非综合征型耳聋相关线粒体12SrRNAA839G基因突变的功能研究

Function study of non-syndromic deafness associated mitochondrial 12S rRNA A839G mutation

目的探讨非综合征型听力损失患者线粒体12srRNA上A839G基因突变对细胞功能的影响。方法根据临床表现、线粒体DNA全序列测序结果，寻找并确定含有线粒体12sr RNAA839G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细胞倍增实验、药敏实验、细胞氧消耗率实验、线粒体膜电位测试和细胞内活性氧（reactive oxygen species，ROS）检测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线粒体12srRNAA839G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839位点的保守性指数高达78．6％。在RPMI．半乳糖培养基中，含有A839G基因突变的细胞株倍增时间明显长于对照组，差异具有统计学意义（P=0．033）；氨基糖苷类抗生素对含有A839G基因突变细胞株的影响并不明显；含有A839G基因突变的细胞株与对照组相比，细胞氧消耗率均降低，差异具有统计学意义（P=0．033）；与对照组相比，含有A839G基因突变的细胞株其ROS水平均出现较大幅度的升高，差异具有统计学意义（P〈0．01）；A839G基因突变细胞株中线粒体膜电位降低细胞群的数量明显多于对照组。结论线粒体12s rRNA A839G基因突变会对细胞内线粒体呼吸链的功能产生影响，突变细胞株的生长速率明显降低。A839G基因突变可能与非综合征型听力损失相关，是一种新的致病性突变。

Objective To investigate the correlation between nonsyndromic deafness and mitochondrial 12s rRNA A839G mutation. Methods According to the clinical manifestations of mitochondrial DNA sequencing and analysis to find and determine family containing mitochondrial 12s rRNA A839G mutation. Harvested its family members blood and transferred their lymphocytes into lymphoblastoid cell lines, followed by cells cultured, cell doubling experiment, susceptibility testing, cellular oxygen consumption rate experiment, ROS and mitochondrial membrane potential experimental tests were progressed to explore the correlation between the A839G mutation and nonsyndromic deafness. Results The mitochondrial 12s rRNA A839G mutation pedigrees were determined through the full sequence detections of the Mitochondrial DNA, further phylogenetic analysis showed that 839 point conservative index （C!） up to 78.6%; in RPMI-galactose medium containing A839G gene mutant cell line, the doubling time was significantly longer than the control group, and the difference was significant （P = 0. 033）. The effect to cell lines containing the A839G mutation of aminoglycoside drugs was not obvious. When compared with the control group, cell lines containing the A839G mutation significantly reduced cellular oxygen consumption rate（P = 0. 033 ）; compared with the control group, the ROS levels of cell lines containing the A839G mutation appeared more substantial elevated with significan difference （ P 〈 0. 01 ）. The mitochondrial membrane potential of cells of experimental group was significantly reduced than the control group. Conclusion The present study proved that the mitochondria 12s rRNA A839G mutations affect the function of the mitochondrial respiratory chain at the cell level, which might reduce the growth rate of the mutant cell lines, result in hearing.