Functional and molecular mitochondrial abnormalities associated with a C->T transition at position 3256 of the human mitochondrial genome - The effects of a pathogenic mitochondrial tRNA point mutation in organelle translation and RNA processing

We have previously identified a mitochondrial DNA polymorphism (a C --> T transition at position 3256, within the mitochondrial tRNA(Leu(UUR)) gene) in a patient with a multisystem disorder, Although there were several indicators suggesting a pathogenetic role for this mtDNA polymorphism, its heteroplasmic nature made functional and molecular studies difficult to interpret, We have now fused enucleated fibroblasts from the patient with a mtDNA-less cell line to generate transmitochondrial cybrids harboring different proportions of mutated and wild-type mtDNA, Individual clones harboring essentially 100% wild-type or >99% mutated mtDNAs were characterized and studied for respiratory capacity, respiratory chain enzymes activity, mitochondrial protein synthesis, and RNA steady state levels and processing. Our results showed that cell lines containing exclusively mutated mtDNAs respire poorly, overproduce lactic acid, and have significantly impaired activity of respiratory complexes I and IV. Molecular studies showed that mutant clones have a decrease in steady-state levels of mitochondrial tRNA(Leu(UUR)), and a partial impairment of mitochondrial protein synthesis and steady-state levels, suggesting that these molecular abnormalities are involved in the pathogenetic mechanism of the mtDNA 3256 mutation.