described examination

      The parents are described as healthy carriers with no clinical signs, and the discussion calls them 'unaffected carriers'. The paper does not report a neurological examination or nerve conduction tests for either parent, so it cannot show that carrying one copy of this variant causes no problems.

      “The parents are heterozygous for the same mutation, without any clinical manifestations.”Case presentation; Discussion

    • ModerateContradiction: 'first case' or 'second case'

      The abstract calls this the first case of this condition caused by p.S94L, but the discussion describes an earlier 2019 report of the same homozygous variant with the same diagnosis and calls this the second case. The novelty claim in the abstract is therefore not supported by the paper's own text.

      “Hence, this presents the first case of CSMAA caused by a TRPV4 mutation (p.S94L)”Abstract (compare Discussion: 'Our patient represents the second case')

    • MinorBackground statement contradicts later text

      The introduction says the TRPV4 channel is impermeable to calcium, while the discussion explains disease through increased calcium entry through the channel. This inconsistency suggests the background section should be read with caution.

      “encodes a cation channel that is selectively impermeable to calcium”Introduction (compare Discussion: 'heightened calcium influx both at resting and stimulated conditions')

    • ModerateLimited genetic work-up

      The test used was a skeletal disorder panel. The paper does not say whether genes for other causes of congenital weakness and contractures were checked, and it relies on an earlier study for evidence that this variant is harmful rather than testing it again.

      “A skeletal disorder panel was ordered for the subject, which included sequence analysis and deletion/duplication testing of 358 genes.”Case presentation

    • MinorOne child, one set of nerve tests

      The conclusion that this is purely a motor neuron disease rests on one study in a toddler, with only two sensory nerves tested. It fits a motor-only picture at that age, but a single early test cannot show what happens later, and one case cannot define a new pattern for the condition.

      “Nerve conduction assessments showed normal sensory nerve parameters in the upper and lower limbs”Case presentation; Table 1

    • MinorFounder effect is speculation

      The founder effect idea is based on two unrelated children from the same population. No shared-ancestry DNA testing was done, so this remains a hypothesis.

      “This suggests the possibility of a founder effect.”Discussion

    How “unaffected” relatives were checked

    The parents are described as 'healthy' and as carriers 'without any clinical manifestations' ('The parents are heterozygous for the same mutation, without any clinical manifestations.', Case presentation). The paper does not report a neurological examination or nerve conduction tests for either parent, so their unaffected status appears to rest on history or general observation.

    Three ways to explain it

    Scientific

    A boy with homozygous TRPV4 c.281C>T (p.S94L) presented with congenital bilateral knee arthrogryposis, talipes equinovarus and ambulatory delay, with normal sensory nerve conduction, reduced peroneal CMAP amplitude and neurogenic EMG changes, and without skeletal dysplasia or vocal fold paralysis. The authors classify this as congenital spinal muscular atrophy and arthrogryposis and, noting a previous Puerto Rican case with the same variant, raise a possible founder effect. Heterozygous parents were reported as clinically unaffected, but their assessment is not described.

    Easier

    This is a report on one young boy. He inherited the same TRPV4 change from both parents, so he has two changed copies. He was born with stiff knees and clubfeet and at two years could not stand or walk on his own, but his arms, speech and hearing were normal. Nerve tests suggested the problem lay in the nerve cells that control muscles, not in the nerves for feeling. His parents each carry one copy and were said to be healthy, but the report does not say how they were checked.

    Simple

    This is the story of one little boy who got the same TRPV4 change from his mom and his dad. He was born with stiff knees and turned-in feet and finds standing and walking hard, but his arms work well. His parents each have one copy and seem well, but we do not know how carefully they were checked.

    Why these ratings

    Relevance 3/5: Describes a recessively inherited, motor-only form of TRPV4 disease present from birth, rather than CMT2C, but touches on variability and on parents who carry one copy.

    Evidence 2/5: A single case report with useful nerve test data, but with limited genetic work-up, no description of how the parents were assessed, and internal contradictions.

    Funding and conflicts of interest

    The authors declared that no competing interests exist. Funding is not stated. The study was reviewed by an institutional review board and judged exempt.

    PubMed 37706131 · DOI

65. #65Full paperCase report (one child, with parental genetic testing)

    Case Report: TRPV4 gene mutation causing neuronopathy, distal hereditary motor, type VIII

    Wang F, Jin X, Zhu Y et al. · 2024 · Frontiers in pediatrics

    Relevance 3/5Evidence 2/51 major · 1 moderate · 3 minor problems

    This report adds one more example of a TRPV4 change looking very different in a parent and child: a child who cannot walk unaided and a father without apparent symptoms. It cannot tell us whether the father has subtle signs, because his examination is only partly reported.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    Case report (one child, with parental genetic testing)

    Who or what

    One 7-year-old boy (born in 2016, 25 kg) seen at a genetics clinic in Jining, China, with bilateral clubfoot and leg weakness, and his parents. He carries the heterozygous TRPV4 variant c.805C>T (p.Arg269Cys), inherited from his father, who is described as having no symptoms; his mother does not carry it.

    What they did

    The doctors described the pregnancy history and the boy's examination, routine blood and urine tests, laryngoscopy and X-ray images. The family had exome sequencing, and the TRPV4 change was confirmed by Sanger sequencing in the boy and both parents. The father had muscle MRI and an electrophysiological examination.

    What they found

    • Inward-turned feet were seen on prenatal ultrasound. At 7 the boy still could not walk independently. He had bilateral clubfoot and wasting of the thigh and calf muscles, and his intelligence was normal.

      “At present, the patient is 7 years old, still unable to walk independently, with normal intelligence but diminished attention span.”Case report

    • Leg strength was graded between 3 and 4, weaker on the left. X-rays showed pelvic tilt, knock-knees (knee valgus) and clubfoot, with no clear abnormality of the long bones or hips.

      “Muscle strength in both lower limbs is graded between 3 and 4.”Case report

    • Laryngoscopy found no vocal cord paresis.

      “Currently, paresis of the vocal cord has been excluded by laryngoscopy.”Case report

    • The boy carries TRPV4 c.805C>T (p.Arg269Cys), classed as pathogenic, inherited from his father, who has no apparent symptoms.

      “This mutation was inherited from the father, who, despite being phenotypically asymptomatic, is presumed to be a non-manifesting carrier.”Case report

    • The father's thigh muscle MRI and electrophysiological examination showed no significant abnormalities.

      “Electrophysiological examination of the patient's father revealed no significant abnormalities in resting potentials, duration, amplitude, and recruitment phase”Case report

    What the authors concluded

    The authors diagnose the boy with neuronopathy, distal hereditary motor, type VIII (congenital distal spinal muscular atrophy) caused by TRPV4 p.Arg269Cys. They suggest the father may be a non-manifesting carrier or have symptoms too mild to detect, and stress the role of genetic testing in early diagnosis.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • MajorNo nerve tests reported for the child

      The diagnosis is a motor-only condition, which the paper defines as having no sensory impairment. Yet no nerve conduction studies, EMG, reflex testing or sensory examination are reported for the boy. Without these, the paper cannot show that his condition is motor-only rather than another form of TRPV4 neuropathy.

      “It is characterized by progressive weakness in distal motor function and atrophy of muscles, without accompanying sensory impairment.”Abstract

    • ModerateFather's unaffected status only partly documented

      The father is called asymptomatic. He had muscle MRI and an electrophysiological test, which is better than history alone. But the main text gives no neurological examination findings (strength, reflexes, sensation, voice), and the electrophysiology is summarized only in general terms, with no nerve conduction values in the main text. The full results are in supplementary material not included here. The authors fairly allow that he may have symptoms too mild to be identified.

      “it is plausible that the patient's father may be a non-manifesting carrier or has symptoms too mild to be identified.”Case report and Discussion

    • MinorSingle case at a single time point

      One child described at one point in time cannot show how the condition will change, and general statements about the disease rely on other studies rather than on this case.

    • MinorContradictory description of the condition

      The abstract calls the condition non-progressive and, in the next sentence, characterized by progressive weakness. This makes it unclear which picture the authors think fits the boy.

      “also known as congenital non-progressive distal spinal muscular atrophy. It is characterized by progressive weakness in distal motor function”Abstract

    • MinorLimited genetic reporting

      The variant is classed as pathogenic under ACMG rules without listing the criteria used, and the paper does not say whether exome sequencing found any other candidate variants.

      “The ACMG classification for this variant is pathogenic.”Case report

    How “unaffected” relatives were checked

    The father, who carries the variant, is described as 'phenotypically asymptomatic'. He had muscle MRI (thighs in the main text; thighs and lower legs in the supplementary figure) and an electrophysiological examination, both reported as without significant abnormalities: 'Electrophysiological examination of the patient's father revealed no significant abnormalities in resting potentials, duration, amplitude, and recruitment phase'. No detailed neurological examination of the father is reported in the main text, and nerve conduction results are not given there.

    Three ways to explain it

    Scientific

    A 7-year-old boy with prenatally detected bilateral clubfoot, distal lower-limb weakness and atrophy, and inability to walk independently carried a heterozygous TRPV4 c.805C>T (p.Arg269Cys) variant inherited from his father. The father had unremarkable muscle MRI and electrophysiology and was considered a non-manifesting carrier or very mildly affected, consistent with incomplete penetrance or variable expressivity. The authors diagnosed distal hereditary motor neuronopathy type VIII, although no electrophysiology or sensory testing of the proband is reported.

    Easier

    A 7-year-old boy was born with both feet turned inward (clubfoot) and has weak, thin leg muscles, so he cannot walk on his own. Genetic testing found a change in the TRPV4 gene that he inherited from his father. His father seems healthy, and his muscle scans and electrical tests looked normal, so he may carry the change without showing it, or have signs too mild to notice. The doctors called the boy's illness a motor-only nerve condition, but the paper does not report the nerve tests that would confirm this.

    Simple

    A boy got a change in a gene called TRPV4 from his dad. TRPV4 is like a door that lets calcium into cells. The boy's legs are weak and he cannot walk alone, but his dad seems fine, so the same gene change can affect people in one family very differently.

    Why these ratings

    Relevance 3/5: About congenital distal spinal muscular atrophy, a condition caused by the same gene as CMT2C. The paper notes that this TRPV4 variant has caused different TRPV4 conditions in another family, and here a carrier parent appears unaffected.

    Evidence 2/5: A single case with good genetic confirmation in the child and both parents, but the specific diagnostic label is not supported by reported nerve tests in the child.

    Funding and conflicts of interest

    Funded by the National Natural Science Foundation of China (82201876), Natural Science Foundation of Shandong Province (ZR2021QH114, ZR2021LZY001), China Postdoctoral Science Foundation (2023M731307), Research Fund for Academician Lin He New Medicine (JYHL2021MS24), Postdoctoral Program in Affiliated Hospital of Jining Medical University (322155), Incubation Programme of High-level Scientific Research Projects in Jining Medical University (JYGC2021KJ007) and Key Research and Development Program of Jining Science (2023YXNS070). The authors declare no commercial or financial conflicts of interest.

    PubMed 38562133 · DOI

66. #66Full paperThesis chapter describing a small case series (muscle MRI)

    Muscle MRI in TRPV4-related congenital distal SMA

    Astrea G, Brisca G, Fiorillo C et al. · 2012 · Neurology

    Relevance 3/5Evidence 2/53 moderate · 2 minor problems

    In this very small study, the man with CMT2C had the same kind of leg muscle MRI pattern as two children with congenital distal SMA, mainly affecting the front of the lower leg, with less thigh involvement. It hints that the conditions sit on one spectrum, but three scans cannot prove a typical CMT2C pattern.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    Thesis chapter describing a small case series (muscle MRI)

    Who or what

    Three people with TRPV4 mutations: two children with congenital distal spinal muscular atrophy (dSMA), a 2-year-old boy and an 11-year-old girl, and one 63-year-old man with CMT2C. The mutations were p.P97R, p.R232C and p.R315W, but the text does not say which patient had which. One patient with a DYNC1H1 mutation, described in another chapter, is used for comparison.

    What they did

    They looked at T1-weighted MRI scans of the thigh and calf muscles to see which muscles had been replaced by fat. They compared the pattern in the two children with congenital dSMA with the pattern in the man with CMT2C.

    What they found

    • The children with congenital dSMA and the man with CMT2C showed a similar pattern, with the front lower-leg muscles (tibialis and peroneal groups) most affected and the medial gastrocnemius relatively spared.

      “A similar pattern of muscle MRI was evident either in patients with congenital distal SMA than in patients with CMT2C with a predominant alteration of anterior calf muscle (tibialis and peroneus groups)”Results and Discussion

    • The thighs showed extensive fatty change in congenital dSMA, and this was less evident in the man with CMT2C.

      “in congenital distal SMA, at thigh level there is an extensive and widespread fatty atrophy”Results and Discussion

    • The authors suggest a continuum of muscle involvement across TRPV4 conditions.

      “This finding suggest the presence of a specific and selective pattern with a continuum of skeletal muscle involvement in the legs between phenotypes”Results and Discussion

    • The TRPV4 pattern looked different from that of one patient with a DYNC1H1 mutation.

      “This pattern is different from non-TRPV4 patients i.e. DYNC1H1 patient presented in chapter 2”Figure 1 legend

    What the authors concluded

    TRPV4-related congenital distal SMA and CMT2C share a recognizable pattern on leg muscle MRI, with the front lower-leg muscles most affected and the medial gastrocnemius spared, while the thighs are more affected in congenital distal SMA. The authors suggest this could help tell the conditions apart when symptoms overlap and could inform thinking about natural history.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • ModerateThesis chapter that differs from the published paper

      This text is a thesis chapter that refers to a paper published in Neurology in 2012. The record for this paper notes that the chapter differs from the published paper (different author list, and the CMT2C patient is added). It is therefore unclear whether the CMT2C case was peer reviewed.

      “manuscript published in Neurology 2012; 78; 364.”Chapter heading

    • ModerateVery few patients

      Three patients with three different mutations, and only one with CMT2C, cannot establish a typical or 'specific' pattern. Which patient carried which mutation is not stated.

      “The three patients was harbouring three different mutation in TRPV4 (p.P97R; p.R232C; p. R315W).”Patients and methods

    • ModerateMethods not described

      The chapter does not describe how scans were scored, who read them, whether readers were blinded, or any healthy or other-neuropathy comparison group beyond one DYNC1H1 patient. Each patient was scanned once, so changes over time cannot be judged.

    • MinorUnclear or conflicting wording about the thigh muscles

      The text speaks of 'selective spread' of biceps femoris, semitendinosus and vastus lateralis, while the figure legend says the fatty atrophy preserves biceps femoris. It is unclear whether these muscles were spared or affected.

      “with selective spread of biceps femoris, semimtendinosus and vastus lateralis”Results and Discussion; compare Figure 1 legend

    • MinorClinical usefulness claimed from three scans

      The suggestion that thigh involvement could help distinguish distal SMA from CMT2C, and inform natural history, goes beyond what three single scans can show.

      “The predominant involvement of thigh’s muscle in distal SMA could be helpful in distinguish this form”Results and Discussion

    Three ways to explain it

    Scientific

    Lower-limb muscle MRI in two children with TRPV4-related congenital distal SMA and one adult with CMT2C showed predominant fatty replacement of the anterior calf compartment (tibialis and peroneal groups) with relative sparing of medial gastrocnemius, and more extensive thigh involvement in congenital distal SMA. The authors propose a continuum of muscle involvement across TRPV4 phenotypes, distinct from a single DYNC1H1 comparison case. With three patients and no described scoring or blinding, this is a preliminary observation.

    Easier

    Doctors scanned the leg muscles of three people with TRPV4 changes: two children with a form of spinal muscular atrophy present from birth and one man with CMT2C. In all three, the muscles at the front of the lower leg had been replaced by fat the most, while one calf muscle was spared. The children's thigh muscles were more affected than the man's. This hints that the conditions form one spectrum, but three people is too few to be sure.

    Simple

    Doctors took special pictures of the leg muscles of three people with TRPV4 changes. The same muscles at the front of the lower leg looked most worn in all three. It is a bit like the same spot wearing out first on three pairs of shoes. But three people are too few to know if this is always true.

    Why these ratings

    Relevance 3/5: It includes one adult with CMT2C, but it is mostly about children with the related condition congenital distal SMA.

    Evidence 2/5: Three patients with one scan each, no scoring system or controls, in a thesis chapter version rather than the peer-reviewed paper.

    Funding and conflicts of interest

    Not stated.

    PubMed 22291064 · DOI

67. #67Full paperCase series (three patients) with literature review

    The Broad Clinical Spectrum of Metatropic Dysplasia: A Case Series and Literature Review

    Robles-Espinoza K, Esparza-García E, González García JR et al. · 2025 · International journal of molecular sciences

    Relevance 3/5Evidence 2/51 major · 3 moderate · 2 minor problems

    This paper is about a severe skeletal TRPV4 condition, not CMT2C. It supports the idea that TRPV4 conditions overlap, but its own patients were not checked for nerve damage, so it adds little direct evidence about CMT2C.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    Case series (three patients) with literature review

    Who or what

    Three unrelated Mexican patients (two girls, one boy) with metatropic dysplasia, a TRPV4 skeletal condition: patient 1 (girl, p.Asn796del), patient 2 (girl, p.Pro799Leu) and patient 3 (boy, p.Pro799Leu). Only TRPV4 exon 14 was sequenced. A literature review combined them with published cases to give 52 patients from 44 families with TRPV4-associated disorders.

    What they did

    The doctors described the three patients' prenatal findings, examinations, X-rays and follow-up. They sequenced exon 14 of TRPV4 by Sanger sequencing and report parental testing for patients 1 and 2. They also reviewed published cases to compare variants and features across skeletal and neurological TRPV4 conditions.

    What they found

    • All three had a bell-shaped chest, flattened vertebrae and short long bones with broad ends, the typical X-ray picture of metatropic dysplasia.

      “All exhibited a bell-shaped thorax, significant platyspondyly, and shortened long bones with broad metaphyses.”Abstract; Figure 1

    • Patients 1 and 3 had more complex courses, including seizures and global developmental delay. Patient 1 also had severe reflux needing a gastrostomy, a neurogenic bladder and narrowing at the junction of the skull and spine, and her prognosis was considered poor.

      “Notably, patients 1 and 3 had more complex clinical courses, including seizures and global developmental delay.”Abstract; Cases description 2.1 and 2.3

    • Patients 2 and 3 carried the same variant, p.Pro799Leu, but patient 2 had a mild course with a favorable outlook, while patient 3 had developmental delay, seizures, spinal dysraphism and repeated fractures.

      “These two patients exemplify the clinical variability of the same variant”Discussion

    • Patient 3 had low bone density (minus 3.6 SD) after several fractures; after a second cycle of zoledronic acid at age 7 his bone density normalized, and no further fractures have occurred to date.

      “A second cycle of zoledronic acid was administered at age 7, resulting in normalization of bone mineral density.”Cases description 2.3

    • In the combined literature data, at least five TRPV4 variants had been linked both to single-system (skeletal-only or neurological-only) and to combined disease, and severity did not follow predictably from the variant.

      “Notably, clinical severity did not correlate predictably with specific variants”Discussion

    What the authors concluded

    The authors conclude that metatropic dysplasia varies widely, even with the same variant, and that TRPV4-related skeletal and neurological conditions form one spectrum of severity rather than separate syndromes, possibly shaped by the specific variant and by genetic modifiers. They call for early diagnosis, thorough clinical and molecular assessment, and multidisciplinary care.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • MajorNo nerve assessment, so the single-spectrum claim goes beyond the cases

      The abstract links the patients' neurological symptoms to the overlap between skeletal and nerve TRPV4 conditions. But the neurological problems described (seizures, developmental delay, narrowing at the skull-spine junction, spinal dysraphism, neurogenic bladder) are not the peripheral nerve problems seen in CMT2C, and no nerve conduction studies, EMG or neuromuscular examination are reported for any of the three patients. The authors point out that other reports lacked formal neurological evaluations, but the same applies to their own patients.

      “the co-occurrence of neurological symptoms and skeletal abnormalities suggests a clinically heterogeneous spectrum consistent with a single disease rather than distinct entities.”Abstract

    • ModerateLink of these variants to CMT2C rests on database entries

      The statement that p.Asn796del has been seen in CMT2C cites reference 13, which is the ClinVar database, not a peer-reviewed case report. The paper itself notes that another ClinVar entry with neuropathy has no published report behind it. Database entries are not checked in the same way as published cases.

      “is a likely pathogenic in-frame deletion that has been previously reported in at least one patient with Charcot-Marie-Tooth disease type 2C [ 13 ]”Discussion; reference 13

    • ModerateVery small series and only one exon sequenced

      Only three patients were studied, and only exon 14 of TRPV4 was sequenced. No other genes were tested, so other causes of the seizures and developmental delay in patients 1 and 3 were not looked for. The authors acknowledge both limits.

      “This study has certain limitations, including the small cohort size and the restriction of molecular analysis to TRPV4 exon 14.”Discussion

    • ModerateLiterature review methods not described

      No search strategy, databases or inclusion rules are given for the combined group of 52 patients from 44 families, and the full list is in a supplementary table not included here. The counts and percentages cannot be checked or assumed to be complete.

      “we identified 52 patients from 44 families with a documented clinical and molecular diagnosis of TRPV4 -associated disorders”Discussion

    • MinorDe novo status of patient 3 not shown

      The Discussion says all three variants were de novo (new in the child), but parental testing is reported only for patients 1 and 2.

      “We report three unrelated patients, each with a de novo heterozygous TRPV4 variant”Discussion; compare Cases description 2.3

    • MinorMixed message on whether the variant predicts severity

      The Discussion says severity did not correlate predictably with specific variants, but the Conclusions say variability appears to be influenced by the specific variant. The patients' own data (one variant, very different courses) fit the first statement better.

      “This variability appears to be influenced by the specific variant involved and potential genetic modifiers.”Conclusions

    Three ways to explain it

    Scientific

    Three unrelated patients with metatropic dysplasia carried de novo or presumed de novo TRPV4 variants in the carboxyl-terminal cytoplasmic domain (p.Asn796del; p.Pro799Leu in two), with marked variability in extra-skeletal features (seizures, developmental delay, spinal dysraphism, fractures) even for the same variant. Combining these with published cases (52 patients, 44 families), the authors propose a single TRPV4 severity continuum spanning skeletal dysplasia and neuropathy, but none of the three had a reported peripheral nerve evaluation, so the neuropathy side of this model is not tested here.

    Easier

    The doctors describe three Mexican children with metatropic dysplasia, a rare condition affecting bone growth that is caused by TRPV4 changes. Two of them had exactly the same gene change, yet one did well and the other had seizures, slow development and broken bones. The authors argue that TRPV4 bone conditions and TRPV4 nerve conditions such as CMT2C are one family of diseases with different levels of severity. However, the children's nerves were not tested, so this paper does not show nerve damage in them.

    Simple

    TRPV4 is like a door that lets calcium into cells, and these three children have changes in it that affect how their bones grow. Two children had exactly the same change but very different health. The doctors think bone and nerve TRPV4 illnesses belong to one big family, but they did not test these children's nerves.

    Why these ratings

    Relevance 3/5: About metatropic dysplasia, a skeletal condition caused by the same gene as CMT2C; the CMT2C link appears only in the literature discussion, and no patient here was assessed for neuropathy.

    Evidence 2/5: Three cases with limited genetic testing and an undescribed literature search; the main single-spectrum claim is not tested by the patients' own data.

    Funding and conflicts of interest

    This research received funding from the IMSS (R-2022-785-001); K.R.-E. received a scholarship from the Secretaría de Ciencia, Humanidades, Tecnología e Innovación. The authors declare no conflicts of interest.

    PubMed 41097048 · DOI

68. #68Detailed summary onlyFamily case report with exome sequencing and cell experiments

    Compound heterozygous TRPV4 mutations in two siblings with a complex phenotype including severe intellectual disability and neuropathy

    Thibodeau ML, Peters CH, Townsend KN et al. · 2017 · American journal of medical genetics. Part A

    Relevance 3/5Evidence 2/54 moderate · 2 minor problems

    This family shows a rare pattern quite different from typical CMT2C: two children who inherited a different TRPV4 variant from each parent had a severe condition affecting nerves, hearing, eyes and development. The cell tests suggested these variants reduce channel activity, which the summary says adds weight to the idea that too little TRPV4 activity, as well as too much, may harm nerves.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    Family case report with exome sequencing and cell experiments

    Who or what

    One family of Northern European ancestry. Two affected children, a brother aged 15 and a sister aged 9, each carry two different TRPV4 variants, Asn833Ser (c.2498A>G) and Glu840Lys (c.2518G>A). Their father, mother, older sister and paternal grandfather each carry one of the variants and are described as well. Each variant was also tested in Chinese hamster ovary cells (six cells each).

    What they did

    The team sequenced the exomes of the two affected children and their healthy mother, looking for rare variants that both children carried in two copies, then confirmed candidate variants by Sanger sequencing in the children, their healthy sister and both parents. They put each TRPV4 variant separately into Chinese hamster ovary cells and measured channel current by patch clamp before and after adding a compound that opens TRPV4, and used antibody staining to see where the protein went. This analysis is based on a detailed secondary summary of the paper written by another reader, not on the paper itself.

    What they found

    • Both affected children inherited a different TRPV4 variant from each parent: Asn833Ser from the father's side and Glu840Lys from the mother. Relatives with only one variant were described as well.

      “This is the first report of a child inheriting a different TRPV4 variant from each parent, and the result was a much more severe, multi-system condition than CMT2C.”Secondary summary, opening summary and table 'The variants and who carries them'

    • Both children had a widespread axonal sensory and motor neuropathy, along with severe sensorineural hearing loss found in infancy, retinal disease, short stature and very limited communication. The brother, who is more severely affected, cannot walk.

      “Widespread axonal sensory and motor neuropathy.”Secondary summary, table 'The two children'

    • In hamster cells, both variant channels reached the cell surface normally but carried about a third of the normal current, so each forms a channel that partly works.

      “Both carried about a third of the normal current: 119.5 pA/pF for Asn833Ser and 107.7 for Glu840Lys, against 371.1 for the normal channel (six cells each, p = 0.0085).”Secondary summary, 'Laboratory findings'

    • The authors argue that reduced channel function inherited from both parents is a new way TRPV4 can cause disease, in contrast to the overactive channel usually proposed for dominant CMT2C.

      “Reduced channel function inherited from both parents is a new disease mechanism for TRPV4. Dominant CMT2C is usually put down to an overactive channel.”Secondary summary, 'What the authors argue'

    • The brother also carried two variants in ABCA4, a gene linked to inherited retinal disease, which may add to his eye disease.

      “The brother also carried two variants in ABCA4, one of them already known to cause disease.”Secondary summary, 'Laboratory findings'

    • The authors note that profound intellectual disability had not been linked to TRPV4 before, and raise the possibility that TRPV4 matters for brain development, which they say needs further study.

      “Profound intellectual disability has not been tied to TRPV4 before.”Secondary summary, 'What the authors argue'

    What the authors concluded

    The authors conclude that inheriting two partly working TRPV4 variants, one from each parent, can cause a severe condition affecting nerves, hearing, eyes and development, and that this reduced channel function is a new disease mechanism for TRPV4. They add that the parents being well does not prove each variant is harmless on its own, and that a possible role for TRPV4 in brain development needs further study.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • ModerateOne family only

      The proposed new disease mechanism rests on two siblings in a single family, with no second family showing the same pattern.

      “One family. No second family with the same pattern is reported.”Secondary summary, 'Limitations'

    • ModerateVariants never tested together

      Each variant was tested alone in hamster cells with six recordings. The children carry both, so the lab results do not directly model their situation, and hamster cells are some distance from human nerves.

      “Each variant was tested alone in hamster cells with six recordings. The two were never expressed together, which is the situation in the children.”Secondary summary, 'Limitations'

    • ModeratePossible second genetic causes

      The brother's ABCA4 variants may explain part of his eye disease, and another undetected gene could explain the intellectual disability, so not every feature can be safely put down to TRPV4.

      “Another undetected gene could explain the intellectual disability. Exome sequencing does not cover everything.”Secondary summary, 'Limitations'

    • ModerateHow the single-variant relatives were assessed is not stated

      The parents, older sister and grandfather are described only as well. The summary does not say whether they had a neurological examination or nerve tests, which matters for any claim that one variant alone causes no disease.

      “The parents being well does not prove each variant is harmless on its own.”Secondary summary, 'What the authors argue'

    • MinorComputer predictions disagree for one variant

      Prediction programs were inconsistent about whether Asn833Ser is harmful, although the cell tests did show reduced current.

      “Computer predictions disagreed on whether Asn833Ser is harmful.”Secondary summary, 'Limitations'

    • MinorKey clinical detail not seen

      The summary writer did not see the supplement holding most clinical detail and the nerve test values, so this analysis cannot check them.

      “Most of the clinical detail, including nerve test values, is in a supplement I did not see.”Secondary summary, 'Limitations'

    How “unaffected” relatives were checked

    The father, mother, older sister and paternal grandfather, who each carry one variant, are described in the summary only as 'Well'. The summary does not say whether they were examined by a neurologist or had nerve conduction tests, so how their unaffected status was decided is unclear. The summary also notes the authors offer reduced penetrance as one reason the parents are well. Quote: “The parents being well does not prove each variant is harmless on its own.”

