Abstract
The 1555A→G mutation in mitochondrial 12S rRNA has been associated with aminoglycoside-induced and non-syndromic deafness in many individuals worldwide. Mitochondrial genetic modifiers are proposed to influence the phenotypic expression of m.1555A→G mutation. Here, we report that a deafness-susceptibility allele (m.4317A→G) in the tRNAIle gene modulates the phenotype expression of m.1555A→G mutation. Strikingly, a large Han Chinese pedigree carrying both m.4317A→G and m.1555A→G mutations exhibited much higher penetrance of deafness than those carrying only the m.1555A→G mutation. The m.4317A→G mutation affected a highly conserved adenine at position 59 in the T-loop of tRNAIle We therefore hypothesized that the m.4317A→G mutation alters both structure and function of tRNAIle Using lymphoblastoid cell lines derived from members of Chinese families (three carrying both m.1555A→G and m.4317A→G mutations, three harboring only m.1555A→G mutation, and three controls lacking these mutations), we found that the cell lines bearing both m.4317A→G and m.1555A→G mutations exhibited more severe mitochondrial dysfunctions than those carrying only the m.1555A→G mutation. We also found that the m.4317A→G mutation perturbed the conformation, stability, and aminoacylation efficiency of tRNAIle These m.4317A→G mutation-induced alterations in tRNAIle structure and function aggravated the defective mitochondrial translation and respiratory phenotypes associated with the m.1555A→G mutation. Furthermore, mutant cell lines bearing both m.4317A→G and m.1555A→G mutations exhibited greater reductions in the mitochondrial ATP levels and membrane potentials and increasing production of reactive oxygen species than those carrying only the m.1555A→G mutation. Our findings provide new insights into the pathophysiology of maternally inherited deafness arising from the synergy between mitochondrial 12S rRNA and tRNA mutations.
Highlights
The 1555A3 G mutation in mitochondrial 12S rRNA has been associated with aminoglycoside-induced and non-syndromic deafness in many individuals worldwide
We showed that MTO1, MSS1/GTPBP3, or MTO2/TRMU genes involved in the biosynthesis of the hypermodified nucleoside 5-methylaminomethyl-2-thiouridine in the wobble position of several mitochondrial tRNAs were the potential nuclear modifier genes for the phenotypic expression of m.1555A3 G and m.1494C3 T mutations (28 –33)
Using genetic and molecular approaches, in combination with functional assays, we demonstrated that a deafness susceptibility allele (m.4317A3 G mutation) in the tRNAIle gene modulated the phenotypic expression of deafness-associated 12S rRNA 1555A3 G mutation
Summary
Identification of the tRNAIle 4317A3 G mutation in a large cohort of hearing-impaired subjects. In the sequence analysis of the entire mtDNA in the proband (WZD91-III-3), three symptomatic affected matrilineal relatives (III-1, III-9, and III-17) and two asymptomatic matrilineal relatives (IV-5 and IV-10) (Fig. 1B) exhibited the presence of both m.1555A3 G and m.4317A3 G mutations and a set of polymorphisms belonging to the Eastern Asian haplogroups B4 (Table S1) [45] These variants included 16 variants in the D-loop region, four known variants in the 12S rRNA gene, three variants in the 16S rRNA gene, the previously identified COII/tRNALys intergenic 9-bp deletion corresponding to mtDNA at positions 8281– 8289, 10 known and one novel silent variant in the protein encoding genes, as well as a missense variant m.8573G3 A (p.16G3 D) in the ATP6 gene [46]. Further analysis showed that both m.4317A3 G and m.1555A3 G mutations were present in homoplasmy in all matrilineal relatives of pedigree WZD91 but was absent in other members of these families (Fig. 1C)
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