Bidooki S K, Johnson M A, Chrzanowska-Lightowlers Z, Bindoff L A, Lightowlers R N
Department of Neurology, Medical School, University of Newcastle upon Tyne, United Kingdom.
Am J Hum Genet. 1997 Jun;60(6):1430-8. doi: 10.1086/515460.
We report the clinical, biochemical, and genetic investigation of a patient with a severe mitochondrial encephalomyopathy. Genetic studies identified 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 oxidase (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%, respectively). A second, heteroplasmic transition (A-->G), at nt 5656, showed a different distribution than the tRNA(Gly) mutation, with very low levels in skeletal muscle (< 3%) but higher levels in blood (22.7% +/- 3%) and skin fibroblasts (21% +/- 2%). These transitions were followed both in vivo, by repeat biopsy and blood sampling, and in vitro, by establishing primary cultures of myoblasts and skin fibroblasts. Repeat muscle biopsy showed a dramatic increase in COX-deficient fibers, but not of the tRNAGly mutation. Indeed, no significant change in heteroplasmy was measured for either substitution in muscle or blood. In vitro analysis gave very different results. The T10010C was not found in cultured myoblasts, even at early passage. In uncloned fibroblasts, the T10010C was stable (approximately 10%) for several passages but then gradually was lost. In contrast, the A5656G rose progressively from 27% to 91%. In cloned fibroblasts, different combinations of both base-pair changes and wild type could be identified, confirming the presence of clonal, intracellular triplasmy.
我们报告了一名患有严重线粒体脑肌病患者的临床、生化及遗传学研究情况。遗传学研究在第10010位核苷酸处鉴定出一种新的异质性tRNA突变。这种T→C转换位于线粒体tRNA（甘氨酸）的二氢尿嘧啶环中。在骨骼肌中，与细胞色素c氧化酶（COX）缺陷纤维（97.3%±2.6%）相比，其在COX正常的纤维中水平较低（87.2%±11%）；在皮肤成纤维细胞和血细胞中也有发现，但异质性水平较低（分别为15%±6%和17%±10%）。在第5656位核苷酸处的第二个异质性转换（A→G）显示出与tRNA（甘氨酸）突变不同的分布，在骨骼肌中水平极低（<3%），但在血液（22.7%±3%）和皮肤成纤维细胞（21%±2%）中水平较高。通过重复活检和采血在体内对这些转换进行了跟踪，并且通过建立成肌细胞和皮肤成纤维细胞的原代培养物在体外进行了跟踪。重复肌肉活检显示COX缺陷纤维显著增加，但tRNAGly突变没有增加。实际上，在肌肉或血液中，两种替代的异质性均未检测到显著变化。体外分析得出了非常不同的结果。即使在早期传代时，培养的成肌细胞中也未发现T10010C。在未克隆的成纤维细胞中，T10010C在几个传代过程中稳定（约10%），但随后逐渐丢失。相比之下，A5656G从27%逐渐上升至91%。在克隆的成纤维细胞中，可以鉴定出碱基对变化和野生型的不同组合，证实了克隆性细胞内三质性的存在。