    Three ways to explain it

    Scientific

    Two siblings who were compound heterozygous for the TRPV4 variants Asn833Ser and Glu840Lys, both located away from the ankyrin repeat domain, had a severe multisystem phenotype including axonal sensorimotor neuropathy, sensorineural hearing loss, retinal disease, short stature and profound intellectual disability, while heterozygous relatives were reported as well. In Chinese hamster ovary cells each variant trafficked normally but produced about a third of wild-type current, suggesting partial loss of function. The authors propose biallelic reduced channel function as a new TRPV4 disease mechanism, but the evidence comes from one family and the variants were never co-expressed.

    Easier

    Two children in one family each inherited two different changes in the TRPV4 gene, one from their father's side and one from their mother. They had a severe condition affecting their nerves, hearing, eyesight, growth and learning, while relatives with only one of the changes were described as well. In lab-grown hamster cells, each changed channel let through only about a third of the normal electrical current. The authors suggest that having too little working channel, inherited from both parents, is a new way TRPV4 can cause disease.

    Simple

    Think of TRPV4 as a door that lets calcium into cells. These two children got a slightly broken door from each parent, and in lab tests each of these doors only opened about a third as much as normal. The children were very unwell, but this is just one family, so scientists need to see more families to be sure.

    Why these ratings

    Relevance 3/5: It describes a rare, much more severe TRPV4 condition that includes axonal neuropathy. This is relevant to how TRPV4 changes harm nerves, but it is not a description of typical CMT2C.

    Evidence 2/5: One family with supporting cell experiments on small numbers, read here only through a secondary summary.

    Funding and conflicts of interest

    Not stated in the summary. The summary says the team worked through the FORGE Canada rare disease consortium but does not describe funding or competing interests.

    PubMed 28898540 · DOI

69. #69Full paperShort narrative review (commentary)

    TRPV4-pathy, a novel channelopathy affecting diverse systems

    Dai J, Cho TJ, Unger S et al. · 2010 · Journal of human genetics

    Relevance 3/5Evidence 1/52 moderate · 2 minor problems

    This early review helped frame CMT2C as one of several conditions caused by changes in the same gene, TRPV4. Its explanations of why one gene can affect nerves in some people and bones in others are ideas, not tested findings.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    Short narrative review (commentary)

    Who or what

    No new patients or experiments. The authors summarize published TRPV4 mutations: 29 different mutations in skeletal dysplasias (brachyolmia, spondylometaphyseal dysplasia Kozlowski type, metatropic dysplasia, spondyloepiphyseal dysplasia Maroteaux type and parastremmatic dysplasia) and four mutations at three positions in neuromuscular disorders (congenital distal SMA, scapuloperoneal SMA and HMSN IIC, also called CMT2C), plus association studies of common TRPV4 variants.

    What they did

    They discussed three Nature Genetics papers that had just linked TRPV4 to nerve and muscle disease, alongside earlier reports of TRPV4 in skeletal dysplasias. They mapped the known mutations onto the TRPV4 protein and discussed whether simple gain or loss of channel function could explain why some mutations affect bones and others affect nerves.

    What they found

    • TRPV4 mutations were known in two separate groups of dominant conditions: 29 different mutations in skeletal dysplasias, and four mutations in nerve and muscle disorders including HMSN IIC (CMT2C).

      “Altogether, 29 different mutations have been found in the ‘family’ of skeletal dysplasias”Introduction, p. 400

    • All four neuromuscular mutations were arginine changes in the ankyrin repeat region, but the authors argue this is not specific to nerve disease, because ankyrin repeat mutations and arginine changes (for example R594H) also occur in skeletal dysplasias.

      “It seems equally unlikely that substitution of arginine may have a special significance, as R594H is a very frequent mutation in the skeletal group.”Main text, p. 401

    • The first lab studies of the neuromuscular mutations disagreed: one suggested less channel reaching the cell surface, another suggested increased calcium channel activity, and a third suggested both loss of normal function and toxic calcium entry.

      “Functional studies would probably shed light on the consequence of distinct mutations; however, present results and conclusion of functional analyses are diverse among the studies.”Main text, p. 401

    • The authors argue that neither simple gain nor simple loss of channel function can explain the different conditions, and suggest partner proteins or faulty signaling from inside the cell instead.

      “At the current state of knowledge, it appears that the phenotypic diversity of TRPV4 mutations cannot be explained by a simple gain/loss-of-function mechanism, but most likely involve additional molecular partners”Main text, p. 401

    • They propose that common TRPV4 variants linked to blood (serum) sodium levels and chronic obstructive pulmonary disease could also belong under the 'TRPV4-pathy' umbrella.

      “These diverse spectrums of diseases compose a novel channelopathy, TRPV4-pathy, which could further include polygenic traits such as serum sodium concentration and a chronic obstructive pulmonary disease.”Abstract

    • At the time of writing, the authors saw no real overlap in features between the bone conditions and the nerve conditions.

      “It is noteworthy that neither the two groups of monogenic diseases nor the polygenic disorders show essential phenotypic overlap among each other.”Introduction, p. 400

    What the authors concluded

    TRPV4 mutations cause a family of conditions the authors call 'TRPV4-pathy', covering skeletal dysplasias and nerve and muscle disorders, and possibly some common traits. In their view the explanations offered so far (gain or loss of channel function) are too simple, and partner proteins or other signaling routes are probably involved. They expect more TRPV4-related diseases to be found.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • ModerateReview with no new data

      The paper offers interpretation and hypotheses, not new observations. It does not describe how the literature was chosen, so the summary cannot be checked for completeness.

      “In this review, we clarified the TRPV4 mutation spectrum, and discussed the phenotypic complexity of TRPV4-pathy and its pathogenic mechanisms.”Abstract

    • ModerateEarly snapshot with very few neuromuscular cases

      Only four neuromuscular mutations from three papers were known when this was written. Statements such as the lack of overlap between the bone and nerve conditions rest on this small base and may change as more patients are described.

      “Four missense mutations at three distinct positions in the TRPV4 protein have been identified in the neuromuscular disorders”Introduction, p. 400

    • MinorProposed mechanisms are untested ideas

      Ideas such as TRPV4 behaving like a related channel, or signaling wrongly from inside the cell, are offered without experiments. They should be read as hypotheses.

      “If the arginine 269 were indeed crucial to the neuromuscular phenotypes, the mechanism might be a functional trans-specification of TRPV4 toward TRPV1 or TRPV2.”Main text, p. 401

    • MinorCommon traits included on association evidence

      Adding serum sodium and chronic obstructive pulmonary disease to 'TRPV4-pathy' rests on statistical associations with common gene variants. That is a different and less direct kind of evidence than disease-causing mutations that track with illness in families.

      “which could further include polygenic traits such as serum sodium concentration and a chronic obstructive pulmonary disease”Abstract

    Three ways to explain it

    Scientific

    This 2010 review groups the autosomal dominant TRPV4 skeletal dysplasias (29 mutations) and the neuromuscular disorders (congenital distal SMA, scapuloperoneal SMA and HMSN IIC; four arginine substitutions at three ankyrin repeat positions) into a single channelopathy, 'TRPV4-pathy'. The authors argue that ankyrin repeat location and arginine substitution are not specific to neuropathy, that published functional data conflict, and that a simple gain-of-function or loss-of-function model cannot explain the phenotypic divergence. They propose interacting proteins or aberrant endoplasmic reticulum signaling as alternatives, without presenting new data.

    Easier

    TRPV4 is a gene that makes a channel, a kind of gate that lets calcium and similar particles into cells. Changes in this gene were first linked to conditions affecting bone growth, and then to nerve and muscle conditions including CMT2C. The authors grouped all of these under the name 'TRPV4-pathy'. They argued that the early explanations of how the faulty channel causes disease were probably too simple, but they offered ideas rather than new evidence.

    Simple

    One gene, TRPV4, makes a door that lets calcium into cells. Changes in this door can cause bone problems in some people and nerve problems, like CMT2C, in others. The scientists who wrote this said nobody yet knew why, and shared their best guesses.

    Why these ratings

    Relevance 3/5: It places CMT2C (HMSN IIC) within the wider family of TRPV4 conditions, but most of its content is about skeletal dysplasias and it adds no new CMT2C data.

    Evidence 1/5: A short opinion review with no new data and no described method for selecting the literature; this reflects the type of paper rather than poor quality.

    Funding and conflicts of interest

    Supported by Grants-in-Aid from the Ministry of Education, Culture, Sports and Science of Japan (No. 21249024), from Research on Child Health and Development (No. 20-S-3), and from the Ministry of Health, Labor and Welfare of Japan (Measures for Intractable Diseases 046 in 2010). Competing interests: not stated.

    PubMed 20505684 · DOI

70. #70Full paperNarrative review with hypotheses (no new data)

    TRPV4-mediated channelopathies

    Verma P, Kumar A, Goswami C · 2010 · Channels (Austin, Tex.)

    Relevance 3/5Evidence 1/53 moderate · 1 minor problems

    This early review collects what was known about TRPV4 mutations in 2010 and offers ideas about how ankyrin domain mutations might cause disease. Its models were guesses to be tested, not findings, so it says little directly about living with CMT2C.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    Narrative review with hypotheses (no new data)

    Who or what

    Published research on the TRPV4 channel up to 2010: a table of 35 naturally occurring TRPV4 mutations (skeletal dysplasias, neuropathies including CMT2C, spinal muscular atrophies and low blood sodium) and a table of 56 artificially made TRPV4 mutations studied in cells, using rat, mouse, chicken and human versions of the channel.

    What they did

    The authors summarized what was known about TRPV4 structure, partner proteins and disease mutations. They then proposed, using two hypothetical diagrams and a preliminary computer prediction, that ankyrin domain mutations may act by changing how TRPV4 is assembled, moved to the cell surface and broken down, and how it binds an unknown 'inhibitory complex'.

    What they found

    • The review identifies two mutation 'hot spots' in TRPV4: the pore region and the ankyrin repeat domain, where the neuropathy mutations known at the time (R269H, R315W, R316C) sit.

      ““hot spots” in TRPV4 sequences are prominent, one at the pore”Page 323, after Table 2

    • Lab results on the same neuropathy mutations conflicted: in HeLa cells they appeared to lower channel function, while in HEK cells they raised it. The review treats this as a clue that cell-specific partner proteins matter.

      “R316C reveals a loss of function (com- ... when expressed in HeLa cells4 while the ... same mutation reveals a gain of function in HEK cells”Page 324, How these mutations can affect function and surface expression

    • The central idea is a hypothesis: changing the arginine at position 269, 315 or 316 might weaken or strengthen binding of an unknown inhibitory complex that normally holds the channel closed.

      “it is tempting to ... speculate that substitution of R with other amino acids ... either weak or strong interaction of at least one molecular component that works as a inhibitory complex for TRPV4”Pages 324 to 325, with Figure 1

    • The authors also suggest that R269 mutations might disrupt a sequence signal (an RXR motif) that controls how much channel reaches the cell surface, but say this needs testing.

      “and R269H) can destroy the function of one RXR motif located ... However, this possibility needs to be verified”Page 327, just before Future direction and conclusion

    What the authors concluded

    The authors argue that TRPV4 disease mutations probably do not act only by changing how much current the channel passes. They propose that partner proteins and the processes that control assembly, transport to the cell surface and breakdown of TRPV4 are involved, and they say these ideas need experimental testing.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • ModerateReview with speculative models and no stated method

      This is a narrative review, not a study. Its two figures are labeled hypothetical and its structural analysis is called preliminary, so they are ideas rather than findings. The paper does not explain how studies were chosen, says some work was left out for space, and states that the authors take no formal responsibility for the data they summarize.

      “Figure 1. A hypothetical model demonstrating how mutations in the ARD region can ... Preliminary bioinformatic analysis (AGADIR pre- ... undertake no formal responsibility for the scientific authenticity/”Pages 324 to 325 (Figure 1, structural analysis); Acknowledgements and notes

    • ModerateErrors in the mutation table and text

      Table 1 gives the same DNA change (G806A in exon 5) for two different protein changes, R269H and R269C, which cannot both be right. The text also refers to an 'R315C' mutant, although elsewhere the mutation at that position is R315W. Readers should not rely on this table for exact variant details.

      “G806A (exon 5) R269C ... rescue the functions of the mutants (R269H ... and R315C) respectively”Table 1 (page 321), rows 6 to 9; page 324 text

    • ModerateLeaps beyond the evidence

      The review states that proteins from other CMT2 genes are 'potential TRPV4 interacting partners' because they cause a similar disease. Causing a similar disease does not show that two proteins bind each other. It also argues that because patients are heterozygous, signaling events rather than conductance must matter, which does not follow directly.

      “As TRPV4 is involved in the Charcot-Marie-Tooth disease ... are also involved in CMT2 disease, are actually potential TRPV4 ... interacting partners”Page 320, Structure of TRPV4 and different interacting proteins

    • MinorWritten early, with conflicting data at the time

      The review was published in 2010, soon after the first TRPV4 neuropathy reports, when no atomic structure of TRPV4 existed and cell studies disagreed. Its picture reflects that moment and may not reflect later research.

      “At present, no crystallographic or nuclear magnetic ... resonance (NMR) data is available”Page 319, Structure of TRPV4

    Three ways to explain it

    Scientific

    This 2010 narrative review catalogs 35 naturally occurring and 56 engineered TRPV4 mutations and highlights conflicting gain-of-function and loss-of-function data for ankyrin repeat domain neuropathy mutations, depending on the cell line (HEK versus HeLa). The authors hypothesize that these mutations alter oligomerization, trafficking, ubiquitination-dependent surface expression or binding of an unidentified inhibitory complex, rather than conductance alone. These models are explicitly hypothetical and untested.

    Easier

    This article reviews research on the TRPV4 channel up to 2010. At that time, lab studies disagreed about whether the neuropathy mutations made the channel more active or less active, and the answer seemed to depend on the type of cell used. The authors suggest the mutations might change how the channel is built, moved to the cell surface or held shut by other proteins, but these were ideas to be tested, not proven results.

    Simple

    TRPV4 is like a door that lets calcium into a cell. In 2010, scientists were not sure whether faulty versions of the door were stuck open or stuck shut. These authors guessed that helper proteins that normally hold the door closed might be part of the problem.

    Why these ratings

    Relevance 3/5: A 2010 review of TRPV4 diseases in general; ankyrin domain neuropathy mutations are a main example, but the CMT2C content is short and mostly hypothesis.

    Evidence 1/5: A narrative review with no new data whose central idea is an untested hypothesis, and whose mutation table contains internal errors.

    Funding and conflicts of interest

    Funding from the National Institute of Science Education and Research (Bhubaneswar, India) is acknowledged. Competing interests are not stated.

    PubMed 20676052 · DOI

71. #71Abstract onlySingle-center case series

    Natural history of TRPV4-Related disorders: From skeletal dysplasia to neuromuscular phenotype

    Ürel-Demir G, Şimşek-Kiper PÖ, Öncel İ et al. · 2021 · European journal of paediatric neurology: EJPN: official journal of the European Paediatric Neurology Society

    Relevance 3/5Evidence 1/51 moderate problem

    This Turkish series describes TRPV4 conditions as a spectrum from bone growth disorders to nerve and muscle disorders such as CMT2C, with a lot of overlap. The abstract does not give the details of the patients.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    Single-center case series

    Who or what

    11 patients from six families with TRPV4-related disorders, seen at a single center in Turkey. The abstract does not give ages, variants or how many had skeletal versus neuromuscular features.

    What they did

    The authors report the clinical and molecular spectrum of patients diagnosed with TRPV4-related disorders at their center. This analysis is based only on the abstract, so the methods cannot be judged.

    What they found

    • The authors describe 11 patients from six families with TRPV4-related disorders, which they say is the largest single-center group with TRPV4 variants in Turkey.

      “we report on the clinical and molecular spectrum of 11 patients from six different families diagnosed with TRPV4-related disorders.”Abstract

    • The abstract groups TRPV4 disorders into skeletal dysplasias and neuromuscular disorders (congenital distal SMA, scapuloperoneal SMA and CMT2C) and stresses considerable overlap within and between the groups. It does not report the results for the 11 patients.

      “Of note, there is considerable phenotypic overlap within and between both groups.”Abstract

    What the authors concluded

    The abstract presents TRPV4-related disorders as one broad spectrum running from skeletal dysplasia to neuromuscular disease, with overlapping features. It does not state specific conclusions drawn from the 11 patients.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • ModerateMethods and results cannot be judged from the abstract

      Only the abstract was available, and most of it is background. It gives no patient ages, variants, examination methods, follow-up length or findings, so nothing about natural history can be checked. It is not stated how relatives were classed as affected or unaffected.

      “This study yet represents the largest cohort of patients with TRPV4 variants from a single center in Turkey.”Abstract

    How “unaffected” relatives were checked

    Cannot be judged: the abstract does not say how members of the six families were classed as affected or unaffected, or whether they were examined or had nerve tests.

    Three ways to explain it

    Scientific

    This single-center series reports 11 patients from six families with TRPV4-related disorders, framed as a spectrum spanning skeletal dysplasias and neuromuscular phenotypes (congenital distal SMA, scapuloperoneal SMA and CMT2C) with considerable overlap within and between groups. The abstract gives no genotype, phenotype or longitudinal data, so its natural history findings cannot be assessed.

    Easier

    Doctors in Turkey describe 11 people from six families with conditions caused by changes in the TRPV4 gene. These conditions range from problems with bone growth to nerve and muscle problems such as CMT2C, and many people have features of more than one. The abstract does not describe what was found in each patient.

    Simple

    Changes in the TRPV4 gene can affect bones, nerves and muscles, and often more than one. This study looked at 11 people with these changes, but the summary does not say what happened to them.

    Why these ratings

    Relevance 3/5: It covers the whole range of TRPV4 disorders, including CMT2C and the related skeletal and spinal muscular atrophy forms, but the abstract does not say how many patients had neuromuscular disease.

    Evidence 1/5: Rated 1 because only the abstract was available and it reports no actual patient results.

    Funding and conflicts of interest

    Not stated in the abstract.

    PubMed 33774370 · DOI

72. #72Abstract onlyFamily report with literature review

    TRPV4 related scapuloperoneal spinal muscular atrophy: Report of an Italian family and review of the literature

    Biasini F, Portaro S, Mazzeo A et al. · 2016 · Neuromuscular disorders: NMD

    Relevance 3/5Evidence 1/51 moderate problem

    In this Italian family the same TRPV4 change, R269H, caused mild weakness in a father and a more severe condition from birth in his son. The abstract's claim that severity rises in later generations rests on this one pair.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    Family report with literature review

    Who or what

    The first Italian family reported with scapuloperoneal spinal muscular atrophy (SPSMA), carrying TRPV4 c.806G>A (p.R269H): a father and his son.

    What they did

    The authors describe the father and son and review the literature on TRPV4-related SPSMA. This analysis is based only on the abstract, so the methods cannot be judged.

    What they found

    • Severity differed between father and son: the father had mild muscle involvement, while the son had skeletal dysplasia from birth and a progressive course.

      “The pattern of expression was variable: the father showed a mild muscular involvement, while the son presented at birth skeletal dysplasia and a progressive course.”Abstract

    • The authors say this supports the idea that the disease can be more severe in later generations.

      “We reinforce the concept that the disease can be more severe in the following generations.”Abstract

    • The authors suggest that SPSMA should be considered in dominantly inherited scapuloperoneal syndromes with nerve-related weakness, and that skeletal deformities strongly support the suspicion.

      “The presence of skeletal deformities strongly supports this suspicion.”Abstract

    What the authors concluded

    TRPV4-related SPSMA varies in severity and can be more severe in later generations. It should be considered in dominantly inherited scapuloperoneal syndromes with a neurogenic pattern, especially with skeletal deformities, and the authors say early diagnosis may be crucial to prevent the more severe congenital form.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • ModerateMethods cannot be judged from the abstract

      Only the abstract was available, so it is not possible to check how the father was examined or whether he had nerve tests. A single father and son cannot establish that the disease gets worse in later generations. The abstract does not explain how early diagnosis could prevent the congenital form.

      “An early diagnosis of SPSMA may be crucial in order to prevent the more severe congenital form.”Abstract

    How “unaffected” relatives were checked

    The father is described as having mild muscle involvement; the abstract does not say how he was examined or whether he had nerve conduction tests, and does not mention other relatives.

    Three ways to explain it

    Scientific

    In the first Italian SPSMA family, the TRPV4 R269H variant showed variable expressivity: mild muscular involvement in the father and congenital skeletal dysplasia with a progressive course in the son. The authors interpret this as supporting greater severity in successive generations and recommend considering SPSMA in autosomal dominant neurogenic scapuloperoneal syndromes, particularly with skeletal deformities. A single parent-child pair cannot establish anticipation, and methods cannot be assessed from the abstract.

    Easier

    Doctors describe an Italian father and son with the same TRPV4 change, R269H, which caused weakness around the shoulder blades and lower legs. The father was only mildly affected, while the son had bone problems from birth and got worse over time. The authors think the illness can be more severe in later generations, but this is based on one father and son.

    Simple

    A dad and his son had the same TRPV4 change. The dad was only a little weak, but the son had more problems from when he was born.

    Why these ratings

    Relevance 3/5: It concerns scapuloperoneal spinal muscular atrophy, another condition caused by TRPV4 changes; here the change is R269H, which is also seen in CMT2C.

    Evidence 1/5: Rated 1 because only the abstract was available; it is also a single father and son.

    Funding and conflicts of interest

    Not stated in the abstract.

    PubMed 26948711 · DOI

73. #73Abstract onlyCase series (four cases)

    Fetal akinesia in metatropic dysplasia: The combined phenotype of chondrodysplasia and neuropathy?

    Unger S, Lausch E, Stanzial F et al. · 2011 · American journal of medical genetics. Part A

    Relevance 3/5Evidence 1/51 moderate problem

    In four very severe cases of a TRPV4 bone condition, the babies did not move before birth, which the authors think may mean their nerves were also affected. This supports the idea that TRPV4 bone and nerve conditions can overlap.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    Case series (four cases)

    Who or what

    Four cases of severe metatropic dysplasia, a TRPV4 skeletal condition, presenting with absent movement before or at birth: three found on prenatal ultrasound and one in a newborn. Each had a new (de novo) TRPV4 mutation: G78W (Case 1), T740I (Cases 2 and 3) and K276E (Case 4).

    What they did

    The authors describe the prenatal and birth findings, X-rays after pregnancy termination or after birth, and TRPV4 sequencing. This analysis is based only on the abstract, so the methods cannot be judged.

    What they found

    • Three cases were picked up on prenatal ultrasound because the fetus was not moving in the second trimester, and the fourth had multiple joint contractures and no limb movement at birth.

      “Three cases were detected on prenatal ultrasound because of absent movements in the second trimester.”Abstract

    • All four had typical X-ray features of metatropic dysplasia and new TRPV4 mutations.

      “Sequencing of the TRPV4 gene confirmed the presence of de novo heterozygous mutations predicting G78W (Case 1), T740I (Cases 2 and 3), and K276E (Case 4).”Abstract

    • Because lack of movement as the leading sign is unusual in skeletal dysplasia, the authors suggest certain TRPV4 mutations cause both a skeletal condition and a peripheral neuropathy.

      “akinesia as leading sign is unusual and suggests that certain TRPV4 mutations produce both chondrodysplasia and a peripheral neuropathy resulting in a severe “overlap” phenotype.”Abstract

    What the authors concluded

    Absent fetal movement can be the presenting sign of severe metatropic dysplasia. The authors suggest that some TRPV4 mutations cause both a skeletal and a nerve condition, a severe overlap form.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • ModerateMethods cannot be judged from the abstract

      Only the abstract was available. It does not mention nerve or muscle tests, so the suggested neuropathy appears to be inferred from the lack of movement rather than shown directly. The authors use cautious wording ('suggests'), which fits the limited evidence.

      “suggesting that certain TRPV4 mutations can cause both a skeletal and a neuropathic phenotype.”Abstract

    Three ways to explain it

    Scientific

    Four cases of severe metatropic dysplasia with de novo heterozygous TRPV4 mutations (G78W, T740I, K276E) presented with fetal akinesia, detected on second-trimester ultrasound or as arthrogryposis with absent limb movements at birth. The authors propose that certain TRPV4 mutations cause both chondrodysplasia and peripheral neuropathy, a severe overlap phenotype. The abstract does not report neurophysiological or pathological confirmation of neuropathy.

    Easier

    Doctors describe four babies with a severe TRPV4 bone growth condition who did not move in the womb or at birth. All had new changes in the TRPV4 gene that were not inherited. The authors think these changes may affect the nerves as well as the bones, but the abstract does not describe nerve tests.

    Simple

    Four babies with a TRPV4 change had bones that grew differently, and they hardly moved before birth. Doctors think their nerves might have been affected too, but they did not show this for sure.

    Why these ratings

    Relevance 3/5: It concerns a TRPV4 skeletal condition with a suggested nerve component, an allelic overlap condition rather than CMT2C.

    Evidence 1/5: Rated 1 because only the abstract was available; it is also a small series, and the nerve involvement is inferred.

    Funding and conflicts of interest

    Not stated in the abstract.

    PubMed 21964829 · DOI

74. #74Abstract onlyFamily study with whole-exome sequencing

    Novel TRPV4 mutation in a large Chinese family with congenital distal spinal muscular atrophy, skeletal dysplasia and scaly skin

    Liu Y, Yan X, Chen Y et al. · 2020 · Journal of the neurological sciences

    Relevance 3/5Evidence 1/51 moderate problem

    In this Chinese family, a new TRPV4 change was linked to leg weakness from birth, bone problems and scaly skin. It adds another feature to the wide range of TRPV4 conditions, though it comes from one family.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    Family study with whole-exome sequencing

    Who or what

    One large Chinese family. Nineteen members were investigated; the proband and four other affected relatives carry a new TRPV4 change, c.2355G > T (p.Trp785Cys).

    What they did

    The authors carried out clinical, electrophysiological and X-ray examinations, used whole-exome sequencing in the proband, and checked whether the TRPV4 change traveled with the disease in the family. This analysis is based only on the abstract, so the methods cannot be judged.

    What they found

    • The proband and four other affected relatives had congenital distal spinal muscular atrophy, skeletal problems including bone death in the head of the thigh bone (osteonecrosis of the femoral head), and scaly skin.

      “The proband and other four affected family members showed signs of congenital distal spinal muscular atrophy, skeletal abnormalities including osteonecrosis of the femoral head, and scaly skin.”Abstract

    • Exome sequencing found a new TRPV4 change, Trp785Cys, in the proband.

      “resulting in a tryptophan to cysteine substitution at amino acid 785 (p.Trp785Cys) of TRPV4.”Abstract

    • The change traveled with the disease in the family.

      “Furthermore, the mutation co-segregated with disease in this family.”Abstract

    What the authors concluded

    The new TRPV4 change Trp785Cys causes congenital distal spinal muscular atrophy with skeletal problems and scaly skin in this family, widening the known range of TRPV4 conditions.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • ModerateMethods cannot be judged from the abstract

      Only the abstract was available. It does not say which relatives had nerve tests or how those called unaffected were assessed, which matters for the co-segregation claim. No laboratory test of the variant is mentioned, and one family cannot show whether scaly skin is truly part of TRPV4 disease.

      “Clinical, electrophysiological, and radiographic examinations were performed.”Abstract

    How “unaffected” relatives were checked

    Unclear: the abstract says 19 family members were investigated with clinical, electrophysiological and X-ray examinations, but not whether every relative classed as unaffected had a neurological examination and nerve tests. Quote: “Nineteen individuals from this family were investigated.”

    Three ways to explain it

    Scientific

    In a large Chinese pedigree, a novel heterozygous TRPV4 variant, c.2355G > T (p.Trp785Cys), identified by whole-exome sequencing, co-segregated with congenital distal SMA, skeletal abnormalities including femoral head osteonecrosis, and scaly skin in five affected members. The authors interpret this as an expansion of the TRPV4 phenotypic spectrum. Phenotyping of unaffected relatives and functional evidence cannot be assessed from the abstract.

    Easier

    Doctors studied a large family in China in which five people had scaly skin, bone problems in the hip and elsewhere, and leg weakness from birth caused by damaged motor nerve cells. They found a new change in the TRPV4 gene in these relatives. The change appeared to travel with the illness through the family.

    Simple

    In one big family, five people had weak legs from birth, bone problems and scaly skin, and they all had the same new change in their TRPV4 gene.

    Why these ratings

    Relevance 3/5: It concerns congenital distal spinal muscular atrophy with skeletal features, an allelic TRPV4 condition.

    Evidence 1/5: Rated 1 because only the abstract was available, so the family data and genetic evidence could not be checked.

    Funding and conflicts of interest

    Not stated in the abstract.

    PubMed 33075594 · DOI

75. #75Abstract onlyReview

    TRPV4-associated skeletal dysplasias

    Nishimura G, Lausch E, Savarirayan R et al. · 2012 · American journal of medical genetics. Part C, Seminars in medical genetics

    Relevance 3/5Evidence 1/51 moderate problem

    This review describes how TRPV4 changes can cause bone growth conditions or nerve conditions, with a few people having both. It notes that most tested TRPV4 changes make the channel more active in the lab, but how this causes disease was still unclear.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    Review

    Who or what

    No new patients. A review of TRPV4-associated skeletal dysplasias, covering over 50 reported TRPV4 mutations and the overlap with TRPV4 nerve conditions.

