Homozygous TRPV4 mutation causes congenital distal spinal muscular atrophy and arthrogryposis

Figures

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  Figure 1. Identification of TRPV4 homozygous mutation (A) Pedigree of the family of the affected patient; the proband is indicated by the arrow. (B) Sanger sequencing pherograms show homozygosity for c.281C>T in TRPV4 in the proband and heterozygosity in both parents. The position of the variant is marked as *. (C) Protein sequence alignment of TRPV4 orthologs showing conservation in the region including p.S94L in higher vertebrates. (D) Schematic diagram of the TRPV4 protein demonstrating localization of p.S94L on the N-terminal intracellular region. Numbers 1–6 correspond to the 6 ankyrin repeats.

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  Figure 2. Structure modeling of TRPV4 mutations Structural model based on the cryoEM structure of Xenopus tropicalis TRPV4 (PDB ID: 6bbj). The structure, corresponding to residues 148–788 (human numbering), does not include disordered N- and C-terminal regions. The N-terminal region is schematized as a dotted line for each subunit, with the phosphoinositide-binding domain (PBD; residues 121–125 in human) and proline-rich region (PRR, residues 135–144) indicated. Residue positions for neuropathy-causing mutations (D62N, P97R, R186Q, R232C/S, R237G/L, R269C/H, R315W, R316C/H, and T701I) and disease mutations with mixed phenotypes (G78W, A217S, E278K, S542Y, V620Y, and T740I) are green and yellow, respectively. Compound heterozygous neuromuscular disease mutations N833S and E840K are not illustrated because they are in the disordered cytoplasmic C-terminal region (residues 789–871). The position of p.S94L is indicated in purple.

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  Figure 3. Influence of TRPV4 mutations on protein expression level and cytotoxicity (A–B) Western blot (A) and quantification based on 3 experimental repeats (B) from HEK293 cells transiently expressing TRPV4-FLAG, p.S94L-FLAG, p.R269C-FLAG, p.R315W-FLAG, and p.T701I-FLAG or empty vector collected 48 hours after transfection and incubated in the absence or presence of HC-067047 (5 μM). Cell lysates were probed with antibodies against FLAG tag to detect transfected wild-type (WT) and mutant TRPV4 proteins and GAPDH as the loading control. (C) Cytotoxicity analysis in cells transfected with control or mutant TRPV4 plasmids. Differences in the protein levels or cytotoxicity were considered significant with *p < 0.05.

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  Figure 4. Subcellular localization of mutant TRPV4 proteins Representative images of HEK293 cells transiently expressing TRPV4-Flag, p.S94L-Flag, p.R269C-Flag, p.R315W-Flag, and p.T701I proteins. Immunofluorescence was performed with anti-Flag tag antibody to detect TRPV4-Flag proteins and Na-K-ATPase to label the plasma membrane. Nuclei were stained with DAPI. Scale bar = 20 μm.