Transformant Cybrids Harboring the tRNALeu(uuR) Mutation Associated with Maternally Inherited Myopathy and Cardiomyopathy

We studied the physiometabolic effects of a mitochondrial DNA (mtDNA) heteroplasmic point mutation, the A Gtransition associated with maternally inherited myopathy and cardiomyopathy. To eliminate the possible influence of the autochthonous nuclear gene set, we fused myoblast-derived cytoplasts of a patient with a human tumoral cell line deprived of mtDNA (Rho0). The presence and amount of the mutant G3260 vs the wild-type A326" were measured by solid phase minisequencing. We observed a marked reduction ofthe percentage of mutant mtDNA in the culture system compared with that measured in the donor's muscle biopsy, suggesting the presence of negative selection against the mutation. Furthermore, stable mitotic segregation of the two mtDNA populations was observed in 18 of 19 transformant clones, suggesting the presence of intraorganelle and possibly intracellular homoplasmy in the precursor cells of the donor. Several indexes of mtDNA-related respiratory capacity, including oxygen consumption, complex Iand complex IV-specific activities, and lactate production, were markedly abnormal in the clones containing a high proportion of mutant mtDNA, as compared with those containing homoplasmic wild-type mtDNA, possibly because of impaired mitochondrial protein synthesis. We conclude that (a) the A G31 transition is indeed responsible for the mitochondrial disorder identified in the donor patient, and (b) transformant cybrid system gives direct evidence of the mitochondrial origin of a genetic disorder and should be adopted for the evaluation of the pathogenic potential of the mtDNA mutations. (J. Clin. Invest. 1994.93:1102-1107.)