    What they did

    The authors reviewed the range of bone growth conditions caused by TRPV4 mutations, rarer variant forms, the overlap with nerve conditions, and what was known about mutation hot spots and channel activity. This analysis is based only on the abstract, so the methods cannot be judged.

    What they found

    • TRPV4 skeletal dysplasias form a continuous spectrum, from lethal metatropic dysplasia to milder forms such as autosomal dominant brachyolmia.

      “form a continuous phenotypic spectrum that includes, in decreasing severity, lethal, and nonlethal metatropic dysplasia (MD), spondylometaphyseal dysplasia Kozlowski type (SMDK), and autosomal dominant brachyolmia.”Abstract

    • A small number of patients have a combination of skeletal dysplasia and peripheral neuropathy from a TRPV4 mutation.

      “a small number of patients have been identified in whom a TRPV4 mutation results in a phenotype combining skeletal dysplasia with peripheral neuropathy.”Abstract

    • Most disease-causing mutations tested so far activate the channel in laboratory tests, but how this leads to bone or nerve disease, and why particular mutations cause particular conditions, remains unclear.

      “While most pathogenic mutations tested so far result in activation of the calcium channel in vitro, the mechanisms through which TRPV4 activation results in skeletal dysplasia and/or peripheral neuropathy remain unclear”Abstract

    What the authors concluded

    TRPV4 skeletal dysplasias form a spectrum, with some overlap with TRPV4 nerve conditions. Most tested mutations activate the channel, but the mechanisms and genotype-phenotype links remain unclear. Careful clinical and X-ray assessment helps guide genetic testing.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • ModerateMethods cannot be judged from the abstract

      Only the abstract was available, so it is not possible to see how the literature was chosen or what the review says about the neuropathy overlap. As a review it presents no new data, and the evidence that most mutations activate the channel comes from laboratory tests.

      “result in activation of the calcium channel in vitro”Abstract

    Three ways to explain it

    Scientific

    This review describes TRPV4 skeletal dysplasias as a continuous spectrum from lethal metatropic dysplasia through spondylometaphyseal dysplasia Kozlowski type to autosomal dominant brachyolmia, with rarer variant phenotypes and a small number of combined skeletal and neuropathic cases. Among more than 50 reported mutations, it notes hot spots at P799 and R594. Most mutations tested show gain of function in vitro, though mechanisms and genotype-phenotype correlations remain unclear. Its handling of the neuropathy overlap cannot be judged from the abstract.

    Easier

    This article reviews the bone growth conditions caused by changes in the TRPV4 gene, which range from very severe to mild. A small number of people have both a bone condition and nerve damage from the same TRPV4 change. In lab tests most of these changes make the channel more active, but how that leads to bone or nerve problems was not yet understood.

    Simple

    TRPV4 is like a door that lets calcium into cells. Many TRPV4 changes cause problems with how bones grow, and a few people also get nerve problems. In lab tests most of these changes left the door stuck too far open, but scientists did not yet know how that causes illness.

    Why these ratings

    Relevance 3/5: It is about allelic TRPV4 skeletal conditions, with a section on overlap with peripheral neuropathy and on channel activation.

    Evidence 1/5: Rated 1 because only the abstract was available, and a review summarizes earlier work rather than adding new data.

    Funding and conflicts of interest

    Not stated in the abstract.

    PubMed 22791502 · DOI

76. #76Abstract onlyCase report

    Spondyloepimetaphyseal dysplasia-Maroteaux type due to dominant TRPV4 mutation: expanding the phenotype with a case report

    Uzman CY, Çankaya T, Güleryüz H et al. · 2023 · Skeletal radiology

    Relevance 3/5Evidence 1/51 moderate problem

    This report describes a boy with a TRPV4 bone growth condition who also had joint contractures from birth. It adds to the wide range of conditions linked to TRPV4 but says nothing directly about CMT2C.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    Case report

    Who or what

    One boy with spondyloepimetaphyseal dysplasia Maroteaux type (SEMD-M), a TRPV4 skeletal condition, and a dominant TRPV4 mutation. The abstract does not name the variant or give his age.

    What they did

    The authors describe the clinical and X-ray findings in one boy with SEMD-M and highlight features not reported before in this condition. This analysis is based only on the abstract, so the methods cannot be judged.

    What they found

    • The boy had the clinical and X-ray signs of SEMD-M together with a dominant TRPV4 mutation.

      “We present a boy who has the clinical and radiological signs of SEMD-M with a dominant TRPV4 mutation.”Abstract, Case presentation

    • He also had features the authors say had not been seen in these patients before: a narrower interpedicular distance in his lower spine (the space between the two bony sides of each lumbar vertebra), contractures from birth and an arachnoid cyst (a fluid-filled sac near the brain or spinal cord).

      “These include a shorter distance between his lumbar vertebrae, congenital contractures, and an arachnoid cyst.”Abstract, Case presentation

    What the authors concluded

    The new features in this boy widen the known picture of SEMD-M caused by TRPV4 and may help doctors recognize the condition.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • ModerateMethods cannot be judged from the abstract

      Only the abstract was available, so the variant, how it was confirmed, and whether nerves and muscles were tested cannot be checked. From one patient it is not possible to tell whether the new features, such as the arachnoid cyst, are caused by TRPV4 or occurred by chance.

      “He also has some striking findings that have not been seen in these patients before”Abstract, Case presentation

    Three ways to explain it

    Scientific

    A boy with spondyloepimetaphyseal dysplasia Maroteaux type and a dominant TRPV4 mutation showed reduced lumbar interpedicular distance, congenital contractures and an arachnoid cyst, which the authors present as previously unreported features. Whether these are TRPV4-related or coincidental cannot be determined from a single case, and neurological assessment is not described in the abstract.

    Easier

    Doctors describe a boy with a bone growth condition caused by a change in the TRPV4 gene. He also had some features not reported before in this condition, including joints that were stiff from birth and a small fluid-filled sac near the brain or spine. With only one patient, it is not clear whether these features are caused by the TRPV4 change.

    Simple

    A boy with a TRPV4 change had bones that grew differently and joints that were stiff from birth. Doctors wrote about him so others can spot the condition.

    Why these ratings

    Relevance 3/5: It concerns an allelic TRPV4 skeletal condition; the abstract mentions contractures from birth but no nerve involvement.

    Evidence 1/5: Rated 1 because only the abstract was available; it is also a single case.

    Funding and conflicts of interest

    Not stated in the abstract.

    PubMed 35776137 · DOI

77. #77Abstract onlyLaboratory cell study

    TRPV4 acts as a mitochondrial Ca2+-importer and regulates mitochondrial temperature and metabolism

    Kanta Acharya T, Kumar A, Kumar Majhi R et al. · 2022 · Mitochondrion

    Relevance 3/5Evidence 1/51 moderate problem

    In lab-grown cells, TRPV4 was found in mitochondria, the cell's energy-makers, and affected how they work. This early lab work does not show what happens in the nerves of people with CMT2C.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    Laboratory cell study

    Who or what

    Various cell lines and several naturally occurring TRPV4 mutations. The abstract does not name the cell lines or the mutations.

    What they did

    The authors studied where TRPV4 sits inside cells and how changing its amount or function affects mitochondria, the parts of the cell that make energy. This analysis is based only on the abstract, so the methods cannot be judged.

    What they found

    • TRPV4 was found in a subgroup of mitochondria in several cell lines.

      “we demonstrate that TRPV4, localizes to a subpopulation of mitochondria in various cell lines.”Abstract

    • Abnormal amounts or function of TRPV4 caused several mitochondrial abnormalities, and TRPV4 was involved in controlling mitochondrial numbers, calcium levels and temperature. Several naturally occurring TRPV4 mutations affected mitochondrial shape and distribution.

      “TRPV4 is also involved in the regulation of mitochondrial numbers, Ca2+-levels and mitochondrial temperature.”Abstract

    • The authors suggest TRPV4 conditions might possibly be classed as mitochondrial diseases.

      “These findings may help in understanding the significance of mitochondria in TRPV4-mediated channelopathies possibly classifying them as mitochondrial diseases.”Abstract

    What the authors concluded

    TRPV4 acts at mitochondria and helps regulate their calcium, number and temperature. The authors suggest this may help explain TRPV4 conditions and that these could possibly be classed as mitochondrial diseases.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • ModerateMethods cannot be judged from the abstract

      Only the abstract was available, so the cell lines, whether TRPV4 was overproduced, which mutations were tested and how effects were measured cannot be checked. The suggestion that TRPV4 conditions might be mitochondrial diseases goes well beyond cell-line findings. The abstract's statement that mitochondrial abnormalities are prominent in all these conditions is also not supported within the abstract.

      “In all these cases, mitochondrial abnormalities are prominent.”Abstract

    Three ways to explain it

    Scientific

    In various cell lines, TRPV4 localized to a subpopulation of mitochondria, and altered TRPV4 expression or function induced mitochondrial abnormalities, with TRPV4 implicated in regulating mitochondrial number, Ca2+ levels and temperature. Several naturally occurring TRPV4 mutations altered mitochondrial morphology and distribution, leading the authors to propose that TRPV4 channelopathies might be considered mitochondrial diseases. The cell systems and mutations studied cannot be assessed from the abstract.

    Easier

    Mitochondria are tiny parts inside cells that make energy. This lab study found that the TRPV4 channel sits on some mitochondria, and that when TRPV4 was abnormal, the mitochondria changed in number, calcium level, temperature and shape. The authors suggest TRPV4 conditions might be partly diseases of the mitochondria, but the findings come only from cells grown in a dish.

    Simple

    Mitochondria are like tiny batteries inside our cells. Scientists found the TRPV4 door on some of these batteries, and when the door was faulty the batteries did not look or work normally in lab cells.

    Why these ratings

    Relevance 3/5: General TRPV4 cell biology that may bear on how TRPV4 changes harm cells, but the abstract does not say whether neuropathy mutations or nerve cells were studied.

    Evidence 1/5: Rated 1 because only the abstract was available; the work was also done in cell lines, not in people.

    Funding and conflicts of interest

    Not stated in the abstract.

    PubMed 36261119 · DOI

78. #78Full paperSingle-center genetic screening study of a CMT2 cohort

    The mutational spectrum in a cohort of Charcot-Marie-Tooth disease type 2 among the Han Chinese in Taiwan

    Lin KP, Soong BW, Yang CC et al. · 2011 · PloS one

    Relevance 2/5Evidence 3/52 moderate · 3 minor problems

    None of the 36 Taiwanese people with CMT2 in this study had a TRPV4 change, so CMT2C was not found in this group. One family in the study also shows that relatives who report no symptoms can still have mild signs when a neurologist examines them.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    Single-center genetic screening study of a CMT2 cohort

    Who or what

    36 unrelated Han Chinese people with CMT2 (one per family), selected from 251 CMT families seen at the neurology clinic of Taipei Veterans General Hospital, Taiwan. Inheritance was autosomal dominant in 20, autosomal recessive in 7 and apparently sporadic in 9. Genes screened included MFN2, RAB7, TRPV4, GARS, NEFL, HSPB1, MPZ, GDAP1, HSPB8, DNM2, AARS and YARS; new variants were checked against 500 local control subjects.

    What they did

    The team sequenced the coding parts of known CMT2 genes, including TRPV4, in each patient. For new variants they checked whether the change tracked with illness in the family, whether the amino acid is conserved across species, and whether controls carried it. They also tested one splice change in a cell-based (minigene) assay.

    What they found

    • A genetic cause was found in 14 of 36 patients (38.9%).

      “Ten disparate mutations were identified in 14 of the 36 unrelated patients (38.9% of our cohort)”Results

    • No one in the group had a TRPV4 change.

      “No mutations were found in RAB7, TRPV4, GARS, MPZ, HSPB8, DNM2, or YARS genes.”Results

    • The most common causes were NEFL changes (6 of 36, 16.7%) and MFN2 changes (5 of 36, 13.9%).

      “In the study cohort, the frequency of NEFL mutations (6/36; 16.7%) was unexpectedly high, and the frequency of MFN2 mutations (5/36; 13.9%) was consistent with other reports”Discussion

    • Most patients (22 of 36, 61.1%) still had no genetic diagnosis after screening.

      “Despite an extensive genetic survey, the genetic causes of CMT2 remain elusive in 22 of the CMT2 patients (61.1%).”Results

    • In one family (with an AARS change), relatives who said they had no symptoms were found to have mild signs when examined by a neurologist.

      “The proband's two younger sisters (II:3 and II:6) and niece (III:3) denied to have any neurological symptoms; however, neurological examinations uncovered mild atrophy, weakness in the intrinsic foot muscles (score 4+/5) and generalized hyporeflexia in all of them.”Results, clinical features of Patient 1

    What the authors concluded

    The study describes which CMT2 genes are involved in this Taiwanese Han Chinese group and adds six new variants. It supports AARS and HSPB1 as causes of axonal CMT, and NEFL changes were found unexpectedly often. The authors suggest NEFL and MFN2 should be tested first in ethnic Chinese people with CMT2. Approximately 60% of patients remained without a genetic answer, suggesting further genes are involved.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • ModerateSmall group, so the TRPV4 result is imprecise

      Finding no TRPV4 changes in 36 people shows CMT2C was not seen in this group, but it cannot show that CMT2C is absent or very rare in Taiwan as a whole.

      “Genomic DNA from 36 unrelated Taiwanese CMT2 patients of Han Chinese descent was screened for mutations in the coding regions”Abstract

    • MinorSelection: one clinic, sensory involvement required

      All patients came from one hospital clinic, and each had to show sensory nerve involvement on nerve tests so that purely motor inherited neuropathies were left out. The paper does not discuss whether these choices could change how often particular genes, including TRPV4, were found.

      “Sensory involvement was demonstrated in all the probands of the 36 CMT2 families by nerve conduction studies (NCS) to exclude hereditary motor neuropathy”Methods, Patients

    • MinorSequencing method could miss some kinds of change

      Direct sequencing of the coding regions finds small spelling changes but would not detect large deletions or duplications, or changes deep in non-coding DNA. The paper does not report testing for these in TRPV4 or the other genes.

      “Mutational analyses of the MFN2, RAB7, TRPV4, GARS, NEFL, HSPB1, MPZ, GDAP1, HSPB8, DNM2, AARS and YARS genes were performed by PCR amplification using intronic primers and direct DNA sequencing.”Methods, Mutation analysis

    • ModerateNew variants supported by small families

      The authors acknowledge that most families with new variants were too small to firmly prove the variants cause disease. The functional evidence for the AARS variant is cited only as a personal communication from an unpublished manuscript.

      “most of these pedigrees are small for segregation analysis to firmly support the pathogenic roles of these mutations.”Discussion

    • MinorCalculation slip and a published correction

      The discussion writes '10/36; 38.9%', but 38.9% matches the 14 patients with a mutation, not the 10 different mutations. The text also shows that a correction to this article was published; its content is not included here, so readers should check it.

      “identified 10 different mutations in 14 patients (10/36; 38.9% of our cohort)”Discussion

    How “unaffected” relatives were checked

    Not a TRPV4 family study. For new variants, segregation was judged from pedigrees with affected and unaffected members, but the paper mostly does not say how unaffected relatives were assessed. In the AARS family, relatives who 'denied to have any neurological symptoms' were examined and found to have mild signs, which shows why examination matters. The NEFL haplotype analysis included '2 unaffected individuals' without describing how they were assessed.

    Three ways to explain it

    Scientific

    Direct (dideoxy) sequencing of CMT2-associated genes in 36 unrelated Han Chinese CMT2 probands identified pathogenic variants in 14 (38.9%), mostly in NEFL (16.7%) and MFN2 (13.9%), with six novel variants supported by conservation, absence from controls and limited segregation. No TRPV4 variants were detected, and 61.1% remained genetically unexplained. The cohort is small and from a single center, so the absence of TRPV4 variants gives only an imprecise estimate of CMT2C frequency in this population.

    Easier

    In 36 unrelated people from Taiwan, the researchers checked the genes known at the time to cause the axonal form of CMT (CMT2). They found the cause in 14 people, most often in two genes called NEFL and MFN2. Nobody had a change in TRPV4, the CMT2C gene, and most people still had no genetic answer.

    Simple

    Doctors looked for spelling mistakes in a set of genes in 36 people with a nerve illness called CMT2. They found mistakes in some genes, but none in TRPV4, the calcium door gene linked to CMT2C. For many people the cause is still a mystery.

    Why these ratings

    Relevance 2/5: A general CMT2 gene screen in which TRPV4 was tested and no TRPV4 changes were found among 36 Taiwanese patients.

    Evidence 3/5: A careful but small single-center gene screen using older sequencing methods, with limited family data for the new variants.

    Funding and conflicts of interest

    Partially funded by the National Science Council (NSC97-2314-B-075A-002-MY3) and the Brain Research Center, National Yang-Ming University (V100-E6-006), Taiwan; the funders had no role in the study. The authors declare no competing interests.

    PubMed 22206013 · DOI

79. #79Full paperRetrospective multicenter case series (chart review) of genetic diagnosis

    Phenotype Presentation and Molecular Diagnostic Yield in Non-5q Spinal Muscular Atrophy

    Fernández-Eulate G, Theuriet J, Record CJ et al. · 2023 · Neurology. Genetics

    Relevance 2/5Evidence 3/52 moderate · 5 minor problems

    In this series of adults with motor-nerve SMA not caused by the usual gene, TRPV4 was one of the three most common causes. The TRPV4 patients typically had shoulder-blade and lower leg weakness starting in adult life, and often vocal cord problems. Only 4 TRPV4 patients were included, all with the motor form rather than CMT2C.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    Retrospective multicenter case series (chart review) of genetic diagnosis

    Who or what

    Seventy-one adults from 65 families with non-5q spinal muscular atrophy (motor nerve weakness with proximal involvement, without the usual SMN1 deletion): 61 from 10 French neuromuscular reference centers and 10 from London. TRPV4 was found in 4 patients from 3 families.

    What they did

    The authors reviewed clinical records, nerve tests and genetic results, grouping patients by weakness pattern: proximal only (P-SMA), proximal and distal (PD-SMA) or scapuloperoneal (SP-SMA). They measured how often gene panels and other tests found a cause and used statistics to see which clinical features predicted a genetic diagnosis. They also summarized the features of the most common genes, including TRPV4.

    What they found

    • Most patients had no family history and were classed as sporadic.

      “Seventy-one patients from 65 different families were included, mostly sporadic cases (60.6%).”Abstract

    • A genetic cause was found in 32 patients (45.1%). Neuropathy gene panels alone found a cause in 19 of 59 patients (32.2%).

      “Neuropathy gene panels were performed in 59 patients with a 32.2% diagnostic yield (19/59).”Abstract

    • The chance of finding a cause was highest in the scapuloperoneal pattern, the pattern most typical of TRPV4.

      “The genetic diagnostic yield was lowest in P-SMA (6/21, 28.6%) compared with PD-SMA (16/35, 45.7%) and SP-SMA (10/15, 66.7%).”Abstract

    • Earlier onset and a family history of the disease or consanguinity independently predicted finding a genetic cause.

      “An earlier disease onset and a family history of the disease or consanguinity were independent predictors of a positive genetic diagnosis.”Abstract

    • TRPV4 was one of the three most common causes. The 4 TRPV4 patients mostly had a scapuloperoneal pattern, weakness starting in adult life (18 to 53 years), absent reflexes, and often childhood orthopedic problems.

      “These patients showed predominantly a SP-SMA (3/4), although 1 patient presented with a PD-SMA without scapula alata.”Results, TRPV4 section

    • Three of the 4 TRPV4 patients had vocal cord paralysis with a weak voice, and three had some breathing weakness, none needing ventilation.

      “Three patients had some degree of respiratory insufficiency, none requiring ventilation. Three of these patients also had vocal cord paralysis with hypophonia.”Results, TRPV4 section

    What the authors concluded

    Non-5q SMA has many genetic causes, and routine neuropathy gene panels find the cause in about one-third of patients. BICD2, DYNC1H1 and TRPV4 account for almost two-thirds of diagnosed patients. Adding neuromuscular and metabolic genes, muscle MRI and careful clinical review can raise the yield, but more than half remain without a diagnosis.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • ModerateRetrospective review at specialist centers

      Patients were identified from records at national reference centers, which tend to see more complex or unusual cases, and only adults were included. The diagnostic yield and features may differ in other settings.

      “we retrospectively reassessed the clinical records of adult patients (18 years or older)”Methods, inclusion and exclusion criteria

    • ModerateTRPV4 group very small

      Only 4 TRPV4 patients from 3 families were included, so the description of TRPV4 features (adult-onset weakness, vocal cord paralysis, breathing problems) is anecdotal and cannot give reliable frequencies.

      “TRPV4 (4 patients from 3 families).”Results, TRPV4 section

    • MinorPeople with sensory involvement excluded by design

      Patients needed near-normal sensory nerve responses to be included. This is appropriate for studying SMA, but it means TRPV4 patients with a CMT2C-like sensory picture were excluded, so the TRPV4 findings describe only the motor form.

      “less than 30% reduction of normal sensory nerve action potential (SNAP) amplitudes”Methods, inclusion and exclusion criteria

    • MinorDifferent genetic tests used

      Panels ranged from 15 to 148 genes across several laboratories, and some patients had exome or genome tests. The overall yield therefore mixes tests of different breadth, although the authors found no significant difference between the main Lyon panel and the others.

      “Eleven additional neuropathy gene panels were sequenced by the National Hospital for Neurology and Neurosurgery (NHNN) in London (n = 4, 15-32 genes)”Methods, neuropathy gene panels

    • Minor'Sporadic' based on family history only

      Patients were classed as sporadic from recorded family history, without examining or testing relatives. Several patients with dominant genes had no clear family history, which could reflect mild or unrecognized disease in relatives, new changes or incomplete history.

      “only 13/20 patients had a clear AD family history of the disease, the rest were therefore categorized as sporadic cases”Results, most common genes

    • MinorUnclear exclusion criterion

      Patients whose disease progressed at 6 months were excluded, but the reason is not explained. This may have been meant to exclude faster-progressing conditions, but it is unclear how it affected who was included.

      “Patients with clinical or neurophysiologic disease progression at 6 months were excluded.”Methods, inclusion and exclusion criteria

    • MinorCorrections published and key tables not in this text

      The article has had two published corrections whose content is not included here. Tables 1 and 2, which hold the clinical data and the predictor statistics, are not reproduced in the text available, so those numbers could not be checked.

      “This article has been corrected.”Article notes; Tables 1 and 2

    How “unaffected” relatives were checked

    Not a family or penetrance study. 'Sporadic' status was based on recorded family history, not on examining or testing relatives. Quote: 'most patients had no family history of neuromuscular disease or consanguinity and were therefore classified as sporadic (60.6%)' (Results).

    Three ways to explain it

    Scientific

    In a retrospective series of 71 adults with non-5q SMA from French and London reference centers, a molecular diagnosis was reached in 32 (45.1%), most often in BICD2, DYNC1H1 and TRPV4; neuropathy gene panels alone yielded 19/59 (32.2%). Yield was highest in scapuloperoneal presentations and was independently predicted by earlier onset and family history or consanguinity. The 4 TRPV4 patients mostly had scapuloperoneal SMA with adult-onset weakness, areflexia and frequent vocal cord paralysis and respiratory involvement, a subgroup too small for firm conclusions.

    Easier

    This study looked back at the records of 71 adults who had weakness caused by damage to the nerves that move muscles but did not have the usual gene change behind spinal muscular atrophy. Doctors found the genetic cause in fewer than half. TRPV4 was one of the three most common causes, found in 4 people from 3 families. Those 4 mostly had weak shoulder-blade and lower leg muscles starting in adult life, and 3 had vocal cord weakness.

    Simple

    Doctors looked at 71 adults whose muscles were weak because of nerve problems and searched their genes for the cause. They found it in a bit less than half of them. TRPV4, the gene for a calcium door in cells, was the cause in only 4 people, so we cannot learn much about TRPV4 from this study alone.

    Why these ratings

    Relevance 2/5: It is mainly about genetic testing in non-5q SMA, with a short section on 4 TRPV4 patients who have the motor (SPSMA-type) rather than CMT2C form.

    Evidence 3/5: A reasonably large multicenter series for a rare condition, but retrospective, from specialist centers, with mixed genetic tests and only 4 TRPV4 patients.

    Funding and conflicts of interest

    The authors report no targeted funding and no relevant disclosures.

    PubMed 37470033 · DOI

80. #80Full paperCross-sectional skin biopsy study with healthy controls

    Charcot-Marie-Tooth disease: New insights from skin biopsy

    Manganelli F, Nolano M, Pisciotta C et al. · 2015 · Neurology

    Relevance 2/5Evidence 3/52 moderate · 3 minor problems

    In three people with CMT2C, fingertip skin biopsies showed loss of touch receptors and nerve endings, apparently more than in CMT1A, but three people is too few to be sure. Skin biopsy may one day help track nerve loss, but this study did not test that over time.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    Cross-sectional skin biopsy study with healthy controls

    Who or what

    31 adults with genetically confirmed CMT (CMT1A 10, CMTX1 9, late-onset CMT1B 5, CMT2B 3, CMT2C 3, AR-CMT2K 1) and 45 healthy controls. 23 patients were recruited at the University Federico II of Naples and 8 at the University of Verona. All patients were older than 18.

    What they did

    Everyone had a 2-mm punch biopsy from a fingertip, and patients also had a neurological examination, nerve conduction tests and a CMT Neuropathy Score (CMTNS). Under a microscope, the researchers counted touch receptors (Meissner corpuscles) and myelinated nerve endings, and measured the insulated segments of nerve fibers (internodes) and the gaps between them (nodal gaps). They compared CMT types with each other and with controls.

    What they found

    • Every CMT type, including CMT2C, had fewer touch receptors (Meissner corpuscles) and myelinated nerve endings in the fingertip than healthy controls.

      “The 2-group comparison (Mann-Whitney U test) showed that a loss (p < 0.005) of MC and IME was present in the fingertip of all patients from each CMT genotype compared to healthy controls”Results, 'Quantification of MCs and myelinated nerve fibers'

    • The axonal CMT types as a group tended to have greater loss than CMT1A, and this was especially evident in the 3 people with CMT2C.

      “The axonal group as whole tended to have a greater loss of MC and IME than CMT1A, and this was especially evident in the CMT2C group”Results, 'Quantification of MCs and myelinated nerve fibers'

    • Severity was mild to moderate overall with no significant difference between CMT types, but the CMT2C group showed a trend toward greater impairment (CMTNS = 17.6, as reported).

      “CMTNS did suggest a trend toward greater impairment in CMT2C (CMTNS = 17.6)”Results, 'Patients'

    • In every CMT type, including TRPV4-related CMT2C, the insulated nerve segments were shorter and the gaps between them longer than in controls. CMT1A had the shortest segments and widest gaps, and there was no difference between CMT caused by myelin genes and by axon genes such as TRPV4.

      “each CMT genotype had shorter internode (p < 0.009) and longer nodal gap (p < 0.0001) compared to healthy controls”Results, 'Morphometric analysis of myelinated nerve fibers'

    • Loss of receptors and nerve endings did not correlate significantly with the CMT Neuropathy Score or with muscle response size in any CMT type.

      “The Spearman rank-order correlation test did not reveal significant correlations between MC or IME loss and CMTNS and compound muscle action potential (CMAP) amplitude (included in CMTNS) for any CMT genotype.”Results, 'Quantification of MCs and myelinated nerve fibers'

    What the authors concluded

    Fingertip skin biopsy can detect axon loss and changes at and between the nodes of nerve fibers in both axonal and demyelinating CMT. Shortened internodes in all types suggest that both myelin and axon gene mutations may disturb how nerve segments form during development. The degree of shortening and gap widening may help explain nerve conduction speeds. Because biopsies can be repeated, they might be used to follow the course of the disease, but larger studies are needed.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • ModerateOnly 3 people with CMT2C

      Any statement about CMT2C rests on three people, whose specific TRPV4 variants are not given in the text. Results from so few people could easily reflect individual differences rather than CMT2C in general.

      “CMT1A (n = 10), CMTX1 (n = 9), late-onset CMT1B (n = 5), CMT2B (n = 3), CMT2C (n = 3), and AR-CMT2K (n = 1)”Methods, 'Patients'

    • ModerateCMT2C differences described, not tested

      The greater loss in CMT2C is described as a tendency, and the higher CMTNS as a trend, without a statistical test showing these differences are reliable. With groups of 3 and 1, such comparisons cannot be firm.

      “CMTNS did suggest a trend toward greater impairment in CMT2C (CMTNS = 17.6)”Results

    • MinorMain results table not in the copy analyzed

      The values for each CMT type are in Table 1, whose contents were not included in the text supplied, so the CMT2C numbers could not be checked here.

    • MinorOne time point and one biopsy site

      Each person had one fingertip biopsy at one time. The idea that skin biopsy could track disease over time is a suggestion, not something this study tested, and the authors call for larger studies.

      “further studies on larger CMT samples are needed to better define the sensitivity of MC density as a measure of axonal degeneration”Discussion

    • MinorBlinding not stated

      The paper does not say whether the people counting receptors and measuring fibers knew which samples came from patients or controls, which can influence measurements.

