Loss of function mutations in HARS cause a spectrum of inherited peripheral neuropathies

Abstract

Inherited peripheral neuropathies are a genetically heterogeneous group of disorders characterized by distal muscle weakness and sensory loss. Mutations in genes encoding aminoacyl-tRNA synthetases have been implicated in peripheral neuropathies, suggesting that these tRNA charging enzymes are uniquely important for the peripheral nerve. Recently, a mutation in histidyl-tRNA synthetase (HARS) was identified in a single patient with a late-onset, sensory-predominant peripheral neuropathy; however, the genetic evidence was lacking, making the significance of the finding unclear. Here, we present clinical, genetic, and functional data that implicate HARS mutations in inherited peripheral neuropathies. The associated phenotypic spectrum is broad and encompasses axonal and demyelinating motor and sensory neuropathies, including four young patients presenting with pure motor axonal neuropathy. Genome-wide linkage studies in combination with whole-exome and conventional sequencing revealed four distinct and previously unreported heterozygous HARS mutations segregating with autosomal dominant peripheral neuropathy in four unrelated families (p.Thr132Ile, p.Pro134His, p.Asp175Glu and p.Asp364Tyr). All mutations cause a loss of function in yeast complementation assays, and p.Asp364Tyr is dominantly neurotoxic in a Caenorhabditis elegans model. This study demonstrates the role of HARS mutations in peripheral neuropathy and expands the genetic and clinical spectrum of aminoacyl-tRNA synthetase-related human disease.

Keywords: RNA processing; hereditary motor and sensory neuropathies; molecular genetics; neurodegeneration; whole-exome sequencing.

Using linkage analysis and whole-exome sequencing, Safka Brozkova et al. reveal missense mutations in the histidyl-tRNA synthetase gene in 23 patients from four families with axonal and demyelinating neuropathies of varying severity. The mutations cause loss of function in yeast complementation assays and neurotoxicity in a C. elegans model.

Figure 1

Pedigrees of the families with HARS mutations. Female family members are indicated with a circle and male family members are indicated by squares. Filled symbols indicate affected individuals, while empty symbols indicate unaffected individuals. The number of the individual is shown in Arabic numerals if the DNA was available for genotyping.

Figure 2

CMT-associated HTS1 variants decrease yeast cell viability. Haploid Δhts1 yeast strains were transformed with a vector containing no insert (pRS315 Empty) or an insert to express wild-type, p.Thr132Ile, p.Thr132Ser, p.Pro134His, p.Asp175Glu or p.Asp364Tyr HTS1 (Supplementary Table 4). Two colonies (indicated by ‘A’ and ‘B’) from transformations with Thr132Ile, Thr132Ser, Pro134His, Asp175Glu or Asp364Tyr HTS1 are shown. Resulting colonies (undiluted, diluted 1:10, or diluted 1:100) were grown on agar plates containing 0.1% 5-FOA. Note the severe depletion of growth associated with p.Asp175Glu HTS1 at 1:10 and 1:100 dilutions.

Figure 3

p.Asp364Tyr (D364Y) motor neuron expression causes axonal pathology and neuromuscular defects in C. elegans. (A) Diagram of the 19 DD and VD GABA motor neuron cell bodies (green dots), which are located in the ventral nerve cord (VNC), and their commissural axonal processes. Ventral motor neuron cell bodies extend dorsal circumferential axons forming the dorsal nerve cord (DNC). Figure adapted with permission from Vester et al. (2013). (B) Whole animal (upper panels) and magnified (lower panels) confocal images of control oxIs12 [Punc-47::GFP] and oxIs12; hars-1 (D364Y) expressing animals. In control animals, commissural axons (white arrowhead) extend dorsally from ventral motor neuron cell bodies forming a continuous dorsal nerve cord. Note the homogeneous GFP expression and lack of axonal blebbing in control animals (lower panel, white arrowhead). Expression of hars-1(D364Y) causes aberrant axonal commissures that fail to reach the dorsal nerve cord (yellow arrowhead), prominent dorsal nerve cord gaps (yellow arrow), and axonal blebbing (yellow asterisks) not observed in controls. Scale bar = 50 µm. (C) Quantification of aberrant GABA motor neuron axonal commissures in HARS-1 (D364Y) expressing animals (n ≥ 100 worms/genotype; n = 5 trials/genotype). (D) Quantification of thrash assays in liquid media (n ≥ 40 animals/genotype). Error bars ± SEM, *P < 0.05, Students t-test.