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

Abstract

Charcot-Marie-Tooth disease type 2C (CMT2C) is an autosomal dominant neuropathy characterized by limb, diaphragm and laryngeal muscle weakness. Two unrelated families with CMT2C showed significant linkage to chromosome 12q24.11. We sequenced all genes in this region and identified two heterozygous missense mutations in the TRPV4 gene, C805T and G806A, resulting in the amino acid substitutions R269C and R269H. TRPV4 is a well-known member of the TRP superfamily of cation channels. In TRPV4-transfected cells, the CMT2C mutations caused marked cellular toxicity and increased constitutive and activated channel currents. Mutations in TRPV4 were previously associated with skeletal dysplasias. Our findings indicate that TRPV4 mutations can also cause a degenerative disorder of the peripheral nerves. The CMT2C-associated mutations lie in a distinct region of the TRPV4 ankyrin repeats, suggesting that this phenotypic variability may be due to differential effects on regulatory protein-protein interactions.

Figure 1

Phenotypic and genetic characteristics of CMT2C. a) 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. Written consent was obtained to publish these photographs. b) Light microscope images of hematoxylin and eosin-stained sections of gastrocnemius muscle biopsy from subject F1.IV.4 demonstrating profound denervation atrophy of muscle. Scale bar=10 µm. c) Toluidine blue-stained section of sural nerve shows mild loss of large, sensory myelinated axons. Scale bar=10 µm. d) Electron microscopy of sural nerve confirms mild loss of large and small myelinated axons and unmyelinated axons. Scale bar=10µm. e) Arrow demonstrates a rare axon undergoing Wallerian-like axonal degeneration. Scale bar=2µm. f) Pedigrees of Families 1 and 2 demonstrating affected subjects in each of 3 generations. White=unaffected, black=affected, and grey=unknown disease status. *Sample collected. g) The haplotypes for subjects F1.V.3 and F2.IV.12 are demonstrated. Family 1 defines the lower border (marker D12S1343) and Family 2 defines the upper border (marker D12S105) of the region of interest. The bracket indicates the linked region. h) Sequencing shows a heterozygous C>T change at position 805 in Family 1 and a G>A change at position 806 in Family 2 in the TRPV4 gene. i) Protein homology of TRPV4 in various species. The mutations result in amino acid substitutions at R269, a highly conserved residue.

Figure 2

Mutant TRPV4 causes neuronal toxicity. a) Quantification of TRPV4 transcript by qRT-PCR in control human lymphoblast lines (n=4), human dorsal spinal cord (n=3), ventral spinal cord (n=3), and tracheal cartilage (n=3) using qRT-PCR primers specific for following exon regions: exons 3–4, exons 5–6, exons 7–8, exon 5, and exon 7. Values are normalized to the first tracheal cartilage sample. Data is averaged; error bars, s.e.m. b) DRG neurons were transfected with WT and mutant forms of TRPV4 (green). At 16 hours, some cells expressing mutant forms of TRPV4 show evidence of early cellular toxicity with a collapsed cytoplasm. The nuclear DAPI stain is blue. Scale bar=40 µm. c) Quantification of propidium iodide uptake in DRG neurons expressing WT and mutant TRPV4 reveals a marked increase of cell toxicity in mutant expressing cells at 48 hours. *p<0.0001. d) HEK293 cells expressing R269C and R269H TRPV4 show an increase in the number of dead cells (red channel is EthD-1 stain) at 48 hours that is prevented by the TRP channel blocker ruthenium red (RR). Green channel is calcein-AM stain for live cells. e) Quantification of cell death in HEK293 cells indicates a time dependent increase in cell death that is blocked by the TRP channel blocker ruthenium red (RR). *p<0.01, **p<0.001. Data in d, f are averaged from three independent experiments; error bars, s.e.m.

Figure 3

The R269C and R269H mutations cause increased TRPV4 currents without a change in membrane localization. a) WT and mutant TRPV4 were expressed in Xenopus oocytes and currents were measured at 18°C and after heating to 38°C. Currents obtained at −100 mV were normalized to currents in WT at 38°C. Basal and stimulated channel activities of both mutants were significantly increased compared to WT, p<0.006. Data are averaged from the number of experiments per condition shown in the columns; error bars, s.e.m. *p<0.006. b) Representative current ramps obtained after pre-incubation in 5 µM 4-α-PDD (obtained from clamping cells from −100 to +100 mV in 500 ms). c) Summary of the data obtained in b). Currents obtained at −100 mV were normalized to WT at 18°C. *p<0.05. d) Immunodetection of TRPV4 proteins shows appropriately sized bands for TRPV4 in 10% of whole cell extract inputs and the biotinylated fraction. There is no difference in the amount of WT compared to mutant TRPV4 in the membrane fraction (biotinylated fraction). Shown is one representative blot of four independent experiments.

Figure 4

The R269C and R269H mutations are located in the ankyrin repeat domain (ARD) of the TRPV4 protein. a) Primary structure of the TRPV4 protein, with the positions of the CMT2C (R269; blue arrowhead) and skeletal dysplasia– (green arrowheads) mutations indicated below. b) Ribbon diagram of the chicken TRPV4 ARD, with the location of the R269 residue (R255 in chicken sequence) depicted as a blue sphere, and the I331 and D333 residues (I317 and D319 in chicken sequence, respectively) previously shown to be mutated in skeletal dysplasia as green spheres. c) Surface representation of the TRPV4 ARD with R269 in blue and I331 and D333 in green. Residues that differ between chicken and human TRPV4 are yellow, demonstrating that the palm and fingers regions are conserved.