    Three ways to explain it

    Scientific

    Fingertip skin biopsies from 31 genetically defined CMT patients, including 3 with CMT2C, showed reduced Meissner corpuscle and intrapapillary myelinated ending densities in every genotype compared with 45 controls, with a tendency to greater loss in axonal forms, especially CMT2C. Dermal myelinated fibers in all genotypes, including TRPV4, had shortened internodes and widened nodal gaps, most marked in CMT1A, suggesting shared developmental and axon-glial abnormalities. CMT2C-specific observations rest on 3 patients and descriptive comparisons.

    Easier

    Researchers took a tiny piece of fingertip skin from people with different types of CMT, including three people with CMT2C, and from healthy people. Everyone with CMT had fewer touch sensors and nerve endings in the skin than healthy people, and the loss tended to be greater in the CMT2C group. The insulated sections of nerve fibers were shorter, and the gaps between them wider, in all types of CMT. With only three people with CMT2C, the CMT2C results are a hint rather than a firm finding.

    Simple

    Doctors looked at tiny bits of fingertip skin under a microscope. People with CMT, including three with CMT2C, had fewer of the little touch sensors than healthy people. Three people is a very small group, so this is only a first clue.

    Why these ratings

    Relevance 2/5: A study of several CMT types in which CMT2C is represented by only 3 patients, with a few CMT2C-specific observations.

    Evidence 3/5: A reasonable controlled study using a standard biopsy method, but small, from a single time point, and with very small subgroups for the rarer CMT types.

    Funding and conflicts of interest

    No targeted funding reported. The authors report no disclosures relevant to the manuscript.

    PubMed 26362287 · DOI

81. #81Full paperGenetic screening study using whole-exome sequencing in families

    Improved inherited peripheral neuropathy genetic diagnosis by whole-exome sequencing

    Drew AP, Zhu D, Kidambi A et al. · 2015 · Molecular genetics & genomic medicine

    Relevance 2/5Evidence 3/53 moderate · 3 minor problems

    Exome sequencing found a known cause in 8% of families still undiagnosed after standard tests. The single TRPV4 change found was judged probably harmless, which illustrates that a rare TRPV4 variant on a genetic test report is not automatically the cause of someone's neuropathy.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    Genetic screening study using whole-exome sequencing in families

    Who or what

    110 unrelated index patients with inherited peripheral neuropathy from families seen at a Sydney neurogenetics clinic (including 43 with CMT2, 27 with X-linked CMT, 15 with motor neuropathy with pyramidal signs and 14 with hereditary motor neuropathy), all without a diagnosis after testing of common genes. 1000 control chromosomes from neurologically normal people. Only one TRPV4 variant (p.E183D) was found, in one patient.

    What they did

    They sequenced the protein-coding parts of all genes (the exome) and examined 75 known neuropathy genes. Variants were confirmed by Sanger sequencing, tested in relatives where possible, checked in controls and scored with conservation and prediction software.

    What they found

    • A previously reported disease-causing mutation was found in 9 of the 110 families (8%), in MFN2, GJB1, BSCL2 and SETX.

      “Nine variants (8%), identified in the genes MFN2 , GJB1 , BSCL2, and SETX , are previously reported mutations and considered to be pathogenic in these families.”Abstract; Results, Table 2

    • Twelve new variants (11%) in five genes, including TRPV4, were possible causes but could not be proven.

      “Twelve novel variants (11%) in the genes NEFL , TRPV4 , KIF1B , BICD2 , and SETX are implicated in the disease but require further evidence of pathogenicity.”Abstract; Results, Table 3

    • The only TRPV4 finding, p.E183D, was in one patient with axonal motor and sensory neuropathy and retinal degeneration. Relatives could not be tested, prediction programs called it probably harmless, and the authors concluded it is probably a rare harmless variant.

      “Collectively, these data suggest that p.E183D is a rare polymorphism.”Results, Mutation in TRPV4

    • Six of the diagnoses were in genes that are routinely tested (MFN2 and GJB1). They had been missed because the inheritance pattern was not obvious, the family had not been retested, or an older test was less sensitive.

      “Six of the mutations identified in this study were in genes that are routinely screened as part of normal diagnostic procedures”Discussion

    • A SETX variant reported elsewhere as a possible cause of disease did not travel with disease in a CMT2 family, and the authors reclassified it as harmless.

      “we identified one SETX sequence variant (c.7640T>C), previously reported as a putative mutation, which we have confirmed as a nonpathogenic rare polymorphism”Abstract; Results

    What the authors concluded

    Exome sequencing found a known genetic cause in 8% of families that had no diagnosis after standard testing and possible causes in another 11%. It was especially useful when the inheritance pattern was unclear. Testing common genes first in clinically clear cases remains important. Many new variants, including the TRPV4 variant, could not be proven to cause disease.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • MinorCounting errors

      Table 1 lists 3 previously reported and 7 novel variants for CMT2 but gives the CMT2 total as 9 (8.2%), and gives the overall 21 variants as 18.2%. The text says 62 variants minus 20 false positives left 41. The phenotype counts in the methods do not reach 110, and Family B is called CMT2 in Table 2 but demyelinating CMT in the text. These slips do not change the main message but reduce confidence in exact figures.

      “WES initially identified 62 sequence variants in 20 known IPN genes. Twenty variants were excluded as false positives by Sanger sequencing. Of the remaining 41 sequence variants”Results; Table 1; Table 2; Methods, Study participants

    • ModerateDiscussion overstates segregation and control testing

      The discussion says all 12 novel variants segregated in families and were absent from controls. But for several families, including the TRPV4 family, no relatives could be tested, and the methods say only variants that segregated were checked in controls. Whether the TRPV4 variant was checked in controls is therefore unclear.

      “A further 12 novel sequence variants (Table 3 ) were identified that segregated in families and were absent from 1000 neurologically normal control chromosomes.”Discussion; compare Methods, Genetic analysis and Results, Mutation in TRPV4

    • ModerateNew variants unproven

      No lab tests of protein function were done and families were too small for linkage, so the 12 new variants remain uncertain. The authors acknowledge this.

      “We therefore cannot conclusively assign pathogenicity to the novel sequence variants.”Results, Identification of variants in known IPN genes

    • MinorTRPV4 variant judged on very little information

      The TRPV4 conclusion rests on a family history given by the patient and on prediction software, with no relatives tested. The authors themselves say more information is needed.

      “Family history, based on the patient's description, suggests an autosomal dominant inheritance, however segregation analysis was not possible”Results, Mutation in TRPV4

    • ModerateHarmless-variant rule assumes high penetrance, and relatives' status is assessed inconsistently

      One rule for calling a variant harmless was that two or more unaffected at-risk relatives carried it, assuming high penetrance. Some relatives' status comes from the patient's report rather than examination, and nerve conduction testing of 'unaffected' relatives is not described. Where some carriers have mild or no symptoms, this rule could wrongly dismiss a real disease variant.

      “assuming high penetrance for CMT, two or more unaffected at-risk individuals carry the variant”Results, Rare polymorphisms

    • MinorGaps in sequencing coverage not filled

      Exome sequencing does not read every part of every gene well. Gaps were not re-sequenced and larger deletions or duplications were not looked for, so some causes may have been missed.

      “As we did not resequence gaps using an alternative method, it is possible that a small number of additional mutations may be identified within these regions.”Discussion

    How “unaffected” relatives were checked

    Varied between families and is often not detailed. Some relatives were examined, for example a KIF1B carrier mother: “The mother of the two affected sisters was clinically examined and found to be asymptomatic suggesting reduced penetrance of the disease.” Other family histories came from the patient's own report. For the TRPV4 family no relatives were tested: “Family history, based on the patient's description, suggests an autosomal dominant inheritance, however segregation analysis was not possible”. Nerve conduction testing of unaffected relatives is not described.

    Three ways to explain it

    Scientific

    Whole-exome sequencing of 110 index patients with genetically unresolved inherited peripheral neuropathy identified previously reported pathogenic mutations in 9 families (8%, in MFN2, GJB1, BSCL2 and SETX) and 12 novel candidate variants (11%) whose pathogenicity remains unproven. The only TRPV4 finding, p.E183D in a patient with axonal sensorimotor neuropathy and retinal degeneration, could not be tested for segregation, was predicted tolerated by SIFT and Polyphen-2, and was considered a probable rare polymorphism. Several internal count discrepancies reduce confidence in exact figures.

    Easier

    The researchers read all the genes of 110 people with inherited nerve disease whose cause had not been found by standard tests. They found a known cause in 9 families and possible new causes in 12 more, but those 12 could not be proven. One person had a rare change in TRPV4, but computer checks suggested it was probably harmless and no relatives could be tested.

    Simple

    Scientists read the whole instruction book of 110 people with nerve problems to look for typing mistakes. They found the cause for some families, and one person had a small change on the TRPV4 page that looked like a harmless typo, though they could not be completely sure.

    Why these ratings

    Relevance 2/5: A general neuropathy gene study whose only TRPV4 content is one variant judged probably harmless.

    Evidence 3/5: A moderate-sized family cohort with confirmation and controls, but the new variants are unproven, families are small and several counts in the paper do not add up.

    Funding and conflicts of interest

    Funded by a National Health and Medical Research Council project grant, a Ministry of Higher Education (MOHE) grant and a 2012 Australia Endeavour Research Fellowship. Conflict of interest: none declared.

    PubMed 25802885 · DOI

82. #82Full paperMulticenter development and validation study of an outcome measure

    Development and validation of the Charcot-Marie-Tooth Disease Infant Scale

    Mandarakas MR, Menezes MP, Rose KJ et al. · 2018 · Brain: a journal of neurology

    Relevance 2/5Evidence 3/54 moderate · 2 minor problems

    This study gives researchers a tested way to measure CMT in children under five, which could matter for future studies that include young children with CMT2C. It says almost nothing about CMT2C itself.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    Multicenter development and validation study of an outcome measure

    Who or what

    128 children aged 0 to 4 years assessed at seven sites in Australia, the USA, Italy and Thailand: 26 with confirmed inherited neuropathy (17 CMT1A, one CMT1B, one CMT1D, one CMT2C, one CMT2S, two CMT4C, one CMTX3, one Riboflavin Transporter Deficiency Type 2 and one unidentified mutation), 7 'at risk' and 95 healthy controls. The one child with CMT2C was 16 months old. Earlier steps involved 12 expert reviewers and a pilot in 22 children.

    What they did

    The team drew up 31 possible test items from a literature review and expert input, piloted them, then used them with 128 young children. Using statistical methods (item, factor and Rasch analysis), they cut the list to 15 items. They then checked whether different testers got the same scores, whether the scale separates children with CMT from healthy children, and how it compares with an existing scale for older children (CMTPedS).

    What they found

    • The final CMT Infant Scale (CMTInfS) has 15 play-based items and takes about 20 minutes.

      “The final version of the CMTInfS requires 20 min to administer and is a reliable and sensitive functional outcome measure for early onset CMT and related neuropathies.”Abstract

    • Scores were highly consistent when the same tester rescored from video and when different testers scored the same videos.

      “The CMTInfS demonstrated high intra-rater reliability [intraclass correlation coefficient (ICC)3,1 0.999, 95% confidence interval 0.996-1.000) and inter-rater reliability (ICC2,1 0.997, 95% confidence interval 0.992-0.999).”Abstract

    • Children with CMT scored worse than healthy children, and the most severe subtypes scored worst.

      “Patients with the most severe neuropathy subtypes (CMT1D, CMT2S and RTD2) had the highest CMTInfS z-scores”Results, 'Discriminant ability'

    • In 10 children aged 3 to 4, scores agreed reasonably well with the existing pediatric scale (CMTPedS).

      “There was good agreement between scores (Pearson’s r = 0.76, P = 0.01)”Results, 'Convergent validity'

    • Change over one year was checked in only four children with CMT1A: two worsened and two stayed relatively stable.

      “Two patients worsened (percentage change 276-343%) and two remained relatively stable”Results, 'Longitudinal responsiveness'

    • Only one child with CMT2C was included among the 26 children with confirmed neuropathy.

      “26 confirmed cases of inherited neuropathy (17 CMT1A, one CMT1B, one CMT1D, one CMT2C, one CMT2S, two CMT4C, one CMTX3, one Riboflavin Transporter Deficiency Type 2, and one unidentified mutation)”Abstract

    What the authors concluded

    The CMTInfS is a reliable 15-item measure of how CMT affects function in children under five, suitable for clinical care, natural history studies and future clinical trials. The authors say larger numbers, more rare subtypes and longer follow-up are still needed.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • ModerateAlmost no CMT2C data

      Only one child with CMT2C was included, and that child's scores are not reported separately in the text. Whether the scale works well for children with CMT2C in particular is not tested.

      “17 CMT1A, one CMT1B, one CMT1D, one CMT2C, one CMT2S, two CMT4C”Abstract

    • ModerateSmall affected group, mostly one subtype

      Of the 26 children with confirmed CMT, 17 had CMT1A. Most other subtypes had one child each, so the scale's behavior in rarer types is uncertain. The authors acknowledge this.

      “a larger sample size of patients, especially with rarer genetic subtypes is required.”Discussion, limitations

    • ModerateReliability tested mainly in healthy children

      The same-tester reliability check used 21 children, of whom 19 were healthy controls, and total scores could be calculated for only 12. The between-tester check used 10 videos, four of children with CMT. Very high reliability in mostly healthy children may not carry over fully to children with CMT.

      “The intra-rater reliability analyses involved 21 infants and young children (two CMT1A, 19 controls)”Results, 'Reliability'

    • ModerateSensitivity to change shown in only four children

      For use in trials, a scale must pick up change over time. This was tested in four children with CMT1A over about a year. The authors say more follow-up is needed, so the word 'sensitive' in the conclusion should be read with this in mind.

      “longer-term follow-up for more patients is necessary to make any definitive statements about progression.”Discussion, limitations

    • MinorInconsistent numbers in the paper

      The abstract gives P = 0.006 for the difference between the CMT group and controls, but the results give P = 0.001 for that comparison and P = 0.006 for CMT1A versus controls. The control group's country counts (71, 27 and 1) add up to more than 95, Table 2 has an age band labeled 21-36 that overlaps others, and some control heights and BMI values in Table 1 look implausible for under-fives (up to 180 cm; BMI as low as 5). A correction notice is listed for this article, but its content is not in the document.

      “independent samples t-test, P = 0.001”Abstract compared with Results 'Participants', 'Discriminant ability', Tables 1 and 2

    • MinorBlinding of live assessments not reported

      The paper does not say whether the people scoring children in person knew whether each child had CMT. Knowing this could influence scoring when comparing children with CMT and healthy children.

    Three ways to explain it

    Scientific

    The authors developed the CMT Infant Scale (CMTInfS), a 15-item, Rasch-built functional outcome measure for children aged 0 to 4 years, from a 31-item pool tested in 128 children (26 with confirmed inherited neuropathy, 7 at risk, 95 controls). Scores are expressed as z-scores against age- and gender-matched normative values. The scale showed good Rasch model fit, high intra- and inter-rater reliability, discrimination between affected children and controls, and correlation with the CMTPedS (r = 0.76). Responsiveness was examined in only four children with CMT1A, and only one child with CMT2C was included.

    Easier

    Researchers need a reliable way to measure how much CMT affects very young children before trials can include them. This team built a play-based test that takes about 20 minutes and has 15 tasks, such as sitting, rolling and tearing paper. Different testers gave very similar scores, and children with CMT scored worse than healthy children. Only one child with CMT2C took part, and change over time was checked in just four children with another type of CMT.

    Simple

    Scientists made a set of play tasks, like sitting up and tearing paper, to measure how CMT affects babies and toddlers. The tasks are a bit like a ruler for movement skills. They worked well in tests, but only one child with CMT2C was included.

    Why these ratings

    Relevance 2/5: A general CMT measurement tool for young children that could be used in future CMT2C studies, but only one child with CMT2C took part and no CMT2C-specific results are given.

    Evidence 3/5: A careful multicenter design, but the affected group is small and mostly CMT1A, reliability was tested mainly in healthy children, and change over time was checked in only four children.

    Funding and conflicts of interest

    Supported by the Inherited Neuropathies Consortium, part of the NIH Rare Diseases Clinical Research Network (NIH grant U54NS065712, via NINDS and the Office of Rare Diseases); the consortium also receives support from the Muscular Dystrophy Association and the Charcot Marie Tooth Association. Also a University of Sydney South East Asia Centre Cluster Research Grant (2015). Competing interests: several authors report support from CMT patient associations; M.E.S. reports support from Inflectis Pharmaceuticals and consultancy for Alnylam, Flex Pharma and Accelerant Pharmaceuticals; K.J.R. has consultancy agreements with Biogen Idec, IONIS Pharmaceuticals, Hoffmann-La Roche and ATOM International; K.E. receives consulting fees from Acceleron Pharmaceuticals.

    PubMed 30476010 · DOI

83. #83Full paperRetrospective single-center case series (brief report)

    Expanding the Genotype and Phenotype Diversity in a Chinese Cohort With TRPV4-Related Dysplasia

    Dong L, Ho KC, Tan Z et al. · 2026 · Clinical genetics

    Relevance 2/5Evidence 3/53 moderate · 2 minor problems

    This study is about TRPV4 bone conditions rather than CMT2C, but it reports the R232C change, also seen in neuropathy, in a patient with a bone condition and no recorded neuropathy. Nerve tests were not reported, so it does not show whether that patient's nerves were truly unaffected.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    Retrospective single-center case series (brief report)

    Who or what

    10 Chinese patients (7 males, 3 females) diagnosed with TRPV4-related skeletal dysplasia at one Shenzhen hospital between 2020 and 2025: 6 with spondylometaphyseal dysplasia Kozlowski type (SMDK) and 4 with metatropic dysplasia (MD). Three different TRPV4 variants were found in the MD group, including one new variant of uncertain significance (p.L543R), and four in the SMDK group, including p.R232C in one patient.

    What they did

    They reviewed medical records, X-rays and whole-exome sequencing results. They described spine, leg bone and pelvis changes, the TRPV4 variants found, and neurological complications. They also discussed treatment in light of two patients who had spinal surgery.

    What they found

    • Curvature of the spine (scoliosis) was found in almost all patients, with severity ranging from mild to severe.

      “Scoliosis was highly prevalent, identified in 9/10 patients (90%), with considerable variability in severity.”Results, Spinal features

    • Two patients with metatropic dysplasia had neurological problems due to spinal cord compression; none with SMDK did.

      “Notably, two MD patients presented with spinal cord compression with neurological deficits, whereas none of the SMDK patients exhibited neurological complications.”Results, Spinal features

    • The R232C variant, previously reported in hereditary motor and sensory neuropathy and congenital distal SMA, was found in a patient with the bone condition SMDK, whose table entry shows no neuropathy.

      “One SMDK patient involved the c.694C>T (R232C) variant previously reported as associated with hereditary motor and sensory neuropathy and congenital distal spinal muscular atrophy”Discussion; Table 1 (Case 6)

    • A new TRPV4 variant, L543R, was found in a patient with metatropic dysplasia and classified as a variant of uncertain significance.

      “According to the ACMG guidelines, this variant was classified as VUS (variant of uncertain significance)”Results, Genotypes presentation; Table 1

    • Of two patients who had cervical spine fusion, the one operated on soon after weakness began partly recovered, while the one operated on after near-complete paralysis did not.

      “one patient who underwent cervical spinal fusion shortly after the onset of weakness showed partial neurological recovery”Discussion, Spinal instability and neurological complications

    What the authors concluded

    TRPV4 skeletal dysplasias in this Chinese group showed varied spine, leg and pelvis problems, and the exact variant did not reliably predict the type of dysplasia. Spinal instability and cord compression were the main neurological risk, especially in metatropic dysplasia, so the authors argue for regular spine surveillance and multidisciplinary care.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • ModerateNeuropathy not formally tested

      No nerve conduction studies or EMG are reported. The table's 'Neuropathy' column marks the two MD patients with cord compression as positive, while the text says their problems came from the spine. The R232C patient is marked as having no neuropathy, apparently on clinical grounds. The authors say telling TRPV4 neuropathy apart from spinal compression is essential, but do not report how they did it.

      “it is essential to differentiate TRPV4 -related peripheral neuropathy from neurological abnormalities secondary to vertebral compression”Discussion; Table 1

    • ModerateSmall, retrospective, single-center group

      Ten patients from one orthopedic service were studied by looking back at records. The authors acknowledge possible ascertainment bias, so the frequencies of features and variants may not apply to other groups.

      “given the small, single-center cohort ( n = 10) and potential ascertainment bias”Discussion, Genotype-phenotype correlations

    • ModerateConflicting statements about the new variant

      L543R is called a novel variant not previously reported, but the discussion says it was identified in prior studies of Wolfram syndrome, citing a study of a different gene (WFS1). These statements conflict, so the variant's status is unclear. No testing of parents or lab study of the variant is described.

      “identified in prior studies as related to Wolfram syndrome, an autosomal recessive neurodegenerative disorder”Discussion, Genotype-phenotype correlations; compare Results, Genotypes presentation

    • MinorInconsistent counts

      The results say atlantoaxial instability and spinal stenosis were found in 5/10 patients, while the discussion gives 5 for instability and 3 for stenosis. The abstract names R594H and P799R as the most recurrent variants, although P799L also occurred in two patients.

      “atlantoaxial instability was identified in 5 patients (50%), and spinal stenosis, affecting either the cervical or thoracic spine, in 3 patients (30%)”Discussion; compare Results, Spinal features and Abstract

    • MinorSpeculation and advice beyond the data

      The discussion links symptoms to 'baseline calcium elevation', which was not measured. Its treatment recommendations (bracing, casting, types of spinal surgery) were not tested in this study and rest on general experience and two surgical cases.

      “suggesting that the degree of baseline calcium elevation caused by TRPV4 mutations correlates with the development of mixed phenotypes in skeleton and peripheral nerves”Discussion, Spinal instability and neurological complications

    Three ways to explain it

    Scientific

    In a retrospective single-center series of 10 Chinese patients with TRPV4-related skeletal dysplasia (6 SMDK, 4 MD), scoliosis (9/10), platyspondyly, metaphyseal changes and joint contractures were common, and two MD patients had neurological deficits attributed to spinal cord compression. Variants associated elsewhere with other phenotypes (P799L, R616Q and the neuropathy-associated R232C) occurred in SMDK, and a novel p.L543R variant was classified as a VUS, consistent with the absence of a clear genotype-phenotype correlation. No nerve conduction studies are reported, so peripheral nerve involvement was not objectively assessed.

    Easier

    Doctors looked back at the records of 10 Chinese patients whose TRPV4 change caused problems with bone growth. Most had a curved spine and stiff joints, and two with the more severe type had nerve problems because the spine pressed on the spinal cord. The same TRPV4 change could go with different conditions: R232C, a change also reported in nerve disease, was found in a patient with a bone condition, though nerve tests were not reported.

    Simple

    Changes in TRPV4 can affect bones as well as nerves. In 10 patients with bone problems, the same gene change did not always cause the same problem, a bit like the same recipe turning out differently in different kitchens.

    Why these ratings

    Relevance 2/5: It is about TRPV4 skeletal dysplasia; its neuropathy content is small, though it reports a patient with the neuropathy-linked R232C variant who had a bone condition.

    Evidence 3/5: A small retrospective single-center series; the descriptions are useful, but nerve testing is not reported and several statements go beyond or conflict with the data.

    Funding and conflicts of interest

    Supported by the National Natural Science Foundation of China and several Shenzhen research programs. Disclosure: the authors state they have nothing to report.

    PubMed 41263626 · DOI

84. #84Full paperNarrative review

    The distal hereditary motor neuropathies

    Rossor AM, Kalmar B, Greensmith L et al. · 2012 · Journal of neurology, neurosurgery, and psychiatry

    Relevance 2/5Evidence 2/51 moderate · 3 minor problems

    This older review places TRPV4 among the genes behind inherited nerve conditions that mainly weaken the hands and feet. It notes that TRPV4 disease often involves the vocal cords and can vary a lot within families. It also shows that, when it was written, scientists were not yet sure exactly how TRPV4 changes damage nerves.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    Narrative review

    Who or what

    A review of the distal hereditary motor neuropathies (dHMN), inherited conditions that mainly weaken the hands and feet. It covers the known genes and loci at the time (the abstract says eleven genes and four loci), with a short section on TRPV4. No new patients or experiments.

    What they did

    Authors from a UK neuromuscular center summarized published clinical features of each genetic type of dHMN and proposed an order for genetic testing. They also reviewed laboratory research on how each gene damages motor nerves, and on possible treatments. The copy analyzed here is a text version without the figures and tables, so the testing algorithm was not available.

    What they found

    • dHMN overlaps with the axonal type of Charcot-Marie-Tooth disease (CMT2); the same gene change can produce either picture.

      “there is an overlap between the axonal forms of CMT (CMT2) and dHMN, where the same mutation in a gene may cause both phenotypes.”Introduction

    • At the time of writing, most people with dHMN had no genetic diagnosis.

      “more than 80% of patients with dHMN have mutations in undiscovered genes.”Diagnosis

    • TRPV4 is listed as one cause of dHMN type VII, the form defined by vocal cord paralysis, and is also linked to scapuloperoneal SMA and CMT2C.

      “Type VII is defined by vocal-cord paralysis and can be due to mutations in DCTN1, TRPV4 or in an as-yet unidentified gene on chromosome 2q14.”Classification

    • Summarizing one large series, the review reports that most TRPV4 carriers had symptoms before age 14 and half had vocal cord palsy, and that severity often varies within families, including an unaffected 70-year-old carrier.

      “In the majority of carriers, symptoms developed before 14 years of age, and vocal-cord palsy was present in 50%.”DHMN causative genes, TRPV4

    • At the time, lab studies disagreed on whether neuropathy-causing TRPV4 changes increase or decrease calcium entry; a study in a nerve-derived cell line favored a toxic gain of function.

      “Using the neuronal-derived cell line, Neuro2a, Fecto et al demonstrated increased intracellular calcium, cytotoxicity and channel open time in TRPV4 mutant transfected cells suggesting that a toxic gain of function appears the most likely pathomechanism.”DHMN causative genes, TRPV4

    • TRPV4 blocking drugs were raised as a possible treatment, but the authors urged caution because mice lacking TRPV4 share some features with the disease.

      “Phenotypic similarities with TRPV4 knockout mice suggest that the use of an antagonist should be approached with caution.”DHMN causative genes, TRPV4

    What the authors concluded

    The authors conclude that dHMN are genetically and clinically varied and overlap with CMT2, juvenile ALS and hereditary spastic paraplegia. The known genes point to shared mechanisms (protein misfolding, axonal transport, RNA metabolism and ion channel function), and possible treatments are emerging. They expect new sequencing methods to find more genes but stress that robust functional models will be needed to show which variants cause disease.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • ModerateReview without new data or a systematic search

      This is a commissioned narrative review. Its statements are only as strong as the studies it cites, and it does not explain how studies were chosen, so it may not cover all the evidence available at the time.

      “Provenance and peer review Commissioned; externally peer reviewed.”Footnotes

    • MinorDated summary of TRPV4 mechanism

      Written in 2011, the review says the evidence on how TRPV4 neuropathy changes act was inconclusive, with studies disagreeing. Its view of the mechanism and of treatment options reflects that time and should not be read as current.

      “In vitro studies of neuropathy causing mutations have been less conclusive”DHMN causative genes, TRPV4

    • MinorTRPV4 figures drawn mainly from one series

      The numbers given for TRPV4 (onset before 14 in most carriers, vocal cord palsy in 50%) come from a single series of seven families. Small series from specialist centers may not represent everyone with TRPV4 neuropathy.

      “In a large case series, Zimon et al identified mutations in seven families with distal weakness in the lower limbs and minimal sensory loss.”DHMN causative genes, TRPV4

    • MinorLocus count differs between abstract and text

      The abstract says four loci have been identified, but the main text describes five other loci. This is a small inconsistency that does not affect the TRPV4 content.

      “Five other separate genetic loci have been reported for both autosomal dominant and recessive forms of dHMN (see table 1).”Other dHMN loci; compare Abstract

    Three ways to explain it

    Scientific

    This narrative review covers the distal hereditary motor neuropathies, a genetically heterogeneous group overlapping with CMT2, in which more than 80% of patients lacked a genetic diagnosis at the time. TRPV4 is listed among the causes of dHMN type VII (vocal cord paralysis) and is also linked to scapuloperoneal SMA and CMT2C, with marked intrafamilial variability. On the in vitro evidence then available, the authors considered a toxic gain of function the most likely mechanism, while urging caution about antagonists given the phenotypes of TRPV4 knockout mice.

    Easier

    This 2012 review describes a group of inherited nerve conditions that mainly weaken the muscles of the hands and feet, called distal hereditary motor neuropathies. They overlap with the axonal type of Charcot-Marie-Tooth disease. The review says TRPV4 changes are one cause, often with weak vocal cords, and that symptoms can differ a lot between relatives. At the time, scientists thought the faulty channel probably let in too much calcium, but the evidence was mixed.

    Simple

    Some families have nerve problems that mostly make the hands and feet weak. TRPV4, a door that lets calcium into cells, is one of many genes that can cause this. Back then, scientists thought the door might be letting in too much calcium, but they were not sure yet.

    Why these ratings

    Relevance 2/5: A broad review of motor neuropathies with a short but meaningful section on TRPV4, including its links to CMT2C and vocal cord paralysis.

