线粒体tRNAHis基因突变相关的母系遗传性耳聋的遗传校正

Deafness is one of the major public health problems worldwide. In China, approximately 27.8 million subjects suffered from deafness. More than 50% of the deafness is caused by genetic factors. Mitochondrial tRNA mutation is a major molecular basis of maternally inherited deafness. Our previous data showed that the m.12201T>C mutation in the tRNAHis gene was associated with maternally transmitted deafness. However, the tissue-specific pathogenesis of maternally transmitted deafness is less understood. The overall aim of this investigation is to elucidate the molecular pathogenesis of maternally transmitted deafness and to explore the strategy for genetic correction of hearing loss caused by mitochondrial DNA mutation. The first aim is to examine if overexpression of HARS2 in cells carrying m.12201C>T mutation can correct the mitochondrial dysfunction associated with deafness. The secend aim is examine if the m.12201C>T mutation affect the the function of the iPSCs differentiated ear hair cell like cells. Finally, we will test if the cell lines overexpressing HARS2 can be induced into iPSCs and these iPSCs can be differentiated into normal inner ear hair cell like cells. Success of this project will give rise to a deepen understanding molecular pathogenesis of maternally transmitted deafness. Also, the success of this research will provide the valuable information for the development of intervention and therapeutic approaches for deafness and other maternally transmitted diseases in China and worldwide.

耳聋是全球性重大公共卫生问题。我国约有2780万耳聋患者。超过50%的耳聋是由遗传因素造成的。线粒体tRNA基因突变如tRNAHis 12201T>C是母系遗传性耳聋的主要分子基础。本项目拟在前期工作基础上，研究母系遗传性耳聋的分子致病机制以及线粒体聋病遗传校正的策略。首先，利用携带线粒体tRNAHis 12201T>C突变的患者特异性细胞模型，通过过表达人类组氨酰tRNA合成酶（HARS2）基因，校正tRNA His突变引起的线粒体功能障碍；其次，构建携带m.12201T>C突变的诱导多能性干细胞（iPSCs），评估线粒体遗传缺陷对患者特异性iPSCs的诱导及定向分化毛细胞的影响；最后，通过向携带m.12201T>C突变的iPSCs中导入HARS2基因，探索内耳毛细胞功能重建的策略。本项目的实施将有助于进一步诠释耳聋的致病机制，为聋病及其他母系遗传性疾病细胞干预治疗提供科学依据。

耳聋是全球性重大公共卫生问题，我国有近3000万耳聋患者。超过50%的耳聋是由遗传因素造成的，线粒体tRNA基因突变是母系遗传性耳聋的主要分子基础。本课题组前期发现一个携带线粒体12201T>C（m.12201T>C）突变的母系遗传非综合征型耳聋大家系，通过构建患者特异性的永生化淋巴细胞及转线粒体细胞模型，初步阐明了m.12201T>C突变的分子致病机制，但由于缺乏组织特异性模型，对于母系遗传性耳聋的分子致病机制以及线粒体聋病遗传校正的策略研究仍有待深入。本项目一方面利用携带m.12201T>C突变的细胞模型，通过过表达人类组氨酰tRNA合成酶