A recurrent loss-of-function alanyl-tRNA synthetase (AARS) mutation in patients with Charcot-Marie-Tooth disease type 2N (CMT2N)

Abstract

Charcot-Marie-Tooth (CMT) disease comprises a heterogeneous group of peripheral neuropathies characterized by muscle weakness and wasting, and impaired sensation in the extremities. Four genes encoding an aminoacyl-tRNA synthetase (ARS) have been implicated in CMT disease. ARSs are ubiquitously expressed, essential enzymes that ligate amino acids to cognate tRNA molecules. Recently, a p.Arg329His variant in the alanyl-tRNA synthetase (AARS) gene was found to segregate with dominant axonal CMT type 2N (CMT2N) in two French families; however, the functional consequence of this mutation has not been determined. To investigate the role of AARS in CMT, we performed a mutation screen of the AARS gene in patients with peripheral neuropathy. Our results showed that p.Arg329His AARS also segregated with CMT disease in a large Australian family. Aminoacylation and yeast viability assays showed that p.Arg329His AARS severely reduces enzyme activity. Genotyping analysis indicated that this mutation arose on three distinct haplotypes, and the results of bisulfite sequencing suggested that methylation-mediated deamination of a CpG dinucleotide gives rise to the recurrent p.Arg329His AARS mutation. Together, our data suggest that impaired tRNA charging plays a role in the molecular pathology of CMT2N, and that patients with CMT should be directly tested for the p.Arg329His AARS mutation.

Figure 1

Segregation, localization, and conservation of AARS variants. A: Genotyping was performed to determine the segregation of AARS variants with disease in families with CMT. Filled symbols represent affected individuals, with black indicating a diagnosis of CMT and red indicating a diagnosis of rippling muscles and cramps. Empty symbols indicate unaffected individuals. Where applicable, the individual’s genotype is indicated with the amino-acid change or + (for a wild-type allele). Slashes indicate deceased individuals, the question mark indicates an unknown diagnosis, and the arrow indicates the proband in family CMT513. All individuals have been designated with a diamond symbol to protect identities. B: Each variant was mapped to the known functional domains of the AARS protein indicated in red (catalytic domain), green (tRNA^Ala binding domain), and orange (editing domain). The position of each domain along the protein is indicated below the cartoon. C: Multiple-species protein alignments were generated to assess the conservation of each affected amino-acid position. For each of the three detected variants, the affected amino acid is shown along with the flanking AARS protein sequence in multiple, evolutionarily diverse species (indicated on the left). Note that each specific amino-acid change is given at the top, with conservation indicated in red for each protein sequence.

Figure 2

Functional characterization of AARS variants. A: Aminoacylation of tRNA^Ala with alanine by AARS enzymes. Analysis of the rate of aminoacylation (pmole/min/pmole of enzyme) as a function of tRNA concentration for the wild-type AARS enzyme (red) and the mutants N71Y (blue), and R329H (green), and E778A (black) after fitting the data to the Michaelis-Menten equation. Values represent the average of two independent experiments, and error bars indicate the standard deviation. B: Five representative cultures of each yeast strain (indicated along the top of the panel) were inoculated and grown on solid growth medium containing 5-FOA. Each strain was previously transfected with a vector containing no insert (pRS315 Empty), wild-type ALA1 (Wt ALA1), or the indicated mutant form of ALA1 that modeled a human AARS mutation (see Table 3). Two independently generated mutant-bearing constructs were analyzed (indicated as ‘A’ and ‘B’ on the left side of the panel). Before inoculating on 5-FOA-containing medium, each strain was resuspended in 100μL water, then diluted 1:10.

Figure 3

R329H AARS is a recurrent mutation. A: Haplotype analysis was performed along chromosome 16 in family CMT243 and compared to the published data for the previously identified families with the R329H AARS mutation (Family 1 and Family 2). The three genotyped markers and AARS locus are indicated on the left, along with the alleles identified on each disease-associated haplotype (in base pairs). The distance between each locus is indicated in megabases (Mb). B: The codon affected by the R329H AARS variant harbors a CpG dinucleotide. A representative trace (in the reverse-complement) of the region surrounding the R329H codon is shown. The CpG is indicated with a line and the affected cytosine is indicated with an arrow. C: The AARS locus was computationally evaluated for CpG dinucleotide content (see methods). Each AARS exon number is indicated along the top of the panel, with vertical white boxes representing coding exons and horizontal lines representing introns. Areas with at least 100 base pairs containing an observed/expected CpG dinucleotide ratio > 60% and GC content >50% are indicated in yellow. Note that AARS exon 8 is the only coding region to meet these criteria. D: The methylation status of CpG dinucleotides in AARS exon 8 was evaluated via bisulfite (NaHSO₃) treated DNA sequencing analysis. The DNA sequence from ten representative clones harboring AARS exon 8 after bisulfite treatment are shown, with the genomic consensus sequence (‘Genomic’) provide along the top. Converted cytosines are indicated with black arrows and non-converted cytosines are indicated with red arrows. Note that all non-CpG cytosines are converted to thymines, while all CpG cytosines remain unchanged indicating that they are methylated. E: AARS exon 8 harbors multiple, methylated cytosines in CpG dinucleotides. A representation of bisulfite sequencing products of the seven CpGs (indicated along the top of the panel) residing in AARS exon 8 is shown for two control individuals (Control 1 and Control 2). Eighteen clones were analyzed for each control. Filled circles indicate methylated CpGs. The arrow indicates the affected CpG giving rise to R329H AARS.