    Evidence 2/5: An expert narrative review that summarizes other studies; it adds no new data and does not describe a systematic search.

    Funding and conflicts of interest

    AMR: fellowship from the 'National Institutes of Neurological Diseases and Stroke and office of Rare Diseases' (U54NS065712); he has also received an IPSEN clinical research fellowship. LG: Brain Research Trust. MMR: Medical Research Council, Muscular Dystrophy Campaign and NINDS/Office of Rare Diseases (U54NS065712). Partly funded through the UCLH/UCL NIHR Biomedical Research Centre. Competing interests: none. Commissioned review, externally peer reviewed.

    PubMed 22028385 · DOI

85. #85Full paperHypothesis review

    TRPV4: A trigger of pathological RhoA activation in neurological disease

    Bagnell AM, Sumner CJ, McCray BA · 2022 · BioEssays: news and reviews in molecular, cellular and developmental biology

    Relevance 2/5Evidence 2/54 moderate · 3 minor problems

    This article is mainly about brain and spinal cord injury, not CMT2C, and puts forward an idea rather than new results. It notes that a TRPV4-blocking drug was well tolerated in trials for other conditions, but it does not show that blocking TRPV4 helps any nerve disease in people.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    Hypothesis review

    Who or what

    No new patients. A narrative review of published cell and animal research (mostly rodent models of stroke, traumatic brain injury and spinal cord injury) and of the authors' own work on TRPV4 and RhoA, plus one figure of unpublished data from TRPV4-expressing HEK293T cells.

    What they did

    The authors summarize their earlier finding that TRPV4 binds and controls RhoA, a protein that tightens the cell's internal scaffolding. They review animal studies linking TRPV4 and RhoA to poor nerve regrowth and to leaky blood vessel barriers in the brain and spinal cord after injury. They propose that drugs blocking TRPV4 could limit this harmful RhoA activity.

    What they found

    • The authors propose that TRPV4 and RhoA work together as a signaling unit that drives harmful changes to cell shape after nervous system injury and in disease.

      “Here we propose that TRPV4 and the cytoskeletal remodeling small GTPase RhoA together constitute an environmentally sensitive signaling complex that contributes to pathological cell cytoskeletal alterations during neurological injury and disease.”Abstract

    • Summarizing their earlier work, they describe a two-way brake: RhoA bound to TRPV4 reduces channel activity, and TRPV4 holds RhoA in its inactive state. Switching TRPV4 on lets calcium in and activates RhoA.

      “RhoA binding to TRPV4 suppressed ion channel activity, and conversely, TRPV4 binding to RhoA inhibited RhoA activation, likely by sequestering inactive RhoA in a manner similar to RhoGDI”Section 'TRPV4-RhoA complex regulates the cytoskeleton'

    • In a fruit fly model of TRPV4 neuropathy, nerve fiber and dendrite damage could be rescued by blocking the fly version of RhoA.

      “Importantly, degeneration of dendrites and axons in this model could be rescued by inhibition of Rho1, the fly homolog of RhoA, further linking TRPV4-mediated cytoskeletal changes with RhoA signaling.”Section 'TRPV4 as a regulator of pathological RhoA activation in the nervous system'

    • How much TRPV4 is present in nerve cells themselves is uncertain. Mouse spinal cord data show little TRPV4 in alpha motor neurons, and the authors say its role in blood vessel cells is better established.

      “the extent to which TRPV4 is expressed within neurons of the brain, spinal cord, and peripheral nerves remains debatable.”Section 'TRPV4 as a regulator of pathological RhoA activation in the nervous system'

    • A TRPV4-blocking drug, GSK2798745, was well tolerated in trials for other conditions (pulmonary edema and chronic cough), although those trials did not meet their efficacy goals.

      “While these trials did not meet efficacy endpoints for these disease indications, the antagonist drug was well tolerated without any significant adverse effects”Section 'TRPV4 inhibition is a promising therapeutic route to treat neurological disease'

    • Results in peripheral nerve injury models conflict: blocking TRPV4 helped in some mouse models, but removing TRPV4 entirely made outcomes worse after a cut nerve.

      “global knockout of TRPV4 worsened outcomes in a mouse model of peripheral nerve transection.”Section 'TRPV4 inhibition is a promising therapeutic route to treat neurological disease'

    What the authors concluded

    TRPV4 may act as a gatekeeper that switches on RhoA and harmful changes to cell scaffolding after nerve injury and when blood-brain barriers break down. Because TRPV4 can be blocked with available drugs, the authors see it as an attractive target, but say more studies are needed on which cell types are involved and when blocking would help.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • ModerateHypothesis review, leaning on the authors' own work

      The article puts forward an idea rather than testing it. Its central support is the authors' own 2021 study, so the review is not an independent check of that work.

      “Supporting this hypothesis is our recent work demonstrating direct physical and bidirectional functional interactions of TRPV4 with RhoA”Abstract

    • ModerateUnpublished data with no numbers

      Figure 3 shows unpublished experiments in HEK293T cells as time-lapse images only. No number of cells, number of repeats or statistics are given, so this result cannot be judged.

      “our unpublished results demonstrate that inhibition of ROCK with the specific inhibitor Y-27632 blocks TRPV4-calcium-mediated cytoskeletal changes in HEK293T cells”Section 'TRPV4-RhoA complex regulates the cytoskeleton' and Figure 3

    • ModerateEvidence mostly from acute injury in animals, not inherited neuropathy

      Most of the supporting studies are rodent models of stroke, brain injury and spinal cord injury, often about blood vessel cells. The authors themselves say TRPV4 levels in nerve cells are debatable and that a similar role at the blood-nerve barriers of the peripheral nervous system has yet to be established. How far this applies to CMT2C is unclear.

      “Particular examples of coincident RhoA and TRPV4 activation have come from animal models of stroke, TBI, and SCI.”Section 'TRPV4 as a regulator of pathological RhoA activation in the nervous system'

    • ModerateTreatment claims run ahead of the evidence

      The abstract calls TRPV4 inhibition 'safe, feasible, and precise', but the human data come from trials in other conditions that did not meet their efficacy goals, and one nerve injury model got worse when TRPV4 was removed. The body of the review is more cautious than the abstract.

      “These insights also suggest that TRPV4 inhibition may represent a safe, feasible, and precise therapeutic strategy”Abstract

    • MinorKnockout mice described as having no overt problems

      The review says mice lacking TRPV4 show no overt harmful features. The titles of the papers it cites for this describe altered temperature preference, impaired osmotic sensation and abnormal osmotic regulation in these mice. The statement is best read as 'no obvious serious problems', not 'no effects'.

      “TRPV4 knockout does not display any overt deleterious phenotypes in mice.”Section 'TRPV4 inhibition is a promising therapeutic route to treat neurological disease' and references 95 to 97

    • MinorInconsistent drug name

      The text names the ROCK inhibitor Y-27632, but the Figure 3 legend calls it Y-26732. This looks like a typing error.

      “addition of the ROCK inhibitor Y-26732 (10 μ M) blocks cytoskeletal changes downstream of TRPV4-mediated RhoA activation.”Figure 3 legend

    • MinorNo stated method for choosing studies

      This is a narrative review. It does not say how papers were searched for or selected, so studies that do not fit the hypothesis may be under-represented.

    Three ways to explain it

    Scientific

    This hypothesis review proposes that TRPV4 and the small GTPase RhoA form a mechanosensitive signaling complex: inactive TRPV4 sequesters GDP-bound RhoA, while TRPV4-mediated calcium influx releases and activates RhoA, driving ROCK-dependent actin remodeling. Drawing mainly on rodent models of stroke, traumatic brain injury and spinal cord injury, the authors argue that this axis impairs axon regeneration and blood-brain barrier integrity, and that TRPV4 antagonists could limit pathological RhoA activation. Evidence relating to inherited TRPV4 neuropathy is limited to cell and Drosophila work, and neuronal TRPV4 expression is described as debatable.

    Easier

    TRPV4 is a channel that senses things like pressure and swelling around a cell. RhoA is a switch protein that makes the cell's internal scaffolding tighten and pull back. The authors suggest that when TRPV4 is switched on too much, for example after a stroke or spinal injury, it turns on RhoA, which may stop nerves from regrowing and make blood vessels in the brain leaky. They suggest TRPV4-blocking drugs as a possible treatment, but this idea rests mostly on animal studies and has not been tested in people with nerve disease.

    Simple

    TRPV4 is like a door in a cell that also works a switch called RhoA. When the door opens too much, the switch can make the cell scrunch up. The authors think blocking the door might help after nerve injuries, but so far this idea has mostly been tested in animals.

    Why these ratings

    Relevance 2/5: Mostly about acute brain and spinal cord injury, with a short section on the TRPV4-RhoA mechanism found in neuropathy research and the fly model of TRPV4 neuropathy.

    Evidence 2/5: A hypothesis review built mainly on cell and animal studies, with one unpublished figure and therapeutic claims not tested in people with nerve disease.

    Funding and conflicts of interest

    Supported by NIH (NINDS) grants K08 NS102509, R01 NS115475 and R35 NS122306, an American Academy of Neurology fellowship, and the Muscular Dystrophy Association (629305). The authors declare no conflicts of interest.

    PubMed 35297520 · DOI

86. #86Full paperPreprint (not peer reviewed); cell, tissue and sperm laboratory study

    TRPV4 interacts with mitochondrial proteins and acts as a mitochondrial structure-function regulator

    Kumar A, Majhi RK, Acharya TK et al. · 2018 · bioRxiv

    Relevance 2/5Evidence 2/51 major · 6 moderate problems

    This unreviewed lab study suggests that normal TRPV4 may also sit in mitochondria, the cell's power stations. It did not test any CMT2C-causing changes, so it does not tell us whether mitochondria are involved in CMT2C.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    Preprint (not peer reviewed); cell, tissue and sperm laboratory study

    Who or what

    Normal (non-mutant) TRPV4 studied in cell lines (HaCaT, F11, COS7, HeLa, and Chinese hamster ovary cells made to carry TRPV4 or an empty vector), human umbilical vein endothelial cells, mitochondria isolated from animal brain (goat, rat and mouse are all named in different places), goat fat tissue, and sperm from fish, duck, bull and human. No patients and no disease-causing TRPV4 changes were studied.

    What they did

    The authors looked for TRPV4 inside mitochondria, the parts of the cell that make energy, using microscopy and by separating cell fractions. They tested whether a drug that switches TRPV4 on (4αPDD) or off (RN1734) changed mitochondrial shape, membrane voltage, calcium and energy enzyme activity. They also tested which mitochondrial proteins a fragment of TRPV4 sticks to in a test tube.

    What they found

    • TRPV4 was seen in some, but not all, mitochondria in several cell types and in mitochondria isolated from brain.

      “TRPV4 seems to be present in some, but not in all mitochondria, indicating that the presence of TRPV4 in mitochondria is heterogeneous in nature.”Results, TRPV4 is localized in mitochondria

    • A purified tail fragment (C-terminus) of TRPV4 bound the mitochondrial proteins Hsp60, Mfn1 and Mfn2 in pull-down tests.

      “Immunoblot results indicate that Hsp60, Mfn1 and Mfn2 interact with MBP-TRPV4-Ct.”Results, the C-terminus of TRPV4 binds with intact mitochondria

    • In hamster ovary cells made to carry TRPV4, switching TRPV4 on made mitochondria round up and clump together; cells without TRPV4 did not change.

      “This indicates that activation of TRPV4 causes aggregation of mitochondria and increases their perimeter as well as their area.”Results, TRPV4-positive mitochondria display an altered mitochondrial morphology

    • Switching TRPV4 on lowered the mitochondrial membrane voltage in TRPV4-carrying cells, but not in cells without TRPV4.

      “In the presence of TRPV4 activator 4αPDD (5μM), mitochondrial potential was significantly decreased in CHOK1-V4 cells as compared to DMSO control.”Results, TRPV4 regulates mitochondrial membrane potential

    • In mitochondria isolated from goat brain, the TRPV4 activator lowered the activity of energy enzymes complex II and IV and caused swelling, while the inhibitor had opposite or no effects.

      “Mitochondrial Complex activity assays suggest that TRPV4 activator or inhibitor largely regulate the function of Complex II and IV activity as comparison to the Complex I and III.”Results, TRPV4 alters mitochondrial Electron Transport Chain activity

    • The authors link their results to CMT2 by noting that both TRPV4 and MFN2 are CMT2 genes, and argue that their test-tube binding result supports a connection between the two proteins.

      “Indeed our experiments confirm that TRPV4 interacts with Mfn1 and Mfn2 directly and thus support the genetic evidence at the protein level.”Discussion, TRPV4 interacts with mitochondrial proteins and is part of MAM

    What the authors concluded

    TRPV4 is present in a subset of mitochondria and helps regulate their shape, calcium, membrane voltage and energy enzymes, and its tail binds the mitochondrial proteins Hsp60, Mfn1 and Mfn2. The authors suggest this may help link TRPV4 channel diseases, including Charcot-Marie-Tooth disease, with mitochondrial problems, and say that disease-causing TRPV4 variants still need to be studied in this context.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • ModerateNot peer reviewed

      This is a preprint. Independent experts have not checked the methods or conclusions, so the findings should be treated as provisional.

      “The copyright holder for this preprint (which was not certified by peer review) is the author/funder.”Page header

    • MajorNo CMT2C or other disease variants tested

      Every experiment used normal TRPV4, sometimes switched on or off with drugs. The paper therefore cannot say whether CMT2C-causing changes affect mitochondria at all. The authors themselves say mutants still need study.

      “Further detailed studies are needed to understand the mitochondrial localization of TRPV4 and importance of different TRPV4 mutants in the context of mitochondrial functions.”Discussion, molecular size of TRPV4 within mitochondria

    • ModerateWeak reasoning for the CMT2 link

      The fact that two genes cause related diseases does not by itself mean their proteins work together. The binding test used a TRPV4 tail fragment and purified Mfn1 and Mfn2 recovered from bacterial 'inclusion bodies' in a test tube, and binding there does not show the proteins meet in living nerve cells.

      “more than 20 genes including TRPV4 and Mfn2 are considered to be involved in the development of Charcot Marie Tooth 2 disease (defects in sensory/motor neurons) suggesting that TRPV4 is physiologically linked with Mfn2”Discussion, TRPV4 interacts with mitochondrial proteins and is part of MAM

    • ModerateInconsistent description of tissue sources

      The methods describe isolating mitochondria from goat brain obtained from a slaughterhouse, and imaging mitochondria from mouse brain. The results and figure legends instead describe mitochondria from rat brain, and the methods do not describe a rat brain preparation. This makes it hard to know exactly what was done.

      “freshly isolated mitochondrial fractions from rat brain were labelled with mitochondria-specific dye MitoTracker Red.”Results, TRPV4 is present in mitochondria endogenously

    • ModerateDrug effects on isolated mitochondria not shown to depend on TRPV4

      In cells, the authors compared TRPV4-carrying cells with cells lacking it, which is a useful control. For the enzyme and swelling tests on isolated goat brain mitochondria, no TRPV4-free comparison is described, so effects of the drugs (used at 5 to 20 μM) cannot firmly be attributed to TRPV4 itself.

      “All enzymatic activity was determined in freshly isolated goat brain mitochondria”Materials and Methods, assay for mitochondrial electron transport chain and MPT

    • ModerateConclusions stated more strongly than the statistics

      One figure legend states that most brain TRPV4 is in the inner membrane while reporting that the difference was not statistically significant. Statistical methods are described only briefly. The results section then concludes that TRPV4 has been shown 'for the first time' to be a mitochondrial protein, which is stronger than the evidence warrants.

      “Majority of the endogenous TRPV4 is present in the inner membrane rather than in outer membrane of mitochondria isolated from brain (ns = non-significant, n = 4, P value = 0.1181).”Figure 2 legend

    • ModerateModel systems far from human nerves

      Much of the work used hamster ovary cells engineered to carry TRPV4, skin and kidney cell lines, endothelial cells and sperm. Engineered cells already had altered mitochondria before any drug was added. None of these systems are human motor or sensory nerve cells.

      “Taken together, these results indicate that TRPV4 modulates mitochondrial morphology, at least in stable cell lines expressing TRPV4.”Results, TRPV4-positive mitochondria display an altered mitochondrial morphology

    Three ways to explain it

    Scientific

    In cell lines, primary endothelial cells, isolated brain mitochondria and sperm, wild-type TRPV4 localized to a subset of mitochondria, and pharmacological activation with 4αPDD altered mitochondrial morphology, lowered membrane potential, increased mitochondrial calcium and reduced complex II and IV activity, whereas RN1734 had opposite or no effects. A recombinant C-terminal fragment bound Hsp60, Mfn1 and Mfn2 in pull-down assays. No neuropathy-associated variants were studied, so any role in CMT2C pathogenesis remains a hypothesis, and the work is an unreviewed preprint.

    Easier

    Mitochondria are tiny parts inside cells that make energy. This study suggests that TRPV4 is found on some mitochondria, and that switching TRPV4 on with a drug changed their shape and how well they worked, in cells grown in a dish. The authors suggest this might connect TRPV4 to nerve diseases like CMT, but they did not test any of the gene changes that cause CMT2C, and the paper has not been checked by independent reviewers.

    Simple

    Cells have tiny power stations called mitochondria. These scientists think the TRPV4 calcium door might also sit on some of the power stations and change how they work. It is an early idea from lab tests, and they did not look at the CMT2C version of the door.

    Why these ratings

    Relevance 2/5: Mainly about normal TRPV4 in mitochondria; the link to CMT2C is a hypothesis and no CMT2C-causing changes were tested.

    Evidence 2/5: Unreviewed preprint of lab work in cell lines, animal tissue and sperm, with internal inconsistencies and no disease variants.

    Funding and conflicts of interest

    Funding from NISER and DBT (Government of India, grants BT-BRB-TF-2-2011 and BT/PR8004/MED/30/988/2013), with the funders said to have had no role in the study; imaging supported by an MRC Next Generation Optical Microscopy Award (MR/K015869/1). The authors declare no conflict of interests.

    DOI

87. #87Full paperRetrospective multicenter case series with gene panel testing

    Hearing loss in inherited peripheral neuropathies: Molecular diagnosis by NGS in a French series

    Lerat J, Magdelaine C, Roux AF et al. · 2019 · Molecular genetics & genomic medicine

    Relevance 2/5Evidence 2/51 major · 4 moderate · 2 minor problems

    This study adds one person with a TRPV4 change whose hearing loss came from the hearing nerve (auditory neuropathy) and who also had weak vocal cords (vocal cord paresis). Because hearing loss was found from records and questionnaires, and the paper does not report hearing tests for everyone, it cannot tell us how common hearing loss is in TRPV4 neuropathy.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    Retrospective multicenter case series with gene panel testing

    Who or what

    A French series of 3,412 people with inherited peripheral neuropathy, screened through medical records and a clinical questionnaire. 44 had recorded hearing loss, and 27 (15 women, 12 men; mean age 60.77, range 10 to 90) had usable data. One of the 27 (patient XVIII, a 68-year-old man) had a TRPV4 variant, c.694C>T, p.(Arg232Cys); the whole series contained five people with TRPV4 variants.

    What they did

    The team searched records of people with inherited neuropathy to find those who also had hearing loss. They tested DNA with a 92-gene neuropathy panel, checked two common deafness genes (GJB2 and GJB6), and used a 63-gene hearing-loss panel in 8 selected patients. They then compared the type, severity and timing of hearing loss with the genes found.

    What they found

    • Hearing loss was recorded in only 44 of 3,412 people with inherited neuropathy (1.30%), and 27 had usable clinical data.

      “Hearing loss (HL) was present in only 44 patients (1.30%). The clinical data of 27 patients were usable.”Abstract; Results 3.1

    • A genetic cause was found in 16 of the 27 people, including seven new variants in five genes.

      “pathogenic variants were identified in 16 patients out of 27 (59.26%)”Results 3.2

    • The one TRPV4 patient in the 27, a 68-year-old man with familial dominant axonal sensorimotor neuropathy and pes cavus, had moderate auditory neuropathy beginning at 62 and bilateral vocal cord paresis; his neuropathy began at 65.

      “XVIII M, 68 Fam/AD Sensori-motor Axonal Y NC 65 Moderate AN 62 Bilateral Vocal cord Paresis”Table 2

    • Of five people with TRPV4 variants in the whole series, only one had been referred with diagnosed hearing loss.

      “In our French series, five patients were detected with a variant in TRPV4 , but only one of them was referred with diagnosed hearing loss.”Discussion

    • Hearing loss could come from the inner ear (cochlea) or from the hearing nerve. Auditory neuropathy, a hearing nerve problem, was found in five patients, one of them the TRPV4 patient.

      “Hearing loss can be due to cochlear impairment and/or auditory nerve dysfunction.”Conclusion

    What the authors concluded

    The authors conclude that PMP22 changes alone do not explain hearing loss in inherited neuropathy, that hearing loss can come from the cochlea and/or the auditory nerve, and that it is underdiagnosed. They recommend hearing tests and broad gene panels for all patients with inherited neuropathy, and say TRPV4 is among the genes showing genotype-phenotype correlation.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • MajorHearing loss identified from records, not by testing

      People were counted as having hearing loss only if it appeared in medical records or a questionnaire. The paper does not report hearing tests for all 3,412 people, so the 1.30% figure, and the finding that only one of five TRPV4 patients had hearing loss, may undercount. The authors themselves say hearing loss is underdiagnosed.

      “A French series of 3,412 patients suffering from IPN has been analysed thanks to medical records and a clinical questionnaire, so as to identify patients presenting IPN and hearing loss.”Materials and Methods 2.1

    • ModerateMuch missing data

      Only 27 of the 44 people with recorded hearing loss had usable data, and many table entries are marked 'not communicated', including the TRPV4 patient's nerve conduction velocity. This limits comparisons between genes.

      “The clinical data of 27 patients, 15 women and 12 men, were usable for this study.”Results 3.1

    • ModerateTRPV4 conclusions rest on one patient

      The abstract lists TRPV4 among genes with genotype-phenotype correlation, but this rests on one person. No audiogram details or family testing are reported for him, and he was not among the 8 patients given the hearing-loss gene panel, so another genetic cause of his hearing loss was not looked for.

      “Patient XVIII’s phenotype corresponds to this description.”Discussion

    • ModerateStatistical claims without tests, and causation claimed from association

      The paper says some hearing loss rates are statistically different from the general population, and concludes that SH3TC2 variants directly cause hearing loss, but reports no statistical test. It also compares record-based counts with a general population rate. An association in a small series cannot show cause.

      “showing that the pathogenic variant in SH3TC2 is directly responsible for hearing loss”Discussion

    • ModerateNumbers and labels that do not match

      The Results count three PMP22 cases among the five auditory neuropathy patients, while the Discussion and Table 2 point to two. Five of 784 PMP22 patients is given as 0.05%, which does not match. The ABHD12 patient is called XVI in one place and XXVI in the table, and some variant names differ between text, table and figure (for example NEFL c.293A>C in the text and c.293A>G in Table 3). These errors lower confidence in the details.

      “auditory neuropathy was found in five cases: three cases of PMP22 (1.4Mb deletion, point pathogenic variant), one case due to TRPV4 and one case due to ABHD12”Results 3.3

    • MinorHearing-loss gene panel used in only 8 patients

      Only 8 selected patients had the full hearing-loss gene panel, so separate genetic causes of hearing loss may have been missed in the others. The authors acknowledge this.

      “However, only eight cases could be tested by HL-NGS because of availability of the analysis.”Discussion

    • MinorUnclear denominators

      The paper moves between the series of 3,412 patients and a group of 350 patients tested with the neuropathy gene panel without explaining how they relate, which makes its rates hard to interpret.

      “as among 350 patients tested with IPN NGS, 13 had a pathogenic variants in this gene”Discussion

    Three ways to explain it

    Scientific

    In a retrospective French series of 3,412 patients with inherited peripheral neuropathy, record-based ascertainment identified hearing loss in 44 (1.30%), of whom 27 had usable data; panel NGS gave a molecular diagnosis in 16 of 27 (59.26%). Hearing loss was cochlear and/or retrocochlear, with auditory neuropathy in five, including one TRPV4 p.(Arg232Cys) carrier with familial axonal sensorimotor neuropathy and bilateral vocal cord paresis. Only one of five TRPV4 carriers in the series had documented hearing loss, but ascertainment bias prevents any estimate of its frequency.

    Easier

    Researchers searched the records of 3,412 French people with inherited nerve disease and found hearing loss noted in only 44. Gene tests found a cause in 16 of the 27 people with enough information. One man had a TRPV4 change; his hearing loss came from the hearing nerve rather than the inner ear, and he also had weak vocal cords. Because hearing loss was found from records rather than by testing everyone's hearing, the study cannot say how often hearing loss happens in TRPV4 disease.

    Simple

    Doctors looked at many people with nerve illnesses to see who also had trouble hearing. One man with a TRPV4 gene change had a problem in the nerve that carries sound signals, like a phone line with a bad connection, and he also had weak vocal cords. The doctors mostly used old records instead of testing everyone's hearing, so we do not know how common this is.

    Why these ratings

    Relevance 2/5: Mostly about other neuropathy genes, but includes one TRPV4 patient with auditory neuropathy and vocal cord paresis and a note on recorded hearing loss among five TRPV4 patients.

    Evidence 2/5: A retrospective series in which hearing loss was found from records rather than by testing everyone, with much missing data, several internal numerical errors, and only one TRPV4 patient.

    Funding and conflicts of interest

    Funding: not stated. Conflict of interest: 'None.'

    PubMed 31393079 · DOI

88. #88Full paperAnimal model study in the nematode worm Caenorhabditis elegans

    Disruption of genes associated with Charcot-Marie-Tooth type 2 lead to common behavioural, cellular and molecular defects in Caenorhabditis elegans

    Soh MS, Cheng X, Vijayaraghavan T et al. · 2020 · PloS one

    Relevance 2/5Evidence 2/51 major · 2 moderate · 2 minor problems

    Worms missing their version of the TRPV4 gene moved less well as adults and had some muscle changes, but showed no changes in the motor nerve cells examined or in nerve-muscle signaling. Because these worms lack the gene rather than carrying a human CMT2C change, the study tells us little directly about CMT2C in people.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    Animal model study in the nematode worm Caenorhabditis elegans

    Who or what

    C. elegans worms carrying loss-of-function or null mutations in nine worm genes that correspond to human CMT2-linked genes, including osm-9, which the authors pair with human TRPV4 (CMT2C). Mutant worms were compared with normal (wild-type) worms; movement tests used at least 30 worms per group, and population tests used 12 wells of 30 to 50 worms.

    What they did

    The team measured how well mutant worms swam and crawled at four ages, and tracked groups of worms automatically. They imaged motor nerve cells and body-wall muscles, tested muscle responses to the drug levamisole, and recorded electrical signaling at the nerve-muscle junction. They also checked whether adding back a normal copy of each worm gene restored swimming.

    What they found

    • Worms lacking seven of the nine genes moved worse than normal worms, and this tended to get worse with age.

      “Mutations in seven of the nine CMT2-associated genes led to progressive deteriorations in locomotion, especially in the older animals”Results, Individual CMT2-associated mutants exhibit movement defects

    • Worms lacking osm-9 (the TRPV4 counterpart) swam less well only as adults and crawled less at all ages, and adding back a normal copy of the gene restored swimming.

      “the swimming ability of lin-41(ma104) and osm-9(ok1677) mutants were only significantly reduced in adult animals.”Results, Individual CMT2-associated mutants exhibit movement defects; S2 Fig

    • In the automated test of groups of worms, osm-9 mutants moved normally.

      “mutation of osm-9 , hint-1 or nep-2 had no effect on movement”Results, Populations of CMT2-associated mutants experience declines in motility over time

    • osm-9 mutants showed moderate muscle structure changes (40 to 50% scored abnormal, larger gaps and slightly shorter muscle fibers) and an altered response to levamisole.

      “the significant but smaller extent of muscle cell degeneration and deposits in hsp-25(tm700) , dyn-1(ky51) and osm-9(ok1677) mutants meant that only 40-50% of animals were scored defective.”Results, CMT2-associated mutants display defective body wall muscles

    • Nerve-muscle junction signaling was reduced in four mutants (lin-41, dyn-1, unc-116 and fzo-1) but not in osm-9 mutants, and osm-9 mutants had no clear defects in the motor nerve cells examined.

      “the frequency of spontaneous PSCs in lin-41(ma104) , dyn-1(ky51) , unc-116(e2310) and fzo-1(cjn020) animals was between 2- and 5-fold lower than the wild-type.”Results, Electrophysiological recordings uncover neuromuscular junction defects; Fig 3C

    What the authors concluded

    The authors conclude that worms lacking CMT2-linked genes share problems with movement, muscle structure and, for some genes, nerve-muscle signaling. They propose C. elegans as a useful model for CMT2 subtypes, including CMT2C, and as a platform for high-throughput drug screening.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • MajorRemoving the gene is not the same as carrying a CMT2C change

      The worms lack a working osm-9 gene, whereas the paper's own table lists CMT2C as autosomal dominant. The authors acknowledge that patient-specific changes still need to be modeled to know whether they act like a loss of function. So the osm-9 results describe what the worm gene normally does, not how CMT2C develops.

