Homozygous TRPV4 mutation causes congenital distal spinal muscular atrophy and arthrogryposis

Abstract

Objective: To identify the genetic cause of disease in a form of congenital spinal muscular atrophy and arthrogryposis (CSMAA).

Methods: A 2-year-old boy was diagnosed with arthrogryposis multiplex congenita, severe skeletal abnormalities, torticollis, vocal cord paralysis, and diminished lower limb movement. Whole-exome sequencing (WES) was performed on the proband and family members. In silico modeling of protein structure and heterologous protein expression and cytotoxicity assays were performed to validate pathogenicity of the identified variant.

Results: WES revealed a homozygous mutation in the TRPV4 gene (c.281C>T; p.S94L). The identification of a recessive mutation in TRPV4 extends the spectrum of mutations in recessive forms of the TRPV4-associated disease. p.S94L and other previously identified TRPV4 variants in different protein domains were compared in structural modeling and functional studies. In silico structural modeling suggests that the p.S94L mutation is in the disordered N-terminal region proximal to important regulatory binding sites for phosphoinositides and for PACSIN3, which could lead to alterations in trafficking and/or channel sensitivity. 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.

Conclusions: This study identifies a novel homozygous mutation in TRPV4 as a cause of the recessive form of CSMAA.

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.

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.

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.

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.