Mutation of the mitochondrial tyrosyl-tRNA synthetase gene, YARS2, causes myopathy, lactic acidosis, and sideroblastic anemia--MLASA syndrome

Abstract

Mitochondrial respiratory chain disorders are a heterogeneous group of disorders in which the underlying genetic defect is often unknown. We have identified a pathogenic mutation (c.156C>G [p.F52L]) in YARS2, located at chromosome 12p11.21, by using genome-wide SNP-based homozygosity analysis of a family with affected members displaying myopathy, lactic acidosis, and sideroblastic anemia (MLASA). We subsequently identified the same mutation in another unrelated MLASA patient. The YARS2 gene product, mitochondrial tyrosyl-tRNA synthetase (YARS2), was present at lower levels in skeletal muscle whereas fibroblasts were relatively normal. Complex I, III, and IV were dysfunctional as indicated by enzyme analysis, immunoblotting, and immunohistochemistry. A mitochondrial protein-synthesis assay showed reduced levels of respiratory chain subunits in myotubes generated from patient cell lines. A tRNA aminoacylation assay revealed that mutant YARS2 was still active; however, enzyme kinetics were abnormal compared to the wild-type protein. We propose that the reduced aminoacylation activity of mutant YARS2 enzyme leads to decreased mitochondrial protein synthesis, resulting in mitochondrial respiratory chain dysfunction. MLASA has previously been associated with PUS1 mutations; hence, the YARS2 mutation reported here is an alternative cause of MLASA.

Figure 1

Identification of a Mutation in YARS2 Associated with MLASA (A) Pedigree of the family used for homozygosity mapping. (B) DNA sequence identifying the c.156C>G (p.F52L) YARS2 mutation. (C) Schematic diagram of YARS2 domains showing location of the variant F52 residue and its conservation among species. The following abbreviations are used: MTS, mitochondrial targeting sequence; catalytic, catalytic domain; ACB, anticodon binding domain; and S4-like, S4 ribosomal protein-like domain.

Figure 2

Effect of the YARS2 Mutation on Skeletal Muscle RC Protein Expression Immunoblot analysis of YARS2 and the RC complexes in (A) patient II:3 muscle (P2) and three controls (C1, C2, C3). A 1 min exposure of specific subunits of the five RC complexes (I–V) detected by the anti-OXPHOS cocktail are shown on one blot. An additional shorter exposure (5 s) of complex V subunit α is shown for clarity. Note that these subunits are labile if the RC complex is not fully assembled. GAPDH and mitofilin were used as loading controls; however, both were affected. Five micrograms of total protein was loaded, and equivalent loadings were confirmed by Coomassie staining. (B) Immunolabeling of patient II:3 and control muscle cryosections with CO1 (complex IV, subunit 1; red) and nuclear staining with TOPRO-3 (blue). The scale bar represents 76 μm.

Figure 3

The Mutation in YARS2 Results in Reduced Mitochondrial Protein Synthesis in Patient Myotubes (A) SDS-PAGE phosphorimaging analysis of [³⁵S]-methionine-labeled mitochondrial proteins from patient (P1 = family 2, P2 = II:3) and control (C1, C2) myotubes. Mitochondrially encoded subunits of complex I (ND1, ND2, ND3), complex III (CYB), complex IV (CO1, CO2, CO3; note that CO2 and CO3 comigrate), and complex V (ATP6, ATP8) are shown. An additional band (^∗) seen in patient samples may represent a degradation or incomplete translation product. (B) Immunoblot of selected mitochondrial- (complex I; ND1, complex IV; CO1, CO2) and nuclear-encoded (complex II; 70 kDa) RC subunits in patient myotubes.

Figure 4

Interaction Model of Human YARS2 with tRNA The model shows the crystal structure of the enzyme (without the S4-like C-terminal domain) on which one tRNA^Tyr molecule has been docked according to its location in the crystal structure of a bacterial tryosyl-tRNA synthetase and tRNA^Tyr complex. The two monomers are distinguished by dark- and light-green color with the first β strand encompassing residue 52 (p.F52L), shown as the mutant L52, highlighted in purple. Note the proximity of L52 with the accepting end of tRNA shown as orange.