      “it will be important to generate C . elegans models carrying patient-specific mutations in the orthologous genes.”Discussion, Limitations of the current study

    • ModerateA worm gene stands in for human TRPV4

      The authors chose the closest worm genes by sequence similarity and describe them as orthologous or paralogous. Rescue was done with worm genes, not human TRPV4, so it is not shown that osm-9 does the same job as TRPV4. Worms are also anatomically very different from people.

      “rescue of the defects in the C . elegans mutants using the orthologous human genes could be carried out to determine if the genes function similarly between the species.”Discussion, Limitations of the current study

    • ModerateClaims for a CMT2C model go beyond the osm-9 results

      osm-9 mutants had no defect in the group movement test, no motor nerve cell defects and no nerve-muscle signaling defect, so the link to motor nerve disease is weak for this gene. Calling the study an important step toward reliable models, including for CMT2C, overstates what the osm-9 data show.

      “this study of both behavioural and cellular consequences of mutating CMT2-associated genes in C . elegans is an important step forward in the establishment of reliable animal models to understand disease mechanisms.”Discussion, The need for viable CMT2 animal models

    • MinorSummary figure disagrees with the results

      The summary figure legend lists hint-1 and nep-2 among mutants with muscle irregularities, but the results say these two were not significantly different from normal worms in muscle scoring or measurements. This does not affect the osm-9 findings.

      “mutations in lin-41, dyn-1, unc-116, fzo-1, osm-9, cua-1, hsp-25, hint-1 and nep-2 resulted in irregularities in the body wall muscle.”Fig 8 legend; compare Results, Fig 4C and Fig 5

    • MinorSome subjectivity in image scoring

      Movement tests were done blind to genotype, but blinding is not stated for the muscle and nerve image scoring. About 20% of normal worms were also scored as having muscle defects, and the marker used makes worms roll, which complicates imaging. The authors acknowledge that bias was reduced but not removed.

      “while a number of steps have been taken to greatly reduce subjective bias, it has not been eliminated completely.”Discussion, Limitations of the current study

    Three ways to explain it

    Scientific

    In C. elegans, loss-of-function alleles of nine CMT2-associated orthologs or paralogs caused locomotor deficits in most strains, myofilament disorganization, and, for lin-41, dyn-1, unc-116 and fzo-1, reduced spontaneous postsynaptic current frequency at the neuromuscular junction. The osm-9 (TRPV4 counterpart) mutant showed adult-onset swimming deficits, reduced crawling, moderate muscle structural changes and altered levamisole responses, but normal DA/DB motor neuron morphology, population motility and neuromuscular junction current frequency. As null models of a gene whose CMT2C form is autosomal dominant, these data inform normal gene function rather than CMT2C pathogenesis.

    Easier

    Researchers used tiny worms to study nine genes linked to the axonal type of Charcot-Marie-Tooth disease. For each gene, they used worms in which the worm's own version of that gene did not work. Worms lacking their TRPV4-like gene, called osm-9, swam and crawled less well as adults and had some muscle changes, but their motor nerves and nerve-to-muscle signals looked normal. People with CMT2C carry a changed TRPV4 gene rather than a missing one, so these worms are only a distant model.

    Simple

    Scientists studied tiny worms that were missing genes linked to CMT. Worms missing their version of the TRPV4 gene moved a bit worse when they were older. But worms are very different from people, and these worms were missing the gene instead of having a changed one, so this does not tell us much about CMT2C yet.

    Why these ratings

    Relevance 2/5: Mostly about other CMT2 genes; the TRPV4-related part is one of nine worm mutants, and it models loss of the gene rather than a human CMT2C change.

    Evidence 2/5: Reasonably careful worm experiments with blinded movement tests and gene rescue, but in an invertebrate model that is far from people with CMT2C.

    Funding and conflicts of interest

    Supported by Charcot-Marie-Tooth Association of Australia research grants (2015 and 2018) and National Health and Medical Research Council Project Grant 1099690; the funders had no role in the study. The authors declare no competing interests.

    PubMed 32294113 · DOI

89. #89Full paperCell biology study (live-cell imaging in lab cell lines)

    Lysosomal Regulation of Inter-mitochondrial Contact Fate and Motility in Charcot-Marie-Tooth Type 2

    Wong YC, Peng W, Krainc D · 2019 · Developmental cell

    Relevance 2/5Evidence 2/51 major · 3 moderate · 2 minor problems

    In lab cells, the CMT2C-linked TRPV4 change R269H made mitochondria stick together longer and move less, similar to changes linked to other CMT2 types. This is an early, single lab observation and has not been shown in human nerves.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    Cell biology study (live-cell imaging in lab cell lines)

    Who or what

    Human cell lines grown in the lab, mainly HeLa cells, with some work in HEK293 and H4 neuroglioma cells. For CMT2C, cells made to produce normal TRPV4 or the R269H variant were compared (50 events from 10 cells each for contact duration; 90 events from 18 cells per condition for motility). Similar comparisons were made for CMT2A (Mfn2 T105M) and CMT2B (RAB7 V162M). No patients, nerve cells or animals.

    What they did

    Using super-resolution and confocal microscopy of living cells, the authors tracked how mitochondria (the cell's power stations) touch each other, separate, fuse and divide, and how lysosomes, the ER and the proteins RAB7, Mfn1/2 and Drp1 influence this. They then tested whether proteins linked to three forms of CMT2, including TRPV4 R269H for CMT2C, changed these contacts and how much mitochondria moved.

    What they found

    • Mitochondria often touch each other and then separate without fusing, about ten times more often than they fuse or divide.

      “inter-mitochondrial contact tethering and untethering events occurred ten times more frequently (23.1 ± 3.8 tethering events/min, 27.4 ± 4.7 untethering events/min)”Results, matrix transfer section; Figure 1M

    • Contact with lysosomes usually marked, and helped trigger, the separation of mitochondria, a process controlled by the protein RAB7.

      “the majority of inter-mitochondrial contact untethering events had at least one of the mitochondria simultaneously in contact with a LAMP1-positive vesicle within 10 s of untethering (94%, n = 50 events)”Results, lysosomes section; Figure 2

    • Mitochondria held in contact with each other moved less than free mitochondria.

      “their motility was significantly decreased compared to that of free mitochondria not in inter-mitochondrial contacts”Results, motility section; Figure 6

    • The CMT2A-linked Mfn2 change and the CMT2B-linked RAB7 change both made mitochondria stay in contact longer and move less.

      “Mutant Mfn2 (T105M) significantly prolonged the duration of inter-mitochondria contacts leading to inefficient untethering compared to wild-type Mfn2”Results, CMT2 mutants section; Figure 7

    • In cells given the CMT2C-linked TRPV4 R269H, mitochondria also stayed in contact longer and moved less than with normal TRPV4.

      “we found that mutant TRPV4 (R269H) also resulted in significantly prolonged inter-mitochondrial contacts which could not efficiently untether (Figure 7K and 7L), and also decreased mitochondrial motility (Figure 7M).”Results, CMT2 mutants section; Figure 7K to 7M

    What the authors concluded

    Contacts between mitochondria are a frequent, regulated event that limits mitochondrial movement, and lysosomes help release them. The authors suggest that faulty release of these contacts, with reduced mitochondrial movement, may be a shared pathway across several forms of CMT2, including CMT2C.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • MajorCMT2C link rests on one small experiment

      The TRPV4 finding comes from one variant, R269H, compared with normal TRPV4 in a few cells (50 contact events from 10 cells per group). The size of the effect is shown only as values normalized to normal TRPV4 in figures, and the result has not been repeated in nerve cells, animals or patient cells here.

      “n = 50 events from 10 cells (TRPV4 (WT))”Figure 7 legend (K to L)

    • ModerateNo explanation of how TRPV4 affects mitochondria

      TRPV4 is a calcium channel, but the paper does not measure calcium, test a TRPV4 blocker, or look at lysosome contacts in the TRPV4 cells. So it is unclear whether the mitochondrial change is a direct effect, a side effect of overproducing the channel, or a general sign of stressed cells.

    • ModerateClaims about disease go beyond the data

      The paper says it demonstrates this pathway's dysfunction in the disease process of several CMT2 types, but it shows only that added mutant proteins change mitochondrial behavior in cell lines. Whether this happens in people's nerves, or contributes to their symptoms, was not tested.

      “Our study thus identifies a crucial role for inter-mitochondrial contacts in modulating mitochondrial homeostasis and demonstrate its dysfunction in the disease pathogenesis of multiple forms of CMT2.”Introduction, final paragraph

    • ModerateNon-nerve cell lines with added proteins

      Experiments were done in lab cell lines (mainly HeLa, also HEK293 and H4 neuroglioma), with proteins added from plasmids. The text does not clearly state which cell line was used for the TRPV4 comparison. Mitochondria in long nerve fibers may behave differently.

      “we performed electron microscopy and super-resolution live cell imaging of mitochondria in human cells”Results, first section; STAR Methods, cell lines

    • MinorEvents from the same cells treated as independent

      Statistics compare many contact events or mitochondria taken from a modest number of cells using t tests. Measurements from the same cell are not fully independent, which can make results look more certain than they are. The authors state at least three independent experiments per condition.

      “For motility analysis in Fig. 6, 7 and S7, the motility of 5 randomly selected mitochondria per cell was tracked over 3 min”STAR Methods, image analysis

    • MinorMethods mislabel a mutant

      The Methods describe the Figure 7A to 7C Mfn2 experiment as using 'V162M', which is the RAB7 mutant; the Results use Mfn2 T105M. This looks like a typing error.

      “bicistronic Mfn2 WT or V162M”STAR Methods, image analysis

    Three ways to explain it

    Scientific

    Using live super-resolution and confocal imaging, mainly in HeLa cells, the authors show that mitochondria frequently form inter-mitochondrial contacts that restrict motility, with untethering promoted by mitochondria-lysosome contacts via RAB7 GTP hydrolysis and by Drp1, and tethering promoted by Mfn1/2. Overexpressed CMT2A (Mfn2 T105M), CMT2B (RAB7 V162M) and CMT2C (TRPV4 R269H) mutants each prolonged inter-mitochondrial contacts and reduced mitochondrial motility. For TRPV4 this rests on a single experiment in cultured cells, with no mechanism tested.

    Easier

    Mitochondria are the cell's power stations. This study found that they often stick to each other for a short time, and while stuck they move around less. Lysosomes, the cell's recycling centers, help them come apart. When the researchers added a CMT2C-linked TRPV4 change to cells grown in the lab, the power stations stayed stuck together longer and moved less. This was one small experiment in lab cells, not in nerves.

    Simple

    Inside cells, little power stations called mitochondria bump into each other and stick for a while. When cells had a faulty TRPV4, the power stations stayed stuck longer and moved less, a bit like cars in a traffic jam. This was only tested in cells in a dish, not in real nerves.

    Why these ratings

    Relevance 2/5: The paper is mainly about basic mitochondrial biology; CMT2C appears as one experiment with the TRPV4 R269H change.

    Evidence 2/5: Detailed imaging work, but entirely in cell lines with added proteins, and the CMT2C claim rests on a single experiment with one TRPV4 variant and no mechanism.

    Funding and conflicts of interest

    Supported by NIH (NINDS) grants to Y.C.W. (T32 NS041234, F32 NS101778 and K99 NS109252) and D.K. (R01 NS076054); imaging facilities supported by NCI and NIH grants. The authors declare no competing interests.

    PubMed 31231042 · DOI

90. #90Full paperSingle case report with patient cell and mouse nerve cell experiments

    A New Mutation in FIG4 Causes a Severe Form of CMT4J Involving TRPV4 in the Pathogenic Cascade

    Gentil BJ, O'Ferrall E, Chalk C et al. · 2017 · Journal of neuropathology and experimental neurology

    Relevance 2/5Evidence 2/51 major · 5 moderate · 1 minor problems

    This paper is mainly about a different form of CMT (CMT4J, caused by the FIG4 gene). Its TRPV4 findings come from one patient's skin cells and from mouse nerve cells in a dish; they hint that TRPV4 may be involved in nerve damage beyond CMT2C but say little directly about people with CMT2C.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    Single case report with patient cell and mouse nerve cell experiments

    Who or what

    One 48-year-old man followed at the Montreal Neurological Hospital with severe CMT4J caused by two FIG4 variants (p.I41T and a new intronic change, IVS17-10 T>G). His immortalized skin cells (fibroblasts) were compared with two control cell lines. Cultured mouse spinal cord motor neurons were used for Fig4 knockout, TRPV4 overexpression and TRPV4-blocker experiments. No people with CMT2C and no TRPV4 variants were studied.

    What they did

    They described the patient's illness, his nerve tests at ages 36 and 43, and his genetic results. They grew his skin cells and measured FIG4 protein, cell bubbles (vacuoles), the movement of small transport sacs (vesicles), and the amount, location and mobility of TRPV4. In cultured mouse motor neurons they knocked out Fig4, overexpressed TRPV4, and tested whether a TRPV4-blocking drug (GSK2193874) kept the neurons alive.

    What they found

    • The patient carried two different FIG4 variants: a known one (p.I41T) and a new intronic one predicted to disrupt splicing.

      “Genetic testing revealed that the patient is heterozygous for 2 FIG4 mutations, p.I41T and a T > G transversion at IVS17-10, the latter predicted to cause a splicing defect.”Abstract; Methods, Genetics Analysis

    • FIG4 protein was severely reduced in the patient's skin cells, which had more large vacuoles and scattered lysosomes and endosomes.

      “In immortalized fibroblasts derived from skin biopsy, FIG4 expression was strongly diminished, as shown by Western blot analysis”Results, Cellular Phenotype; Figures 1 and 2

    • The patient's skin cells had more TRPV4 protein (1.48 ± 0.16 against 0.81 ± 0.03 in controls, n = 3), more of it at the cell surface, and slower movement of TRPV4 at the surface.

      “59% ± 6% of patient fibroblasts, but none of the control fibroblasts, exhibited this distribution”Results, TRPV4 Dynamics Are Affected by the Absence of FIG4; Figure 4

    • Making normal mouse motor neurons produce extra TRPV4 was toxic to them.

      “Overexpression of TRPV4 by plasmid microinjection was toxic, reducing motor neuron viability (Fig. 5).”Results, TRPV4 Is Involved in Toxicity Induced by Fig4 Deficiency; Figure 5

    • A TRPV4-blocking drug partly delayed the death of mouse motor neurons lacking Fig4, but did not correct the vesicle problems in the patient's skin cells.

      “Cell death induced by KO of Fig4 expression in cultured motor neurons was partially prevented by treatment with the TRPV4 inhibitor, GSK2193874.”Discussion; Figure 6; Supplementary Figure S1

    What the authors concluded

    A new intronic FIG4 variant, together with p.I41T, causes loss of FIG4 and severe CMT4J. The authors argue that FIG4 loss slows TRPV4 turnover so the channel builds up at the cell surface, that this contributes to nerve cell death, and that this points to a functional overlap between CMT4J and CMT2C. They describe TRPV4 inhibition as a proof of principle for treatment that needs further study.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • MajorTRPV4 activity was never measured

      The study measured how much TRPV4 protein there was and where it sat, but not whether more calcium or current actually flowed through it in the patient's cells. The idea that TRPV4 is overactive in CMT4J is therefore an assumption, not a finding.

      “This increase in TRPV4 would be predicted to result in overall increase in cation conductance.”Discussion

    • ModerateOne patient and immortalized cells

      All patient cell results come from a single person's skin cells, altered so they keep dividing, compared with two control lines and often only 3 repeats. Skin cells are not nerve cells, and differences could reflect that particular cell line rather than the disease.

      “The fibroblasts were then immortalized as described previously (15).”Methods, Fibroblast Cell Culture

    • ModerateTreatment claim rests on mouse neurons in a dish

      The drug result comes from mouse motor neurons grown in culture and genetically altered to lack Fig4. The toxicity result comes from forcing normal neurons to make extra TRPV4. This is far from showing that blocking TRPV4 would help people with CMT4J or CMT2C.

      “We provide the proof of principle that TRPV4 inhibition could be a strategy for therapeutic development”Discussion

    • ModerateCalculation and editing errors

      A value of 1.48 against 0.81 is less than double the control, so it cannot be a 182% increase. The discussion calls the genotype 'compound homozygosity' when it is compound heterozygous. The Figure 6 legend still contains an unanswered question ('for how long?'), control cell line names differ between methods and figure, and the drug name is misspelled. These suggest the paper was not carefully checked.

      “representing a 182% increase in TRPV4 expression in FIG4 patient fibroblasts over control”Results; Discussion; Figure 1 and Figure 6 legends

    • ModerateGenetic evidence incomplete

      The new variant is said to disrupt splicing, but this was not tested directly on RNA. The paper also does not say whether parents were tested to show the two variants are on different copies of the gene. The low FIG4 protein level supports, but does not prove, that the new variant is harmful.

      “a novel intronic T > G transversion at IVS17-10, postulated to impair splicing”Methods, Genetics Analysis

    • ModerateLink to CMT2C is by analogy

      No TRPV4 variants or cells from people with CMT2C were studied. The claimed overlap with CMT2C is inferred from TRPV4 protein build-up in one CMT4J patient's skin cells.

      “These findings point to a functional overlap between CMT4J and CMT2C due to gain-of-function mutations in TRPV4.”Discussion, conclusion

    • MinorOverstated description as a longitudinal study

      The clinical follow-up consists of a narrative history and nerve tests at two ages, not a structured study over time.

      “provide a longitudinal study”Abstract

    Three ways to explain it

    Scientific

    A compound heterozygous FIG4 genotype (p.I41T with a novel IVS17-10 T>G variant) was associated with severe CMT4J, markedly reduced FIG4 protein, vacuolation and dispersed endolysosomal compartments in patient fibroblasts. TRPV4 protein was increased and retained at the plasma membrane with slower FRAP recovery, and the TRPV4 antagonist GSK2193874 partially delayed death of Fig4-knockout murine motor neurons without correcting vesicular defects. TRPV4 channel activity was not measured, so a TRPV4 gain of function in CMT4J remains inferred.

    Easier

    This paper describes a man with a severe form of CMT caused by a different gene, FIG4. In his skin cells the FIG4 protein was almost missing, the cells filled with bubbles, and more TRPV4 channel than normal sat on the cell surface. In mouse nerve cells grown in a dish, a drug that blocks TRPV4 helped cells without Fig4 live longer, but the researchers never measured whether the extra TRPV4 was actually letting in more calcium.

    Simple

    A different gene, FIG4, helps cells move and recycle their parts. When it was broken, more TRPV4 doors piled up on the cell surface. Blocking those doors helped mouse nerve cells in a dish live a bit longer, but nobody checked whether the doors were really letting in too much.

    Why these ratings

    Relevance 2/5: Mainly about FIG4-related CMT4J, with a meaningful section linking it to the TRPV4 channel.

    Evidence 2/5: One patient's cell line and cultured mouse neurons, with TRPV4 activity never measured and several errors in the text.

    Funding and conflicts of interest

    Not stated. The acknowledgements only thank individuals for technical help and gifts of plasmids; no funding source or competing interests statement appears in this copy.

    PubMed 28859335 · DOI

91. #91Full paperFamily case report with computer modeling of the protein

    Protein informatics combined with multiple data sources enriches the clinical characterization of novel TRPV4 variant causing an intermediate skeletal dysplasia

    Hines SL, Richter JE, Mohammad AN et al. · 2019 · Molecular genetics & genomic medicine

    Relevance 2/5Evidence 2/51 major · 3 moderate · 2 minor problems

    This family has a TRPV4 bone condition, not CMT2C, and no nerve tests are reported. It shows that the same gene can cause quite different conditions, but it tells us little directly about CMT2C.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    Family case report with computer modeling of the protein

    Who or what

    One family seen at Mayo Clinic, Jacksonville: a 47-year-old woman (the proband) and her 22-year-old daughter, both carrying the new TRPV4 variant c.2401A>G (p.K801E), and the proband's father, who died at age 33. He was not tested and is described from report. Computer models of normal and variant TRPV4 protein (871 amino acids per subunit).

    What they did

    The proband had a physical examination, a skeletal X-ray survey, whole exome sequencing and mitochondrial DNA analysis. The authors built computer models of the normal and variant TRPV4 channel and compared calculated stability and how well the four subunits hold together. They compared the family's features with known TRPV4 skeletal dysplasias.

    What they found

    • Exome sequencing found one copy of a new TRPV4 variant, c.2401A>G (p.K801E), in the proband; her daughter also carries it.

      “The TRPV4 variant c.2401A>G (p.K801E) was found in a heterozygous state”Case study

    • The proband's features (short fingers, flattened vertebrae, joint disease, osteoporosis, childhood scoliosis) fall between two TRPV4 skeletal dysplasias, spondyloepiphyseal dysplasia Maroteaux type and spondylometaphyseal dysplasia Kozlowski type.

      “The symptomatology of our proband cannot be clearly defined by a single TRPV4 -associated disorder”Discussion

    • In the computer model, the variant made each subunit less stable and the four-subunit channel less tightly held together, with a more disordered pore.

      “The p.K801E tetramer still forms a complex but is not as tightly held together and the pore becomes more disordered and has less regular structure than the wild-type protein”Results

    • The model's calculated energy per residue was worse for the variant than for the normal protein.

      “shows an increase of nearly 24% in the per-residue energy for worsening of stability in the VUS over that of the wild type”Results

    • The authors could not tell from the clinical picture whether the variant makes the channel more active (gain of function) or less active (loss of function).

      “Due to this conflict of symptoms, we cannot say with confidence whether this TRPV4 variant causes loss of function or gain of function in TRPV4 protein.”Discussion

    • Family members with the same variant had different features. For example, both had childhood scoliosis, but bone and joint pain was reported only in the proband and a large head (macrocephaly) only in the daughter.

      “When we investigate the confirmed family members with c.2401A>G (p.K801E), there is some overlap and discrepancy in phenotype.”Discussion

    What the authors concluded

    The authors conclude that p.K801E causes this family's intermediate skeletal dysplasia, that it is plausibly a gain-of-function variant, and that protein modeling is a useful extra tool for classifying uncertain variants.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • MajorPathogenicity claimed from modeling alone

      The abstract says modeling 'confirms' the variant causes disease, but the methods call it a variant of unknown significance (VUS). A computer prediction of lower stability is not the same as showing the channel works differently. No cell or electrical tests were done, and no known harmless or known disease-causing variants were modeled for comparison.

      “Protein informatics and molecular modeling are used to confirm the pathogenicity of the unique TRPV4 variant found in this family.”Abstract, Conclusions; compare Methods 3.1 ('variant of unknown significance (VUS) is p.K801E')

    • ModerateDiscussion and conclusion disagree on gain or loss of function

      The discussion says the authors cannot tell whether the variant causes gain or loss of function. The conclusion then says gain of function is 'plausibly likely'. The data in the paper do not settle this.

      “we find it plausibly likely that NC_000012.12 :c.2401A>G (p.K801E) is a gain-of-function variant of TRPV4”Conclusions

    • ModerateLimited family testing

      Only the proband and her daughter were tested, and both are affected. The father was not tested and his features are 'per report', yet the authors say it is 'all but certain' he passed on the variant. No unaffected relatives were tested, so segregation with disease is weakly shown.

      “while we were unable to test her father, it seems all but certain that he gave the proband this variant.”Discussion

    • ModerateNo nerve or muscle assessment reported

      The paper itself notes that TRPV4 skeletal and nerve-muscle conditions can occur together, but it reports no neurological examination or nerve conduction tests for anyone in the family. It is unknown whether nerve involvement was absent or not looked for.

      “Pathogenic variants of TRPV4 can cause both skeletal dysplasias and neuromuscular disorders with these phenotypes not always presenting independently of one another”Introduction

    • MinorModeling method checked only against the authors' own past work

      The modeling approach is supported mainly by citations to the senior author's own earlier studies. The paper does not show how well this method predicts disease for other TRPV4 variants.

      “We have used this method in the past hypothesis generation and mutagenesis experiments”Results

    • MinorText and figure describe the nails differently

      The text describes 'hyperplastic' (overgrown) nails, while the Figure 1 legend says 'hypoplastic' (underdeveloped) nails. These mean opposite things, so one is an error.

      “short broad digits, hyperplastic nails, and short nail beds”Case study; compare Figure 1 legend

    How “unaffected” relatives were checked

    Not described. The pedigree colors relatives 'possessing skeletal abnormalities', but the paper does not say how other relatives were assessed or whether they were examined. The father's features are given 'per report' and he was not tested. No neurological examination or nerve conduction tests are reported for anyone. Quote: “Per report, he had abnormal nails, abnormal hips, and short digits.”

    Three ways to explain it

    Scientific

    A heterozygous TRPV4 c.2401A>G (p.K801E) variant, found by exome sequencing in a 47-year-old woman with features between spondyloepiphyseal dysplasia Maroteaux type and spondylometaphyseal dysplasia Kozlowski type, was also present in her daughter. Monte Carlo-refined structural models predicted reduced subunit stability and weaker inter-subunit interaction near the pore. The authors call the variant pathogenic and 'plausibly likely' gain of function, but no functional assay was performed, segregation rests on the proband and her affected daughter, and the discussion states that gain versus loss of function cannot be determined.

    Easier

    This paper describes a woman with an inherited bone-growth condition, her daughter and her late father. Genetic testing found a new change in the TRPV4 gene in the woman and her daughter. The researchers used computer models of the TRPV4 protein and predicted that the change makes the channel less stable. They describe the change as disease-causing, but they did not test how the channel actually works in cells, and they say they cannot tell whether it makes the channel more or less active.

    Simple

    A mom and her daughter share a new spelling change in the TRPV4 gene, and both have bone problems. A computer drawing of the TRPV4 'door' suggests the change makes it wobblier, but nobody tested the real door, so we cannot be sure what it does.

    Why these ratings

    Relevance 2/5: A TRPV4 skeletal dysplasia family with no nerve assessment reported; CMT2C appears only in a summary table of TRPV4 conditions.

    Evidence 2/5: Based on one family, in which only the proband and her daughter were tested, and on computer predictions alone, with no laboratory test of how the variant changes the channel.

    Funding and conflicts of interest

    Funding was provided by the Center for Individualized Medicine, Mayo Clinic, for the molecular modeling. All authors declare that they have no conflict of interest.

    PubMed 30693671 · DOI

92. #92Full paperCase report with brief literature review

    Long-term follow-up of metatropic dysplasia caused by novel mutations in the TRPV4 gene: Case report and literature review

    Liu Y, Dong Y, Xu J et al. · 2025 · Medicine

    Relevance 2/5Evidence 2/51 major · 2 moderate · 4 minor problems

    This report is mainly about a severe bone growth condition caused by a new TRPV4 change. Its suggestion of nerve involvement is not backed by nerve tests, so it says little about CMT2C.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    Case report with brief literature review

    Who or what

    One girl treated at a hospital in Zhengzhou, China, followed from 3 months to 7 years of age, with metatropic dysplasia (a severe TRPV4-related skeletal condition) and a new TRPV4 change (c.1811T > A, p.I604N) that was not found in either parent.

    What they did

    The authors describe the girl's growth, X-rays and treatment over time, including spinal surgery at age 5. A skeletal gene test found the TRPV4 change, and her parents were tested to see whether it was inherited. They briefly discuss earlier reports of TRPV4 bone and nerve conditions.

    What they found

    • The girl carried a new TRPV4 change, I604N, in exon 11, which was not found in either parent.

      “The examiner had no variation at this locus in this parent, so this mutation was spontaneous”Case presentation, Gene sequencing; Figure 5

    • From 6 months of age her spine and joints developed abnormally, with a sideways and forward spinal curve (scoliosis and kyphosis) that steadily worsened; both hips dislocated at age 2.

      “From the age of 6 months, the joints and spine developed abnormally, manifesting as lateral and kyphosis of the spine, which was progressively aggravated”Case presentation

    • At age 7 her leg strength was grade 1 and she could not stand, while arm strength was about grade 3 to 4. She had no problems with breathing, voice or bladder, and spinal MRI showed no spinal cord compression.

      “muscle strength of both lower limbs was grade 1, and the patient was unable to stand.”Case presentation

    • After spinal surgery at age 5 (vertebral column resection, correction and fusion) she could walk with braces at age 6.

      “She was able to walk with the use of braces at the age of 6 years, and the progression of lateral kyphosis was halted.”Case presentation

    • The authors interpret her leg weakness as part of a mixed skeletal and neuromuscular picture rather than the result of spinal cord compression.

      “suggesting that this TRPV4 mutation may result in a mixed phenotype of severe skeletal dysplasia and neuromuscular pathology with motor inability.”Discussion

    What the authors concluded

    A new TRPV4 change, I604N, caused metatropic dysplasia with what the authors interpret as a mixed skeletal and neuromuscular picture. They say this adds to the known range of TRPV4 changes and features, that spinal surgery delayed the course of the disease in this girl, and that the report may help prenatal diagnosis, genetic counseling, research and the development of symptomatic treatment.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • MajorNerve involvement not tested

      The paper calls this a mixed skeletal and neuromuscular picture, but reports no nerve conduction studies, EMG or muscle biopsy. Leg weakness is the main evidence. The authors themselves note that severe skeletal disease can press on nerves, and the girl had major spinal deformity and spinal surgery, so the cause of her weakness is not established.

