Matthews P M, Brown R M, Morten K, Marchington D, Poulton J, Brown G
Department of Neurology and Neurosurgery, Montreal Neurological Institute, Quebec, Canada.
Hum Genet. 1995 Sep;96(3):261-8. doi: 10.1007/BF00210404.
Studies in vitro have shown that a respiratory-deficient phenotype is expressed by cells when the proportion of mtDNA with a disease-associated mutation exceeds a threshold level, but analysis of tissues from patients with mitochondrial encephalomyopathy, lactic acidosis, and strokelike episodes (MELAS) have failed to show a consistent relationship between the degree of heteroplasmy and biochemical expression of the defect. One possible explanation for this phenomenon is that there is variation of heteroplasmy between individual cells that is not adequately reflected by the mean heteroplasmy for a tissue. We have confirmed this by study of fibroblast clones from subjects heteroplasmic for the MELAS 3243 (A-->G) mtDNA mutation. Similar observations were made with fibroblast clones derived from two subjects heteroplasmic for the 11778 (G-->A) mtDNA mutation of Leber's hereditary optic neuropathy. For the MELAS 3243 mutation, the distribution of mutant mtDNA between different cells was not randomly distributed about the mean, suggesting that selection against cells with high proportions of mutant mtDNA had occurred. To explore the way in which heteroplasmic mtDNA segregates in mitosis we followed the distribution of heteroplasmy between clones over approximately 15 generations. There was either no change or a decrease in the variance of intercellular heteroplasmy for the MELAS 3243 mutation, which is most consistent with segregation of heteroplasmic units of multiple mtDNA molecules in mitosis. After mitochondria from one of the MELAS 3243 fibroblast cultures were transferred to a mitochondrial DNA-free (rho0) cell line derived from osteosarcoma cells by cytoplast fusion, the mean level and intercellular distribution of heteroplasmy was unchanged. We interpret this as evidence that somatic segregation (rather than nuclear background or cell differentiation state) is the primary determinant of the level of heteroplasmy.
体外研究表明，当与疾病相关突变的线粒体DNA（mtDNA）比例超过阈值水平时，细胞会表现出呼吸缺陷表型，但对线粒体脑肌病伴乳酸酸中毒和卒中样发作（MELAS）患者组织的分析未能显示异质性程度与缺陷的生化表现之间存在一致关系。对此现象的一种可能解释是，单个细胞之间存在异质性差异，而组织的平均异质性未能充分反映这种差异。我们通过对携带MELAS 3243（A→G）mtDNA突变的异质性受试者的成纤维细胞克隆进行研究，证实了这一点。对来自两名携带Leber遗传性视神经病变11778（G→A）mtDNA突变的异质性受试者的成纤维细胞克隆也进行了类似观察。对于MELAS 3243突变，不同细胞之间突变型mtDNA的分布并非围绕平均值随机分布，这表明针对高比例突变型mtDNA细胞的选择已经发生。为了探究异质性mtDNA在有丝分裂中的分离方式，我们追踪了大约15代克隆之间异质性的分布情况。对于MELAS 3243突变，细胞间异质性的方差要么没有变化，要么有所降低，这与多个mtDNA分子的异质性单位在有丝分裂中的分离最为一致。将其中一种MELAS 3243成纤维细胞培养物中的线粒体通过细胞质融合转移到源自骨肉瘤细胞的无线粒体DNA（rho0）细胞系后，异质性的平均水平和细胞间分布没有改变。我们将此解释为体细胞分离（而非核背景或细胞分化状态）是异质性水平的主要决定因素的证据。
