Through screening 36 unrelated individuals with nondistinct CMT2 and 17 unrelated families with CMT2C phenotype having vocal cord and diaphragmatic involvement, we identified 2 TRPV4
mutations R232C and R316H. R316H mutation has not been reported before and is de novo. R232C is from a CMT2C family, but only the proband has vocal cord involvement whereas other affected do not. This family was previously localized to 12q23–243
and the variability of vocal cord involvement was discussed.19
The findings here emphasize the importance of considering TRPV4
mutation when vocal cord paralysis feature is not present in inherited axonal neuropathy, i.e., nondistinct clinical CMT2. The previous and the current study suggest the location of TRPV4
mutations does not account for this specific neurologic variability.5,–8
This study highlights the genetic heterogeneity of dominantly inherited axonal neuropathies with vocal cord and diaphragmatic involvement. Specifically, 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. Mutations in GDAP1
have been linked to vocal cord paresis with peripheral neuropathy but are uncommon in dominant or axonal cases. Most affected cases caused by GDAP1
mutation have recessive inheritance and demyelination.21
Also highlighted is that the lack of family history should not dissuade from consideration of CMT2C with the proper phenotype as seen in the individual with a de novo mutation R316H. Furthermore, incomplete penetrance or markedly variable expression may also obscure the genetic cause as seen in our original kindred with R269H mutation where repeated clinical examinations over time including specialized nerve conductions were required to identify very mildly affected persons and hence the accurately diagnosed affected status.2,5,8
Therefore penetrance should only be determined close to the end of life as symptoms among some may not occur until the eighth decade of life.2
Scoliosis and small hands were characteristic of our severely affected persons with R316C and R269H mutations5
but were not seen in the individuals described here with R316H and R232C mutations. The fact that TRPV4 can cause nondistinct CMT2 and CMT2C, SPSMA, and distal SMA as well as different bone dysplasias16
implies the existence of complex pathogenic mechanism related to TRPV4 mutation for varied phenotypes, and pathogenic effect of TRPV4 mutation may not exert through cytotoxic calcium influx alone.
To date, 7 mutations in TRPV4 have been identified in 15 families with axonal neuropathies. Six mutations located in the ankyrin-repeat domains, 5 showed segregation with the disease, and 2 de novo mutations. Our newly reported de novo mutation R316H combined with previous studies suggests codon 269R and 316R are both hot spots for axonal neuropathy-linked TRPV4 mutations. The substitutions of 269R or 316R by either cysteine or histidine cause CMT2C.
The R232C mutation was reported in 3 other families with axonal neuropathies8
in addition to the family described here. The TRPV4
mutations in the 3 initial reports were in the third and fourth ankyrin-repeat domains5,–7
while R232C is in the second domain (). The only neuropathy mutation located outside of the ankyrin repeat domain is V620I,8
which was previously reported in brachyolmia.16
Neurologic examination and testing is not reported among the previously reported TRPV4-associated bone dysplasias. The V620I neuropathy case was a de novo mutation, raising the possibility of an additional hot spot in axonal neuropathy. This person had mild skeletal dysplasia features that suggested a possible overlap syndrome.
The previously reported discrepancies in the functional studies are addressed with our current experiments. Two studies showed TRPV4 mutations R269H, R316C, and R269C have normal cellular localization and increased calcium channel activity while another study showed R269H, R315W, and R316C had decreased calcium channel activity and abnormal cellular localization. Since different cell types were used, the cell-type–specific response could explain the conflicting data. To investigate whether gain of function or loss of function of TRPV4 mutations accounted for the abnormality, we performed functional analysis in both HEK 293 and HeLa cells. Using wtTRPV4 as control, we performed subcellular localization, channel activity, and cell viability assay for R232C, R316H, and R269H. All 3 mutations properly localize to the cell membrane and cause gain of function of TRPV4, resulting in increased intracellular calcium with decreased cell viability. The R269H mutant experiments reproduced the prior results. Our data indicate that an increased constitutive function, rather than increased response to agonist stimulation, is probably the key property gained. Significantly increased cell death was only observed in mutant TRPV4-expressing HeLa cells although cell viability was increased in both transfected HeLa and HEK293 cells after using TRPV antagonist ruthenium red to block the mutant channel. The data support that TRPV4 ankyrin repeat mutations exert their effects by dominant gain of function with normal cell membrane localization. Nevertheless we remain open to the possibility that other unidentified factors are involved in pathogenesis and ongoing work is required.
Intracellular hypercalcemia is a common pathway of diverse pathogenesis.10,22
Ruthenium red, the calcium channel blocker, has recently been shown to improve mitochondrial ATP levels and membrane potentials in neurons expressing mutant α-synuclein or PINK-1 associated with Parkinson.23
Optimism of a pharmacologic approach also comes from recent research where l
-type calcium blockers have been suggested to reduce the chance of developing PD.14
Further work will be required to establish whether the observed hypercalcemia cytotoxicity manifests its effect in the cytoplasm or mitochondria. Calcium channel inhibitors provide a rational new approach for investigation of TRPV4-mediated axonal neuropathies.