一个同时携带线粒体tRNAMet4435A>G和YARS2 c.5750A>C基因突变的原发性高血压家系的线粒体功能

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摘要目的：通过构建携带突变的永生化淋巴细胞系，探讨原发性高血压患者线粒体tRNAMet（以下简写为m.）4435A＞G基因突变和YARS2 c.5750A＞C基因突变对线粒体功能的影响。方法：根据临床表现、线粒体DNA全序列以及YARS2外显子测序结果，寻找并确定含有m.4435A＞G以及YARS2基因突变的先证者，并对其家系成员进行拓展筛查。采集确诊家系的外周静脉血标本，并将血液中的淋巴细胞转化为永生化类淋巴母细胞系，经过一段时间的培养后进行线粒体膜电位（MMP）测试和细胞内活性氧（ROS）检测，Northern blot印记杂交实验等一系列反映细胞功能的相关实验。结果：线粒体DNA全序列扩增检测确定含有m.4435A＞G以及YARS2基因突变的先证者和其家庭成员，进一步的种系发生学分析发现4435位点的保守性指数高达100%。成功建立永生化淋巴细胞系，tRNA稳态水平结果显示，携带m.4435A＞G突变的样本tRNAMet稳态水平明显降低，携带YARS2 c.5750A＞C突变样本的tRNATyr稳态水平亦明显降低，且双突变样本的tRNAMet降低量要多于单突变样本；与对照组相比，含有m.4435A＞G和YARS2 c.5750A＞C基因突变的细胞株其ROS水平均升高，且双突变的升高水平大于单突变，差异均有统计学意义（P＜0.05）；m.4435A＞G和YARS2 c.5750A＞C基因突变细胞株中MMP降低水平明显大于对照组（P＜0.05）。结论：m.4435A＞G和YARS2 c.5750A＞C突变分别引起tRNAMet和tRNATyr的代谢异常，造成线粒体翻译缺陷，ROS水平上升，MMP水平下降，并且核基因突变对线粒体突变具有加重作用，最终导致细胞功能障碍。

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	管敏鑫

关键词 ：原发性高血压, 线粒体, tRNAMet, tRNATyr, 突变

Abstract：Objective: To explore the combined effects of m.4435A>G mutation and YARS2 c.5750A>C mutation on the mitochondrial function in patients with essential hypertension. Methods: Based on clinical manifestations, complete mitochondrial DNA sequences and YARS2 exon sequencing results, we searched and identified the proband and screened the extended family members with m.4435A>G gene mutation. Peripheral blood samples of the confirmed pedigree were collected and blood lymphocytes were translated into immortalized lymphoblastoid cell lines. After a period of culture, a series of experiments reflecting cell function were performed, including mitochondrial membrane potential (MMP) detection, intracellular reactive oxygen test (ROS) and Northern blot test. Results: Mitochondrial DNA sequence amplification was used to identify the proband and family members with m.4435A>G gene mutation. Further phylogenetic analysis revealed that the conservatism index of the 4435 loci reached 100%. With the successful establishment of immortalized cell lines, tRNA results showed that the steady-state level of the samples carrying m.4435A>G mutation decreased significantly, and tRNATyr steady-state level of the samples carrying YARS2 c.5750A>C mutation also significantly reduced. The extent to which tRNAMet decreased in the double mutant samples was greater than single mutation samples. Compared with the control group, the ROS levels containing m.4435A>G and YARS2 c.5750A>C gene mutant cell lines increased significantly, and the increased level of double mutants was higher than single mutations, with statistical difference (P<0.05). The decreased level of the mitochondrial membrane potential in cell lines of m.4435A>G and YARS2 c.5750A>C gene mutation was significantly higher than in the control group. Conclusion: The mutation of m.4435A>G and YARS2 c.5750A>C caused abnormal metabolism of tRNAMet and tRNATyr respectively, which finally led to cell dysfunction resulting from mitochondrial translation defects, increased ROS level and decreased MMP plus the increased mitochondrial mutation due to nuclear gene mutation. The results suggest that m.4435A>G and YARS2 c.5750A>C mutation plays an important role in the occurrence of essential hypertension in the family with essential hypertension.

Key words： essential hypertension mitochondrion tRNAMet tRNATyr mutation

收稿日期: 2017-12-05

基金资助:国家自然科学基金青年基金资助项目（31401070，81670944）；浙江省自然科学基金资助项目（LY17C 060004）；温州市科技计划项目（Y20160005，Y20160010）；浙江省教育厅一般科研项目（Y201636759）。

通讯作者: 管敏鑫，教授，Email：gminxin88@gmail.com

作者简介: 任晓燕（1990-），女，山东青岛人，硕士生。

链接本文:

https://xb.wmu.edu.cn/CN/ 或 https://xb.wmu.edu.cn/CN/Y2018/V48/I7/479

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