INTRACELLULAR MITOCHONDRIAL TRIPLASMY IN A PATIENT WITH 2 HETEROPLASMIC BASE CHANGES

We report the clinical, biochemical, and genetic investigation of a pa tient with a severe mitochondrial encephalomyopathy. Genetic studies i dentified a novel, heteroplasmic tRNA mutation at nt 10010. This T-->C transition is located in the DHU loop of mitochondrial tRNA(Gly). In skeletal muscle, it was present at lower levels in cytochrome c oxidas e (COX)-normal (87.2% +/- 11%) compared with COX-deficient fibers (97. 3% +/- 2.6%); it was found in skin fibroblasts and blood cells, but at lower levels of heteroplasmy (15% +/- 6% and 17% +/- 10%, respectivel y). A second, heteroplasmic transition (A-->G), at nt 5656, showed a d ifferent distribution than the tRNA(Gly) mutation, with very low level s in skeletal muscle (<3%) but higher levels in blood (22.7% +/- 3%) a nd skin fibroblasts (21% +/- 2%). These transitions were followed both in vivo, by repeat biopsy and blood sampling, and in vitro, by establ ishing primary cultures of myoblasts and skin fibroblasts, Repeat musc le biopsy showed a dramatic increase in COX-deficient fibers, but not of the tRNA(Gly) mutation. Indeed, no significant change in heteroplas my was measured for either substitution in muscle or blood. In vitro a nalysis gave very different results. The T10010C was not found in cult ured myoblasts, even at early passage. In uncloned fibroblasts, the T1 0010C was stable (similar to 10%) for several passages but then gradua lly was lost. In contrast, the A5656G rose progressively from 27% to 9 1%, In cloned fibroblasts, different combinations of both base-pair ch anges and wild type could be identified, confirming the presence of cl onal, intracellular triplasmy.