Abstract
Nuclear modifier genes have been proposed to modify the phenotypic expression of mitochondrial DNA mutations. Using a targeted exome-sequencing approach, here we found that the p.191Gly>Val mutation in mitochondrial tyrosyl-tRNA synthetase 2 (YARS2) interacts with the tRNASer(UCN) 7511A>G mutation in causing deafness. Strikingly, members of a Chinese family bearing both the YARS2 p.191Gly>Val and m.7511A>G mutations displayed much higher penetrance of deafness than those pedigrees carrying only the m.7511A>G mutation. The m.7511A>G mutation changed the A4:U69 base-pairing to G4:U69 pairing at the aminoacyl acceptor stem of tRNASer(UCN) and perturbed tRNASer(UCN) structure and function, including an increased melting temperature, altered conformation, instability, and aberrant aminoacylation of mutant tRNA. Using lymphoblastoid cell lines derived from symptomatic and asymptomatic members of these Chinese families and control subjects, we show that cell lines harboring only the m.7511A>G or p.191Gly>Val mutation revealed relatively mild defects in tRNASer(UCN) or tRNATyr metabolism, respectively. However, cell lines harboring both m.7511A>G and p.191Gly>Val mutations displayed more severe defective aminoacylations and lower tRNASer(UCN) and tRNATyr levels, aberrant aminoacylation, and lower levels of other tRNAs, including tRNAThr, tRNALys, tRNALeu(UUR), and tRNASer(AGY), than those in the cell lines carrying only the m.7511A>G or p.191Gly>Val mutation. Furthermore, mutant cell lines harboring both m.7511A>G and p.191Gly>Val mutations exhibited greater decreases in the levels of mitochondrial translation, respiration, and mitochondrial ATP and membrane potentials, along with increased production of reactive oxygen species. Our findings provide molecular-level insights into the pathophysiology of maternally transmitted deafness arising from the synergy between tRNASer(UCN) and mitochondrial YARS mutations.
Highlights
Nuclear modifier genes have been proposed to modify the phenotypic expression of mitochondrial DNA mutations
Using a targeted exome-sequencing approach, here we found that the p.191Gly>Val mutation in mitochondrial tyrosyl-tRNA synthetase 2 (YARS2) interacts with the tRNASer(UCN) 7511A>G mutation in causing deafness
9 of 10 matrilineal relatives in a three-generation Chinese pedigree carrying the m.7511AϾG mutation exhibited hearing impairment, in contrast with only a small portion of hearing-impaired matrilineal relatives in two French pedigrees and one Japanese family carrying the same mtDNA mutation [27,28,29,30]. These findings suggest that the nuclear modifier genes, especially those involved in mitochondrial tRNA metabolism, contributed to the phenotypic expression of m.7511AϾG mutation
Summary
To experimentally test the effect of m.7511AϾG mutation on the stability of tRNASer(UCN), we examined the melting temperatures (Tm) of WT (A4) and mutant (G4) tRNASer(UCN) transcripts. These Tm values were determined by calculating the derivatives of the absorbance against a temperature curve. There was no difference of migration pattern between WT (A4) and mutant (G4) tRNASer(UCN) transcripts under denaturing conditions These data indicated that the m.7511AϾG mutation resulted in the conformational change of tRNASer(UCN). There was no evidence that any of the other members of this family had any other causes to account for hearing loss These matrilineal relatives showed no other clinical abnormalities, including cardiac failure, muscular diseases, visual failure, and neurological disorders. Further analysis showed that the m.7511AϾG mutation was present in homoplasmy in all matrilineal relatives but not in other members of this family (Fig. S1B)
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