School of Ophthalmology and Optometry, Wenzhou Medical University, Wenzhou, Zhejiang, China; Attardi Institute of Mitochondrial Biomedicine, Wenzhou Medical University, Wenzhou, Zhejiang, China.
Division of Medical Genetics and Genomics, Zhejiang Children's Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China; Institute of Genetics, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China.
Mitochondrion. 2018 Sep;42:84-91. doi: 10.1016/j.mito.2017.12.003. Epub 2017 Dec 7.
The purpose of this study was to investigate the pathophysiology underlying Leber's hereditary optic neuropathy (LHON)-associated mitochondrial tRNA mutation.
Severn hundred ninety-seven Han Chinese subjects underwent clinical and genetic evaluation and analysis of mitochondrial DNA (mtDNA). The cybrid cell lines were constructed by transferring mitochondria from lymphoblastoid cell lines derived from a Chinese family into mtDNA-less (ρ) cells. These cell lines were assayed by tRNA Northern blot and Western blot analyses, respiratory enzymatic activities, the rate of ATP production and the generation of reactive oxygen species.
The tRNA 15927G>A mutation was identified in eight probands with suggestively maternal inheritance among 352 Han Chinese probands lacking these known LHON-associated mtDNA mutations. The m.15927G>A mutation affected a highly conserved guanine at position 42 at the anticodon-stem of tRNA, destabilizing the conservative base pairing (28C-42G). We therefore hypothesized that the m.15927G>A mutation, and altered the structure and function of tRNA. Northern blot analysis revealed 60% decrease in the steady-state level of tRNA in the mutant cell lines. Western blot analysis showed the variable reductions of 4 mtDNA encoding proteins, especially for marked decrease of ND1 and CYTB observed in mutant cell lines. Furthermore, we demonstrated that the m.15927G>A mutation decreased the activities of mitochondrial complexes I and III, markedly diminished mitochondrial ATP levels, and increased the production of reactive oxygen species in the mutant cells.
Our data demonstrated the first mitochondrial tRNA mutation leading to LHON. Our findings may provide new insights into the understanding of pathophysiology of LHON.
本研究旨在探讨莱伯遗传性视神经病变（LHON）相关线粒体 tRNA 突变的病理生理学基础。
对 797 名汉族受试者进行临床和遗传评估及线粒体 DNA（mtDNA）分析。通过将源自中国家族的淋巴母细胞系的线粒体转移到 mtDNA 缺失（ρ）细胞中，构建细胞杂种系。通过 tRNA Northern 印迹和 Western 印迹分析、呼吸酶活性、ATP 产生率和活性氧生成来检测这些细胞系。
在 352 名缺乏这些已知 LHON 相关 mtDNA 突变的汉族先证者中，有 8 名先证者存在提示母系遗传的 tRNA 15927G>A 突变。m.15927G>A 突变影响 tRNA 反密码子茎上高度保守的鸟嘌呤 42 位，破坏保守碱基配对（28C-42G）。因此，我们假设 m.15927G>A 突变改变了 tRNA 的结构和功能。Northern 印迹分析显示突变细胞系中 tRNA 的稳态水平降低了 60%。Western blot 分析显示 4 种 mtDNA 编码蛋白的可变减少，尤其是在突变细胞系中观察到 ND1 和 CYTB 的明显减少。此外，我们证明 m.15927G>A 突变降低了线粒体复合物 I 和 III 的活性，显著降低了线粒体 ATP 水平，并增加了突变细胞中活性氧的产生。
我们的数据首次证明了导致 LHON 的线粒体 tRNA 突变。我们的发现可能为理解 LHON 的病理生理学提供新的见解。
