Background: Sequence alternations in mitochondrial genomes, especially in genes encoding mitochondrial tRNA (mt-tRNA), were the important contributors to nonsyndromic hearing loss (NSHL); however, the molecular mechanisms remained largely undetermined. Methods:A maternally transmitted Chinese pedigree with NSHL underwent clinical, genetic, and biochemical assessment. PCR and direct sequence analyses were performed to detect mitochondrial DNA (mtDNA), GJB2, and SLC26A4 gene mutations from matrilineal relatives of this family. Mitochondrial functions including mitochondrial membrane potential (MMP), ATP, and ROS were evaluated in polymononuclear leukocytes (PMNs) derived from three deaf patients and three controls from this pedigree.Results: Four of nine matrilineal relatives developed hearing loss at the variable age of onset. Two putative pathogenic mutations, m.5601C>T in tRNA Ala and m.12311T>C in tRNA Leu(CUN) , were identified via PCR-Sanger sequencing, as well as 34 variants that belonged to mtDNA haplogroup G2b2. Intriguingly, m.5601C>T mutation resided at very conserved nucleotide in the TψC loop of tRNA Ala (position 59), while the T-to-C substitution at position 12311 located at position 48 in the variable stem of tRNA Leu(CUN) and was believed to alter the aminoacylation and the steady-state level of tRNA. Biochemical analysis revealed the impairment of mitochondrial functions including the significant reductions of ATP and MMP, whereas markedly increased ROS levels were found in PMNs derived from NSHL patients with m.5601C>T and m.12311T>C mutations. However, we did not detect any mutations in GJB2 and SLC26A4 genes. Conclusion:Our data indicated that mt-tRNA Ala m.5601C>T and tRNA Leu(CUN) 12311T>C mutations were associated with NSHL.

According a recent report by Heidari et al., a mutational screening for candidate pathogenic mitochondrial tRNA (mt-tRNA) mutations were performed in 45 Iranian patients with non-dystrophic myotonia (NDM) and 70 control subjects. Through PCR amplification and direct sequence analysis, nine mt-tRNA mutations were identified: tRNA Met T4454C, tRNA Trp A5568G, tRNA Cys T5794C, tRNA Arg A10438T and T10462C, tRNA Leu(CUN) A12308G, tRNA Thr A15907G, A15924G and G15928A. However, through the database searches and phylogenetic conservation analysis, we noticed that the tRNA Thr A15924G, G15928A and tRNA Leu(CUN) A12308G mutations should be classified 'pathogenic'. Thus, the roles of mt-tRNA mutations in clinical expression of NDM needed to be further experimentally addressed.