Functional analysis of the novel mitochondrial tRNA and tRNA variants associated with type 2 diabetes mellitus.

Background: Mutations in mitochondrial tRNA () genes that result in mitochondrial dysfunction play important roles in type 2 diabetes mellitus (T2DM). We pre-viously reported a large Chinese pedigree with maternally inherited T2DM that harbors novel and variants, however, the effects of these variants on T2DM progression are largely unknown.

Aim: To assess the potential pathogenicity of T2DM-associated and variants at genetic, molecular, and biochemical levels.

Methods: Cytoplasmic hybrid (cybrid) cells carrying both and variants, and healthy control cells without these mitochondrial DNA (mtDNA) variants were generated using trans-mitochondrial technology. Mitochondrial features, including steady-state level, levels of adenosine triphosphate (ATP), mitochondrial membrane potential (MMP), reactive oxygen species (ROS), mtDNA copy number, nicotinamide adenine dinucleotide (NAD)/NADH ratio, enzymatic activities of respiratory chain complexes (RCCs), 8-hydroxy-deo-xyguanine (8-OhdG), malondialdehyde (MDA), and superoxide dismutase (SOD) were examined in cell lines with and without these variants.

Results: Compared with control cells, the variant caused an approximately 35% reduction in the steady-state level of ( < 0.0001); however, the variant did not affect the steady-state level ( = 0.5849). Biochemical analysis revealed that cells with both and variants exhibited more severe mitochondrial dysfunctions and elevated oxidative stress than control cells: ATP, MMP, NAD/NADH ratio, enzyme activities of RCCs and SOD levels were markedly decreased in mutant cells ( < 0.05 for all measures). By contrast, the levels of ROS, 8-OhdG and MDA were significantly increased ( < 0.05 for all measures), but mtDNA copy number was not affected by and variants ( = 0.5942).

Conclusion: The variant impaired metabolism, which subsequently caused mitochondrial dysfunction. The variant did not alter the steady-state level of , indicating that it may be a modifier of the variant. The variant may exacerbate the pathogenesis and progression of T2DM in this Chinese pedigree.