Deficient activity of alanyl-tRNA synthetase underlies an autosomal recessive syndrome of progressive microcephaly, hypomyelination, and epileptic encephalopathy

Abstract

Aminoacyl-transfer RNA (tRNA) synthetases ligate amino acids to specific tRNAs and are essential for protein synthesis. Although alanyl-tRNA synthetase (AARS) is a synthetase implicated in a wide range of neurological disorders from Charcot-Marie-Tooth disease to infantile epileptic encephalopathy, there have been limited data on their pathogenesis. Here, we report loss-of-function mutations in AARS in two siblings with progressive microcephaly with hypomyelination, intractable epilepsy, and spasticity. Whole-exome sequencing identified that the affected individuals were compound heterozygous for mutations in AARS gene, c.2067dupC (p.Tyr690Leufs*3) and c.2738G>A (p.Gly913Asp). A lymphoblastoid cell line developed from one of the affected individuals showed a strong reduction in AARS abundance. The mutations decrease aminoacylation efficiency by 70%-90%. The p.Tyr690Leufs*3 mutation also abolished editing activity required for hydrolyzing misacylated tRNAs, thereby increasing errors during aminoacylation. Our study has extended potential mechanisms underlying AARS-related disorders to include destabilization of the protein, aminoacylation dysfunction, and defective editing activity.

Keywords: AARS; aminoacylation defect; hypomyelination; microcephaly; transfer RNA.

Figure 1. AARS mutations cause loss-of-fuction defects

A: Identification of AARS mutations. Pedigree structure of the family studied is shown. Shaded symbols indicate affected individuals. Sanger sequencing confirmed the segregation of the identified variants in the affected individuals (MC32501 and MC32502). B: Schematic representation of the AARS mutations. Variants acting in dominant and recessive manners are shown in black and red, respectively. The variants in this study are in bold. The residue Tyr690 is in the editing domain and the residue Gly913 is in the C-terminal domain. C: Immunoblot analysis of AARS abundance in lymphoblastoid cell lines. AARS level in cells available from an affected individual (MC32501) showed a marked reduction of AARS, whereas cells from her parents (MC32505 and MC32506) showed a mild reduction. A representative image by using AARS antibody (A303-473A-T, Bethyl), with an epitope near the N-terminus, is shown. Quantification of AARS abundance was performed Relative AARS/GAPDH intensity ratio was normalized to the control. Values represent the average of three independent experiments. Error bars represent standard deviation. ***p<0.001, **p<0.01, ns; not significant. D: Aminoacylation kinetics of AARS protein variants. Relative acitivity is caliculated by ratio to WT activity. E: Hydrolytic editing activity of AARS. Deacylation of the incorrectly charged Ser-tRNA^Ala was monitored with wild type AARS (WT; blue square), p.Tyr690Leufs*3 (p.T690fs*; purple triangle), and p.Gly913Asp variants (p.G913D; black triangle). p.Cys723Ala (p.C723A; green circle) is served as a positive control of editing defect. F: Misacylation of AARS variants. Accumulation of misacylated Ser-tRNA^Ala was measured on wild type AARS (WT; blue square) and p.Tyr690Leufs*3 (p.T690fs*; purple triangle). Error bar shows the standard deviation. Values represent the average of at least three independent experiments.