A major determinant for binding and aminoacylation of tRNA(Ala) in cytoplasmic Alanyl-tRNA synthetase is mutated in dominant axonal Charcot-Marie-Tooth disease

Abstract

Charcot-Marie-Tooth disease (CMT) is the most common cause of inherited peripheral neuropathy, with an estimated frequency of 1/2500. We studied a large family with 17 patients affected by the axonal form of CMT (CMT2). Analysis of the 15 genes or loci known to date was negative. Genome-wide genotyping identified a CMT2 locus in 16q21-q23 between D16S3050 and D16S3106. The maximum two-point LOD score was 4.77 at theta = 0 for marker D16S3050. Sequencing of candidate genes identified a unique mutation, c.986G>A (p.Arg329His), affecting a totally conserved amino acid in the helical domain of cytoplasmic alanyl-tRNA synthetase (AlaRS). A second family with the same mutation and a different founder was then identified in a cohort of 91 CMT2 families. Although mislocation of mutant Arg329His-AlaRS in axons remains to be evaluated, experimental data point mostly to a quantitative reduction in tRNA(Ala) aminoacylation. Aminoacylation and editing functions closely cooperate in AlaRS, and Arg329His mutation could also lead to qualitative errors participating in neurodegeneration. Our report documents in 18 patients the deleterious impact of a mutation in human cytoplasmic AlaRS and broadens the spectrum of defects found in tRNA synthetases. Patients present with sensory-motor distal degeneration secondary to predominant axonal neuropathy, slight demyelination, and no atypical or additional CNS features.

Figure 1

Patient IV.5 of Family 1

Figure 2

Pedigrees and Haplotypes Filled symbols indicate affected individuals. Empty symbols indicate unaffected individuals. Identical alleles on the disease-causing haplotypes are boxed for each family. Closest recombinants in family 1 are shown in red (centromeric side) or blue (telomeric side).

Figure 3

Modular Organization of H. sapiens and E. coli AlaRS Abbreviations are as follows: AD, aminoacylation domain; HD, helical domain; ED, editing domain. In E. coli, the C-Ala domain brings together AD and ED in docking models, and an N-terminal fragment of 461 amino acids (AD+HD) is sufficient for complete and specific aminoacylation of tRNA^Ala.

Figure 4

Alignment of AlaRS Helical Domains from E. coli to H. sapiens GenBank access for proteins were as follows: NP_001596 (Homo sapiens), ACT28094 (Escherichia coli), NP_001093987 (Rattus norvegicus), NP_001005836 (Gallus gallus), NP_001037775 (Danio rerio), and AAF05593 (Drosophila melanogaster). Fully conserved amino acids are indicated with an asterisk (^∗) below the alignment. Arrowheads (▾) on top of the alignment indicate amino acids that have been studied by mutagenesis.E. coli D285 and R314 (H. sapiens D300, and R329) are indicated in yellow. E. coli R314 interacts with E362, F366, and G402, shown in blue (H. sapiens E378, F382, and G419).