A recurrent WARS mutation is a novel cause of autosomal dominant distal hereditary motor neuropathy

Abstract

Distal hereditary motor neuropathy is a heterogeneous group of inherited neuropathies characterized by distal limb muscle weakness and atrophy. Although at least 15 genes have been implicated in distal hereditary motor neuropathy, the genetic causes remain elusive in many families. To identify an additional causal gene for distal hereditary motor neuropathy, we performed exome sequencing for two affected individuals and two unaffected members in a Taiwanese family with an autosomal dominant distal hereditary motor neuropathy in which mutations in common distal hereditary motor neuropathy-implicated genes had been excluded. The exome sequencing revealed a heterozygous mutation, c.770A > G (p.His257Arg), in the cytoplasmic tryptophanyl-tRNA synthetase (TrpRS) gene (WARS) that co-segregates with the neuropathy in the family. Further analyses of WARS in an additional 79 Taiwanese pedigrees with inherited neuropathies and 163 index cases from Australian, European, and Korean distal hereditary motor neuropathy families identified the same mutation in another Taiwanese distal hereditary motor neuropathy pedigree with different ancestries and one additional Belgian distal hereditary motor neuropathy family of Caucasian origin. Cell transfection studies demonstrated a dominant-negative effect of the p.His257Arg mutation on aminoacylation activity of TrpRS, which subsequently compromised protein synthesis and reduced cell viability. His257Arg TrpRS also inhibited neurite outgrowth and led to neurite degeneration in the neuronal cell lines and rat motor neurons. Further in vitro analyses showed that the WARS mutation could potentiate the angiostatic activities of TrpRS by enhancing its interaction with vascular endothelial-cadherin. Taken together, these findings establish WARS as a gene whose mutations may cause distal hereditary motor neuropathy and alter canonical and non-canonical functions of TrpRS.

Keywords: WARS; dHMN; distal hereditary motor neuropathy; exome sequencing; tryptophanyl-tRNA synthetase.

Figure 1

Genetic analysis of families with distal hereditary motor neuropathy (dHMN). (A) Pedigrees of the two Taiwanese and one Belgian dHMN families harbouring the WARS c.770A > G (p.His257Arg) mutation. Haplotype analysis of seven SNP markers flanking the WARS variant revealed two distinct disease-associated haplotypes in the two Taiwanese families. Open symbol: unaffected; filled symbol: affected; symbol with a diagonal line: deceased; arrow: proband. (B) Sanger sequencing traces confirming the c.770A > G (p.His257Arg) variant. (C) Alignment of multiple TrpRS orthologues showing conservation of the His257 residue throughout vertebrate species, but not yeast. The domain diagram of human TrpRS shows that the His257 residue is localized in the connective polypeptide 1 insertion (CP1). WHEP: a helix-turn-helix motif, responsible for protein–protein interaction. (D) The structural model of human TrpRS homodimers (PDB ID: 2AKE) showing that the His257 residue is situated at the dimer interface (left) as well as in the catalytic domain (green) which is close to the recognition sites (Thr259 and Asn261) for the tRNA acceptor arm (grey) (middle and right).

Figure 2

Clinical and muscle MRI features in patients with WARS p.His257Arg mutation. (A) Atrophy of the intrinsic hand muscles and muscles in the feet and legs seen in Patient A-III-3. (B) Atrophy of the intrinsic hand muscles seen in Patient C-II-1. (C) T₁-weighted MRI of Patient A-III-3 revealing distal lower limb muscle atrophy with fatty infiltration (arrowheads). (D) T₁-weighted lower limb MRI of a 30-year-old healthy male for comparison. Arrow: corresponding cross-section level of the axial view images.

Figure 3

In vitro characterization of wild-type and His257Arg (H257R) mutant TrpRS proteins. (A) β-Gal reporter assay and (B) firefly luciferase reporter assay demonstrating that H257R TrpRS has a dominant-negative effect on protein synthesis. HEK293 cells co-transfected with β-Gal or firefly luciferase reporter plasmids, along with different ratios of wild-type and H257R TrpRS expression plasmids were lysed and assayed for β-Gal or firefly luciferase activities at 48 h after transfection. The error bars indicate standard errors of the mean (n = 3) and asterisks indicate statistically significant differences (**P < 0.01, *P < 0.05). Vector: empty vector control. (C) Aminoacylation of tRNA^trp mediated by TrpRS generating adenosine monophosphate (AMP) and pyrophosphate (PPi), which can be used to assess aminoacylation activities. (D) Analyses of aminoacylation activities of TrpRS in HEK293 cells. Cells with different ratios of wild-type and mutant TrpRS constructs as well as the siRNAs specifically depleting endogenous WARS were lysed at 48 h post-transfection, and assayed for aminoacylation activities by assessing the amount of generated pyrophosphate. The data were displayed relative to the values detected in the control vector-transfected cells (n = 4). (E) Analyses of aminoacylation activities of purified TrpRSs. His-tagged wild-type and H257R TrpRS proteins were produced in E. coli and purified by Ni-NTA affinity chromatography. Both pyrophosphate and AMP generated through aminoacylation were quantified to estimate the aminoacylation activities (n = 3). (F) Investigating the dimerization properties of wild-type and H257R TrpRSs by co-immunoprecipitation (Co-IP) assay. HEK293 cells co-transfected with Myc- or/and GFP-tagged wild-type or/and H257R TrpRS expression plasmids were lysed at 48 h post-transfection. Dimerization was demonstrated via co-immunoprecipitation pull-downs of GFP-tagged wild-type or H257R TrpRS with the Myc-tagged TrpRS using anti-Myc antibody. These data showed that the H257R TrpRS could form dimers with wild-type TrpRS in cells. (G) Cell viability assays. Knockdown of endogenous WARS in HEK293 cells resulted in a significantly compromised cell viability, which could be rescued by expressing exogenous wild-type TrpRS but worsened by H257R TrpRS. Percentage of viable cells was displayed relative to cells transfected with empty vector along with non-targeting siRNA control (Ctrl siRNA) (n = 7). WT = wild-type.

Figure 4

His257Arg (H257R) TrpRS inhibits neurite outgrowth and leads to neurite degeneration. Neuro-2a (N2a) (A) or SH-SY5Y (B) cells were transfected with expression vector containing wild-type (WT) or H257R TrpRS or empty vector (vector control), grown under differentiation conditions, and immunostained against Myc (exogenous TrpRS staining) and neurofilament heavy polypeptide (NFH; neurite staining) at 72 h post-transfection. The representative fluorescence-immunostaining and phase-contrast images were shown, along with statistical results of per cent of cells bearing neurites and average primary neurite length. Scale bar = 50 μm. At least 100 cells from three independent experiments were measured for each preparation and data were expressed as the mean ± standard error of the mean (SEM). *P-value < 0.05; **P-value < 0.01 when compared to wild-type. (C) Rat motor neurons (embryonic Day 14) were transfected with expression vector containing wild-type or H257R TrpRS or empty vector, and processed for immunostaining using antibodies against Myc and SMI-32 (staining for motor neurons and their neurites) at 72 h post transfection. The representative fluorescence-immunostaining and phase-contrast images were shown. Neurites of motor neurons were traced and measured, and the average primary neurite length and per cent of cells bearing beaded neurites were estimated. Error bars represent the mean ± SEM of three independent experiments with at least 50 cells. (D) Rat motor neurons expressing above TrpRS constructs were processed for immunofluorescence analysis using antibody against amyloid precursor protein (APP; neurodegeneration staining) at 72 h post-transfection. Motor neurons expressing wild-type TrpRS showed only a faint or punctate staining of APP in the neurites, while motor neurons expressing TrpRS appeared to accumulate APP at neurites (arrow).

Figure 5

Pull-down assay and in vitro angiogenesis assays showing that human His257Arg (H257R) mutant TrpRS has an augmented angiostatic effect through binding to VE-cadherin. (A) An in vitro pull-down assay used to assess the ability of His-tagged TrpRS to bind to Flag-tagged VE-cadherin. The immunoblots showed that both the full-length (FL) and the T2-H257R TrpRSs had a substantial increase in affinity to VE-cadherin compared to wild-type (WT) TrpRS. (B) Effect of the p.His257Arg mutation on the angiostatic activity of TrpRS evaluated by transwell migration assay and Matrigel angiogenesis assay using HUVECs under 16 h of VEGF₁₆₅ stimulation with or without TrpRS treatment. Top: Representative microscopic images of the underside of the transwell assay membranes; Bottom: representative microscopic images of tube formation on Matrigel^®. (C) Quantification of migration of HUVECs after 16 h treatment with VEGF₁₆₅ and the indicated conditions of three independent experiments. Treatment with full-length H257R TrpRS, T2-H257R TrpRS, or T2-wild-type TrpRS significantly reduced in vitro cell mobility, while full-length wild-type TrpRS application did not further inhibit the cell migration compared to a control treatment of VEGF₁₆₅ alone. The error bars represent standard errors of the mean (n = 3) and asterisks indicate statistically significant differences (**P < 0.01, *P < 0.05, NS = non-significant). (D) Quantification of vessel coverage (the total microscopic area covered by HUVEC vasculature) after 16 h treatment with VEGF₁₆₅ and the indicated conditions of three independent experiments. The data revealed that full-length H257R TrpRS, T2-wild-type or T2-H257R TrpRS could significantly inhibit angiogenesis. Treatment of full-length wild-type TrpRS did not show inhibitory effect on tube formation.