      “It might be difficult to differentiate between neurological symptoms caused by TRPV4 variations in patients with severe skeletal dysplasia because severe skeletal manifestations can compress nerves and result in neurological symptoms.”Discussion

    • ModerateLimited genetic evidence

      The change is new and not inherited from the parents, which supports a causal role, but no lab test of the channel, population database check or formal variant classification is reported. The abstract mentions sequencing of the 'whole exon gene', while the case section describes a skeletal gene check, so it is unclear whether genes for nerve or muscle disease were looked at.

      “Gene sequencing: skeletal gene check (MyGenostics)”Case presentation; Abstract, Outcomes

    • ModerateClaims go beyond one case

      The abstract says surgery 'can effectively delay the course of the disease', and the paper says it provides a reference for prenatal diagnosis and treatment development. One child cannot show that surgery changes the course of the disease.

      “Surgical intervention can effectively delay the course of the disease.”Abstract, Interventions

    • MinorConflicting account of progress after surgery

      The case text says progression of the spinal curve was halted after surgery, but the Figure 3 legend says the kyphosis worsened again 2 years after the operation.

      “the kyphosis gradually worsened again with growth and development 2 years after the operation”Figure 3 legend

    • MinorInconsistent variant description

      The change is given as c.1811T > A in the abstract, case section and Figure 5, but as position 1881 in the conclusion. This looks like a typing error, but exact variant names matter for genetic records.

      “a missense mutation in exon 11 of the TRPV4 gene at position 1881 (c. 1881T > A, p. I604N)”Discussion, conclusion paragraph

    • MinorFunding statement contradicts author roles

      The paper states the authors had no funding, yet two authors are listed under 'Funding acquisition'. This is probably a form-filling error but leaves the funding unclear.

      “The authors have no funding and conflicts of interest to disclose.”End matter; Author contributions

    • MinorLiterature review is brief and not systematic

      The 'literature review' in the title is a short discussion of selected papers with no search method, so it does not give a complete picture of TRPV4 mixed phenotypes.

    Three ways to explain it

    Scientific

    A de novo heterozygous TRPV4 missense variant in exon 11 (c.1811T > A, p.I604N) was identified in a girl with radiologically typical metatropic dysplasia followed from 3 months to 7 years, with progressive kyphoscoliosis treated by vertebral column resection and fusion at age 5. The authors attribute her severe lower limb weakness to a combined skeletal and neuromuscular phenotype, but no electrophysiology or muscle pathology is reported, so neuromuscular involvement is not established. As a single case, it extends the TRPV4 variant spectrum but cannot establish genotype-phenotype correlations or treatment effects.

    Easier

    Metatropic dysplasia is a rare condition in which bones grow abnormally, causing short limbs and a spine that curves more and more over time. This report follows one girl with the condition from babyhood to age 7 and finds a new change in her TRPV4 gene that her parents did not have. She also developed very weak legs, which the authors think may mean her nerves or muscles are affected too, but they did not do nerve tests to check this. She had spinal surgery at age 5 and could later walk with braces.

    Simple

    TRPV4 is like a door that lets calcium into cells, and a change in it can affect how bones grow. This girl had a new change in her door gene, and her bones and spine grew in an unusual way. Her legs became very weak too, but the doctors did not test her nerves, so we do not know why.

    Why these ratings

    Relevance 2/5: It is mainly about a severe skeletal condition caused by TRPV4; the nerve and muscle part is short and not backed by nerve tests.

    Evidence 2/5: A single case report; the skeletal diagnosis is documented with imaging and genetics, but the claimed nerve involvement was not tested and there are internal inconsistencies.

    Funding and conflicts of interest

    The paper states: 'The authors have no funding and conflicts of interest to disclose.' The author contributions nonetheless list two authors under 'Funding acquisition'.

    PubMed 40258774 · DOI

93. #93Full paperCell study (protein interaction screen and lab experiments)

    The Zinc-Finger Domain Containing Protein ZC4H2 Interacts with TRPV4, Enhancing Channel Activity and Turnover at the Plasma Membrane

    Vangeel L, Janssens A, Lemmens I et al. · 2020 · International journal of molecular sciences

    Relevance 2/5Evidence 2/52 moderate · 4 minor problems

    This lab study adds a new piece to the picture of how TRPV4 is controlled. It did not look at CMT2C mutations or nerve cells, so it has no direct meaning yet for people with CMT2C.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    Cell study (protein interaction screen and lab experiments)

    Who or what

    Normal human TRPV4 and human ZC4H2, both introduced into HEK-293T cells (a human embryonic kidney cell line). A screen of 14,817 human protein clones against the TRPV4 N terminus (amino acids 1 to 466). mRNA levels were measured in mouse bone cells (osteoclasts, osteoblasts, chondrocytes), dorsal root ganglia and embryonic spinal motor neuron tissue. TRPV3 was used as a comparison channel.

    What they did

    The authors screened for proteins that bind the inside-facing start of TRPV4 (the N terminus, which carries most disease mutations) and chose ZC4H2 for further study. They confirmed binding, measured calcium levels in cells with and without ZC4H2, and used special microscopy (TIRF and TIR-FRAP) to follow how TRPV4 moves to and from the cell surface.

    What they found

    • A screen identified ZC4H2, a zinc-finger protein linked to a group of rare neurological conditions (ZARDs), as a protein that binds TRPV4.

      “We discovered the zinc-finger domain-containing protein ZC4H2 as a TRPV4-interacting protein.”Abstract

    • In cells making both proteins, ZC4H2 raised resting calcium levels and increased TRPV4 responses to different triggers, including cell swelling.

      “we found that ZC4H2 increases both the basal activity of human TRPV4 as well as Ca 2+ responses evoked by ligands or hypotonic cell swelling.”Abstract

    • ZC4H2 had no effect on TRPV3, the closest related channel, suggesting the effect is specific to TRPV4.

      “ZC4H2 did not have an effect in cells expressing the closest homologue TRPV3, excluding a general effect on TRP channel activity or on cellular Ca 2+ handling.”Discussion

    • ZC4H2 did not change how much TRPV4 was made or how much sat at the cell surface at any one time.

      “these data indicate that ZC4H2 does not increase the expression levels of TRPV4 nor its steady-state presence at the plasma membrane.”Results, section 2.3

    • ZC4H2 appeared to speed up the movement of TRPV4 to and from the cell surface.

      “we found that ZC4H2 accelerates both the bleaching and the recovery from bleaching of TRPV4-GFP, indicative of a faster delivery to and retrieval from the plasma membrane.”Discussion

    • In mouse tissues, ZC4H2 and TRPV4 were both present in bone and cartilage cells, while TRPV4 levels were low in sensory and motor nerve tissue.

      “the qPCR experiments showed substantial co-expression of TRPV4 and ZC4H2 in osteoblasts, osteoclasts, and chondrocytes.”Results, section 2.1

    What the authors concluded

    ZC4H2 is a newly identified partner of TRPV4 that binds its N terminus, increases its activity and speeds up its turnover at the cell surface. The authors suggest a possible link between TRPV4-related diseases and ZC4H2-associated rare disorders, which share some neuromuscular features.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • ModerateNo disease mutations tested

      The study was motivated by the idea that disease mutations change how TRPV4 binds partner proteins, but only normal TRPV4 was tested. Whether CMT2C mutations change ZC4H2 binding or its effects is unknown.

      “it has been hypothesized that disease-causing mutations affect interaction with regulatory proteins.”Abstract

    • ModerateOne cell line with greatly overproduced proteins

      All functional work used HEK-293T kidney-derived cells in which both proteins were raised far above natural levels. Effects seen under these conditions may not match what happens in nerve or bone cells in the body.

      “At the mRNA level, expression of TRPV4 and ZC4H2 was very low in non-transfected HEK-293T, and increased by more than three orders of magnitude upon transfection”Results, section 2.2

    • MinorReplication not clearly reported

      Numbers are given as counts of cells (for example 16 and 14 cells for the microscopy tests), but the number of separate experiments behind them is not stated. Cells from one dish are not fully independent, so the strength of the statistics is hard to judge.

      “Number of cells in ( D - G ): control: n = 16; ZC4H2: n = 14.”Figure 4 legend

    • MinorMethods may miss smaller changes

      The authors acknowledge that their surface-level tests might not detect moderate changes in TRPV4 amount that could still explain the calcium results, and that recovery in the microscopy test had not finished in some cells. So the proposed mechanism is not settled.

      “we acknowledge that these approaches may not be sensitive enough to pick up changes in protein levels in the range of 20%”Discussion

    • MinorDrug concentration typo

      The Figure 2 legend gives 4α-PDD at 10 mM, while the Methods and Figure A3 give 10 μM. This is very likely a typing error but should be noted.

      “4α-PDD (10mM)”Figure 2 legend; compare Methods 4.5

    • MinorLink to ZC4H2-related disorders is speculative

      The suggested link between TRPV4 diseases and ZC4H2-associated rare disorders rests on overlapping symptoms and cell work. No patient data were studied. The authors word this as a suggestion.

      “Our results thus describe a newly identified TRPV4 interactor, and suggest a potential link between ZARD and TRPV4-pathies.”Introduction

    Three ways to explain it

    Scientific

    Using a MAPPIT two-hybrid screen with the TRPV4 N terminus (amino acids 1 to 466) as bait, the authors identified ZC4H2 as a TRPV4 interactor and confirmed binding by co-immunoprecipitation in HEK-293T cells. Co-expressed ZC4H2 raised basal intracellular calcium and enhanced TRPV4 responses to arachidonic acid, 4α-PDD and hypotonic swelling, without affecting TRPV3 or altering TRPV4 mRNA, total protein or steady-state surface levels. TIR-FRAP suggested faster TRPV4 delivery to and retrieval from the plasma membrane. No disease-causing TRPV4 variants were tested.

    Easier

    TRPV4 does not work alone: other proteins attach to it and change how it behaves. This team searched thousands of human proteins and found one called ZC4H2 that sticks to the inside end of TRPV4. In kidney-derived cells grown in a dish, adding ZC4H2 made TRPV4 let in more calcium and made TRPV4 move on and off the cell surface faster. They did not test any of the TRPV4 changes that cause disease, so any link to CMT2C is still unknown.

    Simple

    TRPV4 is like a door that lets calcium into a cell. The scientists found a helper protein, ZC4H2, that makes the door let more calcium through and get swapped in and out faster, at least in cells in a dish. They have not yet checked whether this matters for CMT2C.

    Why these ratings

    Relevance 2/5: Basic research on a new TRPV4 partner protein; CMT2C is mentioned only as background and no neuropathy mutations or nerve cells were tested.

    Evidence 2/5: Reasonable lab work, but all functional tests were done in one kidney-derived cell line with both proteins overproduced, which is far from human nerves.

    Funding and conflicts of interest

    Supported by the KU Leuven Research Council (C1-TRPLe), the Belgian Science Policy BELSPO (IUAP P7/13) and the VIB. The authors declare no conflict of interest, and the funders had no role in the study.

    PubMed 32443528 · DOI

94. #94Abstract onlyReview

    Recent advances in the genetics of hereditary axonal sensory-motor neuropathies type 2

    Ajroud-Driss S, Deng HX, Siddique T · 2011 · Current neurology and neuroscience reports

    Relevance 2/5Evidence 1/51 moderate problem

    This review places CMT2C among more than 10 known types of axonal CMT, each linked to a different gene. The abstract does not say what the review reports about CMT2C itself.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    Review

    Who or what

    No new patients. A review of the genetics of CMT type 2 (axonal CMT), covering more than 10 genes classified from CMT2A to CMT2N.

    What they did

    The authors reviewed recent advances in the genetics of axonal CMT, including the genes found in the previous decade and the biological pathways they may share. This analysis is based only on the abstract, so the methods cannot be judged.

    What they found

    • The gene causing CMT2C and SPSMA (written as CMT2C/SPSMA in the abstract) was the most recent CMT2 gene to be discovered at the time of writing. The abstract does not name the gene.

      “the gene causing CMT2C/SPSMA being the last one discovered.”Abstract

    • More than 10 genes were linked to CMT2 subtypes, and some appear to act in common biological pathways, which may help explain how these conditions arise.

      “More than 10 genes are now associated with different subtypes of CMT2, which are classified from CMT2A to CMT2N.”Abstract

    What the authors concluded

    The number of CMT2 genes has grown quickly, and although they have different functions, some seem to share pathways that give clues to how axonal CMT develops.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • ModerateMethods cannot be judged from the abstract

      Only the abstract was available, so it is not possible to see how the literature was chosen or what the review says about CMT2C. As a review it presents no new data.

    Three ways to explain it

    Scientific

    This review summarizes the expanding genetics of autosomal dominant and recessive CMT2, with more than 10 genes defining subtypes CMT2A to CMT2N, the CMT2C/SPSMA gene being the most recently identified. The authors note that despite distinct functions, some CMT2 genes converge on common biological pathways relevant to pathogenesis. Its coverage of CMT2C cannot be judged from the abstract.

    Easier

    This article reviews what was known about the genes behind axonal CMT, the type where the long nerve fibers themselves are damaged. More than 10 genes had been found, and the one causing CMT2C was the newest. Some of these genes seem to work in the same processes inside the cell, which may help scientists understand the disease.

    Simple

    There are many kinds of CMT, and each is caused by a different gene, like different keys for different locks. This article lists them, and the gene for CMT2C was the newest one found when it was written.

    Why these ratings

    Relevance 2/5: A broad review of axonal CMT genetics in which CMT2C is named as the newest gene discovery; how much it covers CMT2C cannot be told from the abstract.

    Evidence 1/5: Rated 1 because only the abstract was available, and a review summarizes earlier work rather than adding new data.

    Funding and conflicts of interest

    Not stated in the abstract.

    PubMed 21327736 · DOI

95. #95Abstract onlyReview proposing a clinical classification

    Charcot-Marie-Tooth disease (CMT): distinctive phenotypic and genotypic features in CMT type 2

    Gemignani F, Marbini A · 2001 · Journal of the neurological sciences

    Relevance 2/5Evidence 1/51 moderate problem

    This older review lists CMT2C as a distinct type of axonal CMT marked by vocal cord weakness. The abstract gives no further detail about it.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    Review proposing a clinical classification

    Who or what

    No new patients. A review of the clinical types and known genes or gene regions of CMT type 2, including CMT2C.

    What they did

    The authors proposed a way to classify CMT2 by its clinical features and reviewed what was known about each genetic type at the time. This analysis is based only on the abstract, so the methods cannot be judged.

    What they found

    • The authors propose grouping CMT2 into classical CMT2, variants with atypical features, and 'CMT2 plus' forms involving other parts of the nervous system.

      “We propose a clinical classification of the CMT2 phenotypes, and review the features of the identified CMT2 genotypes.”Abstract

    • CMT2C is listed among the recognized CMT2 genetic types, identified by vocal cord paresis; unlike most others in the list, no chromosome location is given for it.

      “CMT2C (with vocal cord paresis)”Abstract

    What the authors concluded

    The identified CMT2 genetic types may match earlier clinical subtypes, but the match is imperfect, and finding more CMT2 genes should clarify how axonal degeneration happens.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • ModerateMethods cannot be judged from the abstract

      Only the abstract was available, so it is not possible to see what the review says about CMT2C or how the classification was developed. As a review it presents no new data, and the abstract gives no gene location for CMT2C, so the review reflects knowledge from before the CMT2C gene location was known.

      “Further cloning of the CMT2 genes will ultimately shed light on the pathogenic mechanism(s)”Abstract

    Three ways to explain it

    Scientific

    This review proposes a phenotypic classification of CMT2 into classical CMT2, atypical variants and complex 'CMT2 plus' forms, and surveys the genotypes then recognized, including CMT2C, defined by vocal cord paresis and listed without a mapped locus. The authors note an imperfect correspondence between genotypes and clinical subtypes. Its specific treatment of CMT2C cannot be judged from the abstract.

    Easier

    This article sorts the axonal type of CMT, CMT2, into groups based on symptoms, and lists the genetic types known at the time. CMT2C is listed as the type that comes with weakness of the vocal cords. The authors say finding more genes should help explain why the nerves are damaged.

    Simple

    Doctors tried to sort the different kinds of CMT2 into groups, like sorting books onto shelves. CMT2C went on the shelf for the kind that can make the voice weak.

    Why these ratings

    Relevance 2/5: A broad CMT2 review in which CMT2C is one of several types covered; how much it says about CMT2C cannot be told from the abstract.

    Evidence 1/5: Rated 1 because only the abstract was available, and a review summarizes earlier work rather than adding new data.

    Funding and conflicts of interest

    Not stated in the abstract.

    PubMed 11231025 · DOI

96. #96Abstract onlyReview

    Respiratory muscle weakness in peripheral neuropathies

    Burakgazi AZ, Höke A · 2010 · Journal of the peripheral nervous system: JPNS

    Relevance 2/5Evidence 1/51 moderate problem

    This review lists CMT2C among the few CMT types linked to weakness of the diaphragm, the main breathing muscle. It notes that ordinary nerve tests often do not reflect how well someone is breathing.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    Review

    Who or what

    No new patients. A review of breathing muscle weakness across peripheral neuropathies, including Guillain-Barré syndrome, chronic inflammatory demyelinating polyneuropathy, multifocal motor neuropathy and some CMT subtypes such as CMT2C.

    What they did

    The authors reviewed how often and how peripheral neuropathies weaken the diaphragm and other breathing muscles, and how breathing problems are assessed and managed. This analysis is based only on the abstract, so the methods cannot be judged.

    What they found

    • Common neuropathies do not usually weaken the diaphragm, but certain CMT subtypes, including CMT2C, have been linked to diaphragm weakness.

      “The linkage has been described between certain subtypes of Charcot-Marie-Tooth (CMT) disease such as CMT2C and CMT4B1 and diaphragmatic weakness.”Abstract

    • Nerve test results generally do not match breathing symptoms or lung function results in peripheral neuropathies, except for the size of responses from the phrenic nerve, the nerve to the diaphragm.

      “A correlation usually has not been found between electrophysiologic findings and clinical respiratory signs or spirometric abnormalities in peripheral neuropathies except in amplitudes of evoked phrenic nerve responses.”Abstract

    • The authors stress careful and frequent assessment of breathing function by a qualified team, and suggest that ventilation criteria developed for Guillain-Barré syndrome may be applied to other neuropathies with breathing problems.

      “Careful and frequent assessment of respiratory function by a qualified team of healthcare professionals and physicians is essential.”Abstract

    What the authors concluded

    Breathing muscle weakness is uncommon in most neuropathies but is linked to some CMT subtypes such as CMT2C. Breathing function needs careful, frequent assessment, and approaches developed for Guillain-Barré syndrome may be applied to other neuropathies.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • ModerateMethods cannot be judged from the abstract

      Only the abstract was available, so it is not possible to see how the literature was selected or what evidence lies behind the CMT2C link. The management approach is largely drawn from Guillain-Barré syndrome, a different condition, and the abstract does not show how well it fits CMT2C.

      “Experience in GBS has led to a standardized approach to manage respiratory problems in peripheral neuropathies.”Abstract

    Three ways to explain it

    Scientific

    This review notes that diaphragmatic weakness is uncommon in most peripheral neuropathies but is described in Guillain-Barré syndrome and in certain CMT subtypes, including CMT2C and CMT4B1. Electrophysiological findings generally do not correlate with respiratory signs or spirometry, except for the amplitudes of evoked phrenic nerve responses. The authors recommend structured respiratory assessment and suggest applying Guillain-Barré ventilation criteria to other neuropathies, though the evidence base cannot be judged from the abstract.

    Easier

    This article reviews breathing muscle weakness in nerve diseases. Most nerve diseases do not weaken the diaphragm, the main breathing muscle, but some types of CMT, including CMT2C, have been linked to diaphragm weakness. The usual nerve tests often do not show how well someone is breathing, apart from tests of the nerve that runs to the diaphragm.

    Simple

    The diaphragm is a muscle under your lungs that works like a pump to help you breathe. In a few kinds of CMT, including CMT2C, this pump can get weak, so doctors check breathing carefully.

    Why these ratings

    Relevance 2/5: Mostly about other neuropathies, especially Guillain-Barré syndrome, but it names CMT2C among the CMT types linked to diaphragm weakness.

    Evidence 1/5: Rated 1 because only the abstract was available, and a review summarizes earlier work rather than adding new data.

    Funding and conflicts of interest

    Not stated in the abstract.

    PubMed 21199102 · DOI

97. #97Abstract onlyReview

    The genetics of spinal muscular atrophies

    Wee CD, Kong L, Sumner CJ · 2010 · Current opinion in neurology

    Relevance 2/5Evidence 1/51 moderate problem

    This review names TRPV4 as one of the genes newly linked to spinal muscular atrophy at the time. The treatment progress it describes was for a different form of SMA, not for TRPV4 disease.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    Review

    Who or what

    No new patients. A review of the clinical, genetic and treatment advances across at least 16 forms of spinal muscular atrophy, including those caused by TRPV4.

    What they did

    The authors reviewed progress in describing the different spinal muscular atrophies, the genes behind them, cell and animal models, and treatment development. This analysis is based only on the abstract, so the methods cannot be judged.

    What they found

    • Genes for 14 forms of spinal muscular atrophy had been found in the previous decade, and TRPV4 was one of four found in the year before the review.

      “The genes associated with 14 of these disorders have been identified in the last decade, including four within the last year: TRPV4, ATP7A, VRK1, and HSPB3.”Abstract

    • The treatment progress described, using gene therapy, antisense oligonucleotides and small molecules, was in preclinical work for proximal SMA, not for TRPV4 disease.

      “there has also been remarkable progress in preclinical therapeutics development for proximal SMA using gene therapy, antisense oligonucleotides, and small molecules.”Abstract

    What the authors concluded

    Better clinical and genetic description of the spinal muscular atrophies matters for how patients are assessed and managed, and new genes should improve understanding of motor neuron disease and may give new treatment targets.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • ModerateMethods cannot be judged from the abstract

      Only the abstract was available, so it is not possible to see what the review says about TRPV4. As a review it presents no new data, and the treatment advances it describes are for proximal SMA, a different condition.

    Three ways to explain it

    Scientific

    This review summarizes clinical and genetic characterization of at least 16 spinal muscular atrophies, with genes identified for 14, including TRPV4, ATP7A, VRK1 and HSPB3 in the preceding year. It also covers preclinical therapeutic advances in proximal SMA using gene therapy, antisense oligonucleotides and small molecules. Its specific coverage of TRPV4 cannot be judged from the abstract.

    Easier

    This article reviews the many inherited conditions in which the motor nerve cells that control muscles are lost. TRPV4 was one of several genes newly linked to these conditions at the time. The treatment progress described was for a different, more common form, not for TRPV4 disease.

    Simple

    There are many kinds of spinal muscular atrophy, and TRPV4 was one of the genes newly found to cause some of them when this article was written.

    Why these ratings

    Relevance 2/5: A broad review of spinal muscular atrophies in which TRPV4 is named among the newly found genes; how much it covers TRPV4 cannot be told from the abstract.

    Evidence 1/5: Rated 1 because only the abstract was available, and a review summarizes earlier work rather than adding new data.

    Funding and conflicts of interest

    Not stated in the abstract.

    PubMed 20733483 · DOI

98. #98Abstract onlyReview

    Molecular genetics and mechanisms of disease in distal hereditary motor neuropathies: insights directing future genetic studies

    Drew AP, Blair IP, Nicholson GA · 2011 · Current molecular medicine

    Relevance 2/5Evidence 1/51 moderate problem

    This review lists TRPV4 among the genes that cause inherited motor neuropathies, a group that overlaps with CMT2. The abstract does not describe TRPV4 disease in detail.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    Review

    Who or what

    No new patients. A review of the genes and disease mechanisms of distal hereditary motor neuropathies (dHMN), covering 11 causative genes including TRPV4.

    What they did

    The authors reviewed the known dHMN genes, what these genes do, possible shared disease mechanisms, and the overlap between dHMN, CMT2 and spinal muscular atrophy. This analysis is based only on the abstract, so the methods cannot be judged.

    What they found

    • At the time, 11 causative genes were known for dHMN, including TRPV4, with five more gene regions where the gene had not been found.

      “There are now 11 causative genes described for dHMN, and an additional five genetic loci with unidentified genes.”Abstract

    • Shared disease mechanisms are emerging, including disrupted calcium channel activity, though the abstract does not say which genes each mechanism applies to.

      “These include disruption of axonal transport, RNA processing defects, protein aggregation and inclusion body formation, disrupted calcium channel activity, and loss of neuroprotective signalling.”Abstract

    • Some dHMN genes also cause CMT and spinal muscular atrophy, so these conditions overlap.

      “Some of these dHMN genes are also mutated in Charcot-Marie-Tooth (CMT) disease and spinal muscular atrophy (SMA).”Abstract

    What the authors concluded

    dHMN is genetically varied and overlaps with CMT2 and spinal muscular atrophy. How the mutations cause disease is not fully understood, but common mechanisms are emerging.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • ModerateMethods cannot be judged from the abstract

      Only the abstract was available, so it is not possible to see how the literature was chosen or what the review says about TRPV4. As a review it presents no new data, and the mechanisms are described as still emerging.

      “The pathogenesis of mutations remains to be fully elucidated”Abstract

    Three ways to explain it

    Scientific

    This review describes 11 causative genes for distal hereditary motor neuropathies, including TRPV4, and five further loci, and outlines emerging shared pathogenic mechanisms such as impaired axonal transport, RNA processing defects, protein aggregation, disrupted calcium channel activity and loss of neuroprotective signaling. It highlights genetic overlap between dHMN, CMT2 and SMA. Its treatment of TRPV4 cannot be judged from the abstract.

    Easier

    This article reviews inherited conditions that weaken muscles by damaging the motor nerves, with little or no effect on feeling. It lists 11 genes known to cause them, including TRPV4, and describes several ways these genes may damage nerves, one being problems with channels that let calcium into cells. Some of the same genes also cause types of CMT.

    Simple

    Some genes, including TRPV4, can make the nerves that move muscles stop working well. Scientists are still finding out exactly how this happens.

    Why these ratings

    Relevance 2/5: Mostly about distal hereditary motor neuropathies in general, with TRPV4 listed as one of the 11 genes reviewed.

    Evidence 1/5: Rated 1 because only the abstract was available, and a review summarizes earlier work rather than adding new data.

    Funding and conflicts of interest

    Not stated in the abstract.

    PubMed 21902652 · DOI

99. #99Abstract onlyReview

    Distal hereditary motor neuropathies

    Tazir M, Nouioua S · 2024 · Revue neurologique

    Relevance 2/5Evidence 1/51 moderate problem

    This review places TRPV4 among the few genes linked to inherited motor neuropathy with vocal cord weakness, and among genes shared between this group and CMT2. The abstract does not discuss CMT2C itself.

    Full analysis: findings, problems and plain explanations

    The study

    Type

    Review

    Who or what

    No new patients. A review of distal hereditary motor neuropathies (dHMN), a group of inherited conditions causing slowly progressive weakness without sensory loss, covering more than 30 genes, including TRPV4.

    What they did

    The authors reviewed the clinical features, nerve test findings, genes, overlap with other conditions and differential diagnosis of dHMN. This analysis is based only on the abstract, so the methods cannot be judged.

    What they found

    • dHMN with vocal cord palsy is associated with three genes, one of which is TRPV4.

      “dHMN with vocal cord palsy is associated with SLC5A7, DCTN1 and TRPV4 genes.”Abstract

    • Some dHMN genes, including TRPV4, also cause CMT2, so the two groups overlap genetically.

      “a genotypic overlap with some other neurogenetic disorders and other hereditary neuropathies such as CMT2, mainly with the HSPB1, HSPB8, BICD2 and TRPV4 genes of AD-inherited transmission”Abstract

    • Around 60 to 70% of people with dHMN still have no genetic diagnosis, and recent studies put prevalence at 2.14 and 2.3 per 100,000.

      “around 60 to 70% of cases of dHMN remain uncharacterized genetically.”Abstract

    What the authors concluded

    dHMN is genetically varied, with many cases still unexplained. It overlaps with CMT2 and other conditions through shared genes such as TRPV4, and some causes, such as riboflavin transporter deficiency, are important to recognize because they can be treated.

    Problems with the research

    Every study has limits. Listing them shows how much weight each finding can bear.

    • ModerateMethods cannot be judged from the abstract

      Only the abstract was available, so it is not possible to see how the literature was chosen or what the review says about TRPV4 in detail. As a review it presents no new data.

    Three ways to explain it

    Scientific

    This review of distal hereditary motor neuropathies notes that more than 30 genes are implicated, yet around 60 to 70% of cases remain genetically uncharacterized, with prevalence estimates of 2.14 and 2.3 per 100,000. TRPV4 is cited among autosomal dominant genes overlapping with CMT2 and, with SLC5A7 and DCTN1, as a cause of dHMN with vocal cord palsy. Coverage of TRPV4 cannot be judged from the abstract.

    Easier

    This article reviews inherited conditions that slowly weaken muscles by damaging motor nerves, without affecting feeling. More than 30 genes are involved, but most people with these conditions still have no genetic answer. TRPV4 is one of three genes linked to the form that also weakens the vocal cords, and it is also a cause of CMT2.

    Simple

    Some inherited nerve conditions make muscles weak but do not change how things feel. TRPV4 is one of the genes that can cause the kind that also makes the voice weak.

    Why these ratings

    Relevance 2/5: Mostly about distal hereditary motor neuropathies in general, but it names TRPV4 as one of three genes associated with the form with vocal cord palsy and as a gene shared with CMT2.

    Evidence 1/5: Rated 1 because only the abstract was available, and a review summarizes earlier work rather than adding new data.

    Funding and conflicts of interest

    Not stated in the abstract.

    PubMed 38702287 · DOI

100. #100Abstract onlyReview

     Adult-onset spinal muscular atrophy: An update

     Juntas Morales R, Pageot N, Taieb G et al. · 2017 · Revue neurologique

     Relevance 2/5Evidence 1/51 moderate problem

     This review includes TRPV4 among the genes that can cause spinal muscular atrophy starting in adulthood. The treatments in trials that it mentions are for other forms, not for TRPV4 disease.

     Full analysis: findings, problems and plain explanations

     The study

     Type

     Review

     Who or what

     No new patients. A review of adult-onset genetic spinal muscular atrophies with a known gene (SMN1-related SMA, Kennedy disease, CHCHD10, TRPV4, DYNC1H1 and BICD2), plus sporadic lower motor neuron disease.

     What they did

     The authors gave an overview of the clinical features and disease mechanisms of adult-onset genetic spinal muscular atrophies. This analysis is based only on the abstract, so the methods cannot be judged.

     What they found

     • TRPV4 is one of the genes covered as a cause of adult-onset spinal muscular atrophy.

       “This report provides a general overview of the clinical features and pathophysiological mechanisms in adult-onset genetic SMA disorders in which the causative gene has been identified (SMN1-related SMA, Kennedy disease, CHCHD10, TRPV4, DYNC1H1 and BICD2).”Abstract

     • Treatments being tested in clinical trials are described for the most frequent forms, SMN1-related SMA and Kennedy disease; the abstract does not mention treatments for TRPV4.

       “has led to the development of therapeutic strategies currently being tested in clinical trials.”Abstract

     What the authors concluded

     Many new genes and phenotypes have been described in adult-onset spinal muscular atrophy, shedding light on motor neuron degeneration, and progress in the commonest forms has led to treatments now in clinical trials.

     Problems with the research

     Every study has limits. Listing them shows how much weight each finding can bear.

     • ModerateMethods cannot be judged from the abstract

       Only the abstract was available, so it is not possible to see how the literature was chosen or what the review says about TRPV4. As a review it presents no new data, and the treatments it mentions are for other forms of spinal muscular atrophy.

     Three ways to explain it

     Scientific

     This review summarizes clinical features and pathophysiology of adult-onset genetic SMA with identified genes, including SMN1-related SMA, Kennedy disease, CHCHD10, TRPV4, DYNC1H1 and BICD2, and discusses progressive muscular atrophy as a likely ALS variant. Therapeutic trials are mentioned only for SMN1-related SMA and Kennedy disease. Its coverage of TRPV4 cannot be judged from the abstract.

     Easier

     This article reviews inherited conditions in which the motor nerve cells that control muscles are lost, starting in adulthood. TRPV4 is one of six genes it covers. The new treatments it mentions are for other, more common forms, not for TRPV4 disease.

     Simple

     Some people's muscles get weak as adults because the nerve cells that move them stop working. TRPV4 is one of several genes that can cause this.

     Why these ratings

     Relevance 2/5: Mostly about other forms of spinal muscular atrophy, but TRPV4 is one of the six genes the review covers.

     Evidence 1/5: Rated 1 because only the abstract was available, and a review summarizes earlier work rather than adding new data.

     Funding and conflicts of interest

     Not stated in the abstract.

     PubMed 28456383 · DOI

101. #101Abstract onlyRetrospective single-center case series (children)

     [Experience in molecular diagnostic in hereditary neuropathies in a pediatric tertiary hospital]

     Fernández-Ramos JA, López-Laso E, Camino-León R et al. · 2015 · Revista de neurologia

     Relevance 2/5Evidence 1/51 moderate problem

     Among 36 children with CMT at one hospital, one had a TRPV4 change, with joint stiffness from birth and vocal cord paralysis; the child's mother had weakness of the shoulder and lower leg muscles. This shows TRPV4 disease can appear among children referred for CMT.

     Full analysis: findings, problems and plain explanations

     The study

     Type

     Retrospective single-center case series (children)

     Who or what

     36 children diagnosed with CMT at one tertiary hospital between 2003 and 2015, including one child with a TRPV4 mutation.

     What they did

     The authors looked back at the records of children diagnosed with CMT at their hospital and summarized which genetic diagnoses they had received. This analysis is based only on the English abstract, so the methods cannot be judged.

     What they found

     • CMT1A (a duplication in PMP22, found in 16 patients) was the most common diagnosis at 44%, and 12 patients had no molecular diagnosis.

       “CMT1A predominated in our series (44%), as previous studies.”Abstract, Conclusions

     • One child born with stiff joints in many parts of the body (multiple congenital arthrogryposis) and vocal cord paralysis had a TRPV4 mutation, with congenital spinal muscular atrophy and a mild distal axonal neuropathy. The child's mother had a scapuloperoneal syndrome.

       “a patient with multiplex congenital arthrogryposis and vocal cord paralysis, whose mother had a scapular-peroneal syndrome, had a congenital spinal muscular atrophy with mild distal axonal neuropathy by mutation in gene TRPV4”Abstract, Results

     What the authors concluded

     CMT1A was the most common type, as in earlier studies. The authors highlight the child with a TRPV4 mutation, a gene recently described as a cause of CMT2C, and note that the share of patients without a molecular diagnosis is similar to the main European series.

     Problems with the research

     Every study has limits. Listing them shows how much weight each finding can bear.

     • ModerateMethods cannot be judged from the abstract

       Only the abstract was available, so it is not possible to check how patients were selected, which genetic tests were used, or which TRPV4 variant the child carried. The abstract does not say whether the mother with a scapuloperoneal syndrome was examined or genetically tested. It is a retrospective review from one referral center.

       “Retrospective study of 36 pediatric patients diagnosed with CMT in a tertiary center in 2003-2015.”Abstract, Patients and methods

     Three ways to explain it

     Scientific

     In a retrospective series of 36 pediatric CMT patients from one tertiary center (2003-2015), CMT1A due to PMP22 duplication was the most frequent diagnosis (44%), and 12 patients remained without a molecular diagnosis. One child with multiple congenital arthrogryposis and vocal cord paralysis had a TRPV4 mutation and a phenotype of congenital spinal muscular atrophy with mild distal axonal neuropathy, and the mother had a scapuloperoneal syndrome. Selection, methods and the specific variant cannot be assessed from the abstract.

     Easier

     Doctors looked back at the records of 36 children with CMT seen at one specialist hospital between 2003 and 2015. The most common type was CMT1A. One child, born with stiff joints and paralyzed vocal cords, had a change in the TRPV4 gene, and the child's mother had weakness of the shoulder and lower leg muscles.

     Simple

     A children's hospital checked which CMT genes its young patients had. Many had the most common type, and one child had a change in the TRPV4 gene, which can cause CMT2C.

     Why these ratings

     Relevance 2/5: Mostly about other CMT types in children, but it highlights one child with a TRPV4 mutation, vocal cord paralysis and a mother with a scapuloperoneal syndrome.

     Evidence 1/5: Rated 1 because only the abstract was available; it is also a small retrospective series from one center.

     Funding and conflicts of interest

     Not stated in the abstract.

     PubMed 26602803 · DOI

102. #102Abstract onlyReview

     Molecular basis of axonal dysfunction and traffic impairments in CMT

     Gentil BJ, Cooper L · 2012 · Brain research bulletin

     Relevance 2/5Evidence 1/51 moderate problem

     This review suggests that many different CMT2 genes, possibly including TRPV4, may harm nerves through problems with transport inside nerve fibers or with mitochondria. The abstract presents this as a possibility, not as something shown for TRPV4.

     Full analysis: findings, problems and plain explanations

     The study

     Type

     Review

     Who or what

     No new patients. A review of how genes causing axonal CMT (CMT2), including TRPV4, may share a link through transport inside nerve fibers and mitochondrial dynamics.

     What they did

     The authors reviewed evidence that genes causing axonal CMT, including MFN2, KIF1B, DYNC1H1, Rab7, TRPV4, NEFL and others, may act through common pathways such as transport inside the cell and mitochondrial dynamics. This analysis is based only on the abstract, so the methods cannot be judged.

     What they found

     • The review focuses on what links the genes that cause axonal CMT, and TRPV4 is one of the genes covered.

       “Our review will focus on the common link between genes causing axonal phenotypes like MFN2, KIF1B, DYNC1H1, Rab7, TRPV4, ARSs, NEFL, HSPB1, MPZ, and HSPB8.”Abstract

     • Growing evidence suggests transport inside cells and mitochondrial dynamics might be faulty in CMT2 and might link very different CMT2 genes, although for most of these genes (apart from two motor protein genes) the role in transport is described as less obvious.

       “intracellular trafficking and mitochondrial dynamics might be dysfunctional in CMT2, and these mechanisms might present a common link between dissimilar CMT2-causing genes.”Abstract

     What the authors concluded

     Impaired transport inside nerve cells and disturbed mitochondrial dynamics might be a common link between the different genes that cause axonal CMT.

     Problems with the research

     Every study has limits. Listing them shows how much weight each finding can bear.

     • ModerateMethods cannot be judged from the abstract

       Only the abstract was available, so it is not possible to see what evidence the review gives for TRPV4 specifically. The authors themselves word the shared-mechanism idea cautiously and say the link to transport is less obvious for most of these genes.

       “the involvement of the other CMT2-causing genes in this function is less obvious.”Abstract

     Three ways to explain it

     Scientific

     This review examines whether genes causing axonal CMT, including MFN2, KIF1B, DYNC1H1, Rab7, TRPV4, NEFL and HSPB1, converge on impaired intracellular trafficking and mitochondrial dynamics. Only KIF1B and DYNC1H1 are directly linked to axonal transport, and the authors present a shared trafficking defect as a possible common mechanism rather than an established one. Evidence specific to TRPV4 cannot be judged from the abstract.

     Easier

     Nerve fibers are very long, and cells must move materials and mitochondria, the cell's energy-makers, along them. This review asks whether the many different genes that cause axonal CMT, including TRPV4, all disrupt this transport. The authors present this as a possible common link, not a proven one.

     Simple

     Long nerves need a delivery system to move supplies from one end to the other, like trains on a long track. Scientists think many CMT genes might upset this delivery system, but they are not sure yet.

     Why these ratings

     Relevance 2/5: A general review of axonal CMT mechanisms in which TRPV4 is one of ten genes covered; how much it says about TRPV4 cannot be told from the abstract.

     Evidence 1/5: Rated 1 because only the abstract was available, and a review summarizes earlier work rather than adding new data.

     Funding and conflicts of interest

     Not stated in the abstract.

     PubMed 22595495 · DOI

103. #173Full paper (scan)Family study with autopsy, muscle MRI, muscle pathology and genetic linkage

     Autosomal dominant congenital spinal muscular atrophy: a true form of spinal muscular atrophy caused by early loss of anterior horn cells

     Oates EC, Reddel S, Rodriguez ML et al. · 2012 · Brain: a journal of neurology

     Relevance 1/5Evidence 2/53 moderate · 4 minor problems

     This family did not have a TRPV4 change, so the findings concern a different, unidentified cause of congenital SMA rather than CMT2C. It is mainly a reminder that TRPV4 is only one of several genetic causes of similar symptoms.

     Full analysis: findings, problems and plain explanations

     The study

     Type

     Family study with autopsy, muscle MRI, muscle pathology and genetic linkage

     Who or what

     One four-generation family with autosomal dominant congenital spinal muscular atrophy (SMA), studied through the Children's Hospital at Westmead, Australia. TRPV4 was ruled out in this family. DNA came from nine members of the third and fourth generations. Key people: the proband (IV.2), a girl born to parents of Caucasian ancestry, who died aged 14 months of meningococcal septicemia and had a full autopsy; her severely affected brother (IV.5, aged 4.5 years); her mother (III.2, leg muscle MRI at 32 years); and an affected sister (IV.6). Clinical and nerve test summaries were in supplementary tables that were not part of the document analyzed.

     What they did

     Affected family members were examined, muscle samples were studied, and the paper shows leg muscle MRI findings for the mother. The team tested whether the condition was linked to the TRPV4 region, sequenced TRPV4, and ran a genome-wide linkage study to check other known SMA, motor neuropathy and CMT genes. When the youngest affected girl died of an unrelated infection, they examined her spinal cord, nerve roots, nerves and muscles at autopsy.

     What they found

     • TRPV4 was ruled out: affected children had inherited different copies of the TRPV4 region from their mother, and no TRPV4 mutation was found in the severely affected brother.

       “Microsatellite analysis showed that affected siblings in generation IV had inherited different maternal haplotypes at the TRPV4 locus, which was inconsistent with linkage to this region.”Results, Linkage analysis and sequencing results, journal p. 1717 (PDF p. 7)

     • Known genes for dominant SMA, distal hereditary motor neuropathy and dominant CMT were excluded or made unlikely, but no new gene location reached significance (highest LOD score 1.81).

       “the logarithm (base 10) of odds (LOD) scores did not reach statistical significance for any of these regions (maximum LOD score 1.81)”Results, Linkage analysis and sequencing results, journal p. 1717 (PDF p. 7)

     • At autopsy the girl had fewer motor nerve cells (anterior horn cells) in the lower (lumbar and sacral) spinal cord and, less so, the neck (cervical) region, with thin motor nerve roots. The thoracic and upper lumbar cord looked normal.

       “There was a reduction in anterior horn cell number in the lumbar and, to a lesser degree, the cervical spinal cord, and atrophy of the ventral nerve roots at these levels”Abstract (PDF p. 2); Results, Autopsy findings, journal p. 1718 (PDF pp. 9 to 10)

     • Sensory nerve roots and the sural, phrenic and tibial nerves looked normal, so there was no sign of a sensory or peripheral nerve disorder.

       “The sural, phrenic and tibial nerves contained a normal number and distribution of myelinated fibres”Results, Autopsy findings, journal pp. 1718 to 1719 (PDF pp. 10 and 12)

     • There were no signs of ongoing nerve cell damage in the spinal cord at 14 months, which the authors take to mean the nerve cells were lost early in life.

       “there was no pathological evidence of ongoing loss or degeneration of anterior horn cells suggesting that anterior horn cell loss in dominant congenital spinal muscular atrophy occurs in early life”Abstract (PDF p. 2)

     • The authors contrast this early loss with CMT2C, which they describe as a deterioration of motor nerves after birth, while noting that TRPV4-related congenital SMA, scapuloperoneal SMA and CMT2C are allelic (caused by changes in the same gene).

       “Nevertheless, TRPV4-related dominant congenital SMA, scapuloperoneal SMA and Charcot-Marie-Tooth disease type 2C are clearly allelic disorders”Discussion, journal p. 1722 (PDF pp. 17 to 18)

     What the authors concluded

     Dominant congenital SMA is a true form of spinal muscular atrophy, caused by loss of motor nerve cells in the lumbar and cervical spinal cord early in development; this loss is largely complete by the end of infancy. Because TRPV4 and other known genes were excluded, this family probably has a new genetic form. The authors suggest that muscle MRI may help diagnosis and that other genetic causes of CMT2 are sensible candidates to test in dominant congenital SMA.

     Problems with the research

     Every study has limits. Listing them shows how much weight each finding can bear.

     • ModerateConclusions drawn from one autopsy

       All the spinal cord findings come from one 14-month-old child who died of an unrelated infection. The abstract says these findings 'confirm' the nature of the disease in general, which is more than one case can show.

       “These findings confirm that dominant congenital spinal muscular atrophy is a true form of spinal muscular atrophy caused by a loss of anterior horn cells localized to lumbar and cervical regions early in development.”Abstract (PDF p. 2)

     • ModerateUnknown gene, so findings may not carry over

       TRPV4 was excluded and no gene was found. The family may have a distinct condition, so the autopsy results cannot be assumed to apply to TRPV4-related congenital SMA, to CMT2C, or to other families.

       “suggesting this family has a new genetic form of dominant congenital SMA”Discussion, journal p. 1720 (PDF p. 13)

     • ModerateAffected and unaffected status not fully assessed

       Two relatives were counted as affected from verbal report only, and the paper does not say how relatives were judged unaffected. The linkage analysis assumed a fully penetrant model, so a mildly affected relative wrongly classed as unaffected could distort the gene exclusions.

       “Subjects I.4 and II.2 could not be examined, but by verbal report had life-long difficulty walking long distances or standing for long periods, due to rapid fatigue.”Results, journal p. 1717 (PDF p. 7)

     • MinorToo few DNA samples for gene mapping

       DNA was available from nine relatives, which the authors say was not enough to reach a significant result for any new gene location.

       “However insufficient family DNA samples were available to achieve a significant LOD score to define a new disease locus.”Discussion, journal p. 1720 (PDF p. 13)

     • MinorNerve cell loss described, not counted

       The reduction in motor nerve cells is described from microscope sections compared with one age-matched control. No cell counts are given in the text, so the size of the loss cannot be judged.

       “showing reduction of anterior horn cell bodies in dominant congenital SMA proband, compared with control, especially the medial ventral horn in A.”Figure 4 legend, journal p. 1719 (PDF p. 12)

     • MinorTiming inferred from a single time point

       The claim that the loss is largely complete by the end of infancy is inferred from the absence of active damage at one moment in one child. The authors also note scattered signs that some loss may continue.

       “suggests there may be a small amount of ongoing denervation that may account for the mild clinical progression after birth observed in some individuals.”Discussion, journal p. 1721 (PDF p. 16)

     • MinorBrief, indirectly sourced description of CMT2C

       The short description of CMT2C used for comparison is supported by references whose titles, in the reference list, are about congenital lower motor neuron disorders and dominant congenital SMA rather than CMT2C. This paper should not be used as a description of CMT2C.

       “Charcot-Marie-Tooth disease type 2C is an axonal form of Charcot-Marie-Tooth disease characterized by absence of congenital contractures, onset of weakness and wasting in the lower limb after infancy”Discussion, journal p. 1722 (PDF p. 17)

     How “unaffected” relatives were checked

     Not clearly described. The Methods state that 'All affected family members, except for Subjects I.4 and II.2, were examined by experienced neurologists'. I.4 and II.2 were classed as affected 'by verbal report'. For unaffected relatives the paper says only that 'Features of the condition were not present in Subjects III.1, IV.1, IV.3 or IV.4', without saying whether they were examined or had nerve, muscle or MRI tests. This matters because the linkage analysis assumed full penetrance.

     Three ways to explain it

     Scientific

     In a four-generation family with autosomal dominant congenital SMA, TRPV4 was excluded by haplotype analysis and sequencing, and known dominant SMA, distal HMN and CMT loci by linkage. Autopsy of a 14-month-old proband showed reduced anterior horn cells in the lumbosacral and, less so, cervical cord with atrophic ventral roots, but normal dorsal roots and normal sural, phrenic and tibial nerves. The absence of active neuronal degeneration led the authors to conclude that anterior horn cell loss occurs early and is largely complete by the end of infancy. These conclusions rest on a single autopsy in a family whose causative gene is unknown.

     Easier

     This study looked at a family with a rare inherited condition in which children have weak, wasted leg muscles and stiff joints from birth or early childhood. The family's condition was not caused by TRPV4 or by other known genes for similar conditions. When one affected baby died of an unrelated infection, an examination after death showed that she had fewer of the spinal cord nerve cells that control muscles, while the nerves that carry feeling looked normal. As there were no signs of ongoing damage, the authors think the nerve cells were lost early in life, though this is based on one child.

     Simple

     In this family, children have weak legs from very early in life. Looking at one child's spine after she died of an unrelated illness showed that some of the nerve cells that tell leg muscles to move were missing. It looked as if they were lost early, like a wall built with some bricks missing rather than bricks falling out later. The cause was not the TRPV4 gene.

     Why these ratings

     Relevance 1/5: TRPV4 was ruled out in this family, so its findings do not apply directly to CMT2C; CMT2C appears only in a short comparison in the Discussion.

     Evidence 2/5: Careful work in one family, but the main conclusions about the disease rest on a single autopsy of one child.

     Funding and conflicts of interest

     Funding: NHMRC grant APP1022707 (to K.N.N. and N.F.C.). Competing interests: not stated in the document.

     PubMed 22628388 · DOI

104. #174Abstract onlyGenetic linkage study in two families (about CMT2A)

     Linkage mapping of the gene for Charcot-Marie-Tooth disease type 2 to chromosome 1p (CMT2A) and the clinical features of CMT2A

     Saito M, Hayashi Y, Suzuki T et al. · 1997 · Neurology

     Relevance 1/5Evidence 1/51 moderate problem

     This paper is about a different type of CMT, CMT2A. It mentions CMT2C only as a type whose gene region had not yet been found.

     Full analysis: findings, problems and plain explanations

     The study

     Type

     Genetic linkage study in two families (about CMT2A)

     Who or what

     Two Japanese families with CMT2. CMT2C is mentioned only as background.

     What they did

     The authors ran linkage analysis using markers near the CMT2A, CMT2B and CMT2D gene regions and described the clinical features of the families. This analysis is based only on the abstract, so the methods cannot be judged.

     What they found

     • Both families were linked to the CMT2A region on chromosome 1p, with a highest combined multipoint LOD score of 3.69, and the CMT2B and CMT2D regions were excluded.

       “The highest cumulative multipoint lod score of 3.69 was obtained at D1S244.”Abstract

     • At the time, CMT2C had not been linked to any known gene region.

       “CMT2C (not linked to any known loci)”Abstract

     What the authors concluded

     The two Japanese families have CMT2A linked to chromosome 1p, with similar weakness of the front and back lower leg muscles, reduced or absent leg reflexes and mild sensory disturbance in only 20% of affected people.

     Problems with the research

     Every study has limits. Listing them shows how much weight each finding can bear.

     • ModerateMethods cannot be judged from the abstract

       Only the abstract was available. It does not give family sizes or say how relatives were classed as affected or unaffected, so neither the linkage nor the clinical description can be checked. The paper is not about CMT2C.

     How “unaffected” relatives were checked

     Cannot be judged: the abstract does not say how family members were classed as affected or unaffected. In any case these families have CMT2A, not CMT2C.

     Three ways to explain it

     Scientific

     Linkage analysis in two Japanese CMT2 families gave a maximum cumulative multipoint LOD score of 3.69 at D1S244, assigning them to CMT2A on 1p35-36, with CMT2B and CMT2D excluded. CMT2C is cited only as a CMT2 form then unlinked to any known locus.

     Easier

     Researchers traced the cause of CMT in two Japanese families to a region of chromosome 1 known as the CMT2A region. The paper mentions that the gene region for CMT2C had not been found at the time.

     Simple

     This study found which part of the DNA caused CMT in two families, and it was a different kind of CMT from CMT2C.

     Why these ratings

     Relevance 1/5: It is about CMT2A, and CMT2C appears only in passing as a type not yet linked to any gene region.

     Evidence 1/5: Rated 1 because only the abstract was available, so the family data and clinical methods could not be checked.

     Funding and conflicts of interest

     Not stated in the abstract.

     PubMed 9409358 · DOI

105. #175Abstract onlyRetrospective observational single-center cohort

     Charcot-Marie-Tooth disease: experience from a large Italian tertiary neuromuscular center

     Gentile L, Russo M, Fabrizi GM et al. · 2020 · Neurological sciences: official journal of the Italian Neurological Society and of the Italian Society of Clinical Neurophysiology

     Relevance 1/5Evidence 1/51 moderate problem

     At this large Italian center, TRPV4 changes were found in only 2 of 566 people with CMT, so they were rare there. The figure reflects one center's patients and testing.

     Full analysis: findings, problems and plain explanations

     The study

     Type

     Retrospective observational single-center cohort

     Who or what

     566 people with CMT cared for at the Neuromuscular Center of Messina University Hospital, Italy, between 1994 and 2016, of whom 352 had a genetic diagnosis and 2 had TRPV4 mutations.

     What they did

     The authors reviewed clinical records, nerve tests and genetic results to see how many patients received a genetic diagnosis and how common each genetic type was. This analysis is based only on the abstract, so the methods cannot be judged.

     What they found

     • About 352 of 566 patients (62.19%) received a genetic diagnosis, most often CMT1A due to PMP22 duplication (51.13%).

       “About 352/566 (62.19%) received a genetic diagnosis.”Abstract, Results

     • TRPV4 mutations were rare in this center, found in 2 patients.

       “TRPV4 mutation (n. 2 pts)”Abstract, Results

     What the authors concluded

     The study adds data on how often each CMT gene and subtype occurs in a large Mediterranean area, to help clinicians plan genetic testing.

     Problems with the research

     Every study has limits. Listing them shows how much weight each finding can bear.

     • ModerateMethods cannot be judged from the abstract

       Only the abstract was available, so it is not possible to check which genes were tested in whom or how testing changed over the years. It is a retrospective review from a single referral center, so the figures describe that center's patients, and nothing is reported about the 2 TRPV4 patients themselves.

       “In this retrospective, observational study”Abstract, Methods

     Three ways to explain it

     Scientific

     In a retrospective single-center cohort of 566 CMT patients followed between 1994 and 2016, 352 (62.19%) received a genetic diagnosis, with PMP22 duplication (CMT1A) the most frequent (51.13%). TRPV4 mutations were identified in 2 patients. Testing strategy and selection cannot be assessed from the abstract.

     Easier

     Doctors in Sicily looked back at the records of 566 people with CMT treated at their center over 22 years. Of these, 352 got a genetic diagnosis, and the most common type by far was CMT1A. Only 2 people had a change in the TRPV4 gene.

     Simple

     A big hospital looked at which CMT genes its patients had. Very few, only 2 people, had a change in the TRPV4 gene, so this kind of CMT is uncommon there.

     Why these ratings

     Relevance 1/5: TRPV4 is mentioned only as a rare finding in 2 of 566 patients.

     Evidence 1/5: Rated 1 because only the abstract was available, so the selection and testing methods could not be checked.

     Funding and conflicts of interest

     Not stated in the abstract.

     PubMed 31902012 · DOI

106. #176Abstract onlyDiagnostic genetic study using a multigene panel

     Frequent genes in rare diseases: panel-based next generation sequencing to disclose causal mutations in hereditary neuropathies

     Dohrn MF, Glöckle N, Mulahasanovic L et al. · 2017 · Journal of neurochemistry

     Relevance 1/5Evidence 1/51 moderate problem

     In a large group of people tested with a gene panel for inherited neuropathy, TRPV4 changes were found but were uncommon. The abstract gives no detail about those patients.

     Full analysis: findings, problems and plain explanations

     The study

     Type

     Diagnostic genetic study using a multigene panel

     Who or what

     612 index patients with a CMT-like condition, hereditary sensory neuropathy, familial amyloid neuropathy or small fiber neuropathy, tested with a custom next generation sequencing panel. TRPV4 is one of many genes with findings; the abstract does not say how many TRPV4 cases there were.

     What they did

     The authors tested patients with a customized multigene panel and reported how often a likely cause was found and in which genes. This analysis is based only on the abstract, so the methods cannot be judged.

     What they found

     • At least one putative disease-causing mutation was found in 121 of the 612 patients (19.8%).

       “In 121 cases (19.8%), we identified at least one putative pathogenic mutation.”Abstract

     • TRPV4 was among the less frequently affected genes with likely or known disease-causing variants.

       “We further detected likely or known pathogenic variants in HINT1, HSPB1, NEFL, PRX, IGHMBP2, NDRG1, TTR, EGR2, FIG4, GDAP1, LMNA, LRSAM1, POLG, TRPV4”Abstract

     • The authors conclude that panel-based sequencing is a useful, time- and cost-effective way to reach a diagnosis.

       “We conclude that panel based next generation sequencing is a useful, time- and cost-effective approach”Abstract

     What the authors concluded

     Panel-based next generation sequencing is a useful, time- and cost-effective approach that helps clinicians reach the correct diagnosis in hereditary neuropathies.

     Problems with the research

     Every study has limits. Listing them shows how much weight each finding can bear.

     • ModerateMethods cannot be judged from the abstract

       Only the abstract was available, so it is not possible to check how patients were selected, how variants were classed as pathogenic, or how many TRPV4 cases there were. The abstract gives no cost data, so the cost-effectiveness claim cannot be checked.

     Three ways to explain it

     Scientific

     A customized next generation sequencing panel applied to 612 index patients with hereditary neuropathies identified at least one putative pathogenic mutation in 121 (19.8%), most often in PMP22, GJB1, MPZ, SH3TC2 and MFN2. TRPV4 was among the less frequent genes with likely or known pathogenic variants. The authors judge panel sequencing time- and cost-effective, though the abstract gives no cost data.

     Easier

     Researchers used a test that reads many neuropathy genes at once in 612 people with inherited nerve conditions. A likely cause was found in 121 of them. TRPV4 was one of the less common genes found.

     Simple

     Doctors used a test that checks lots of nerve genes at once, like searching many drawers at the same time. A few people had a change in TRPV4, but most answers were in other genes.

     Why these ratings

     Relevance 1/5: TRPV4 is mentioned only as one of many genes found in a large diagnostic panel study.

     Evidence 1/5: Rated 1 because only the abstract was available, so the panel design, patient selection and variant classification could not be checked.

     Funding and conflicts of interest

     Not stated in the abstract.

     PubMed 28902413 · DOI

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