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. 2010 Oct 8;87(4):560-6.
doi: 10.1016/j.ajhg.2010.09.008.

Compound Heterozygosity for Loss-Of-Function lysyl-tRNA Synthetase Mutations in a Patient With Peripheral Neuropathy

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Compound Heterozygosity for Loss-Of-Function lysyl-tRNA Synthetase Mutations in a Patient With Peripheral Neuropathy

Heather M McLaughlin et al. Am J Hum Genet. .
Free PMC article

Abstract

Charcot-Marie-Tooth (CMT) disease comprises a genetically and clinically heterogeneous group of peripheral nerve disorders characterized by impaired distal motor and sensory function. Mutations in three genes encoding aminoacyl-tRNA synthetases (ARSs) have been implicated in CMT disease primarily associated with an axonal pathology. ARSs are ubiquitously expressed, essential enzymes responsible for charging tRNA molecules with their cognate amino acids. To further explore the role of ARSs in CMT disease, we performed a large-scale mutation screen of the 37 human ARS genes in a cohort of 355 patients with a phenotype consistent with CMT. Here we describe three variants (p.Leu133His, p.Tyr173SerfsX7, and p.Ile302Met) in the lysyl-tRNA synthetase (KARS) gene in two patients from this cohort. Functional analyses revealed that two of these mutations (p.Leu133His and p.Tyr173SerfsX7) severely affect enzyme activity. Interestingly, both functional variants were found in a single patient with CMT disease and additional neurological and non-neurological sequelae. Based on these data, KARS becomes the fourth ARS gene associated with CMT disease, indicating that this family of enzymes is specifically critical for axon function.

Figures

Figure 1
Figure 1
Characterization, Conservation, and Localization of KARS Variants (A) Representative sections of sequence chromatograms are shown for the regions encompassing each identified KARS variant in the indicated individuals. Arrows denote the variant (present in the heterozygous state), with the predicted amino acid changes depicted above. (B) Chromatograms from allele-specific sequencing of an ∼3.7 kb PCR-generated genomic segment spanning the two KARS variants (p.Leu133His and p.Tyr173SerfsX7) identified in patient BAB564. Arrows indicate each mutation. Note that each variant was identified on separate alleles, indicating that this patient is a compound heterozygote. (C) For each of the four detected variants, the affected amino acid is shown along with the flanking KARS protein sequence in multiple, evolutionarily diverse species. Note that each specific amino acid change is given at the top, with the relevant position depicted in red for each protein sequence. Dashes indicate gaps in the sequence alignment. (D) The known functional domains of the KARS protein are indicated in yellow (tRNALys-binding domain) and blue (core catalytic domain).
Figure 2
Figure 2
Functional Consequences of KARS Variants (A–C) An illustration of the KARS protein crystal structure is shown for the monomer (A), dimer (B), and tetramer (C). The anti-codon binding and catalytic domains are indicated in (A). The position of residues L133 and I302 are indicated in red and black, respectively. Note that L133 resides at the tetramer interface in (C). (D) Initial aminoacylation rates, V0 (pmol/s), of wild-type KARS (blue squares) and p.Leu133His KARS (red circles) were plotted against tRNA concentration and fit to the Michaelis-Menten equation. Error bars indicate standard deviation. (E) Representative cultures of the indicated yeast strains were inoculated and grown on solid growth medium containing 5-FOA. Each strain was previously transfected with a vector containing no insert (pRS315), wild-type KRS1 (WT KRS1), or the indicated mutant form of KRS1 that modeled a human KARS mutation (see Table 4). Before inoculating on 5-FOA-containing medium, each strain was diluted 1:10 or 1:50 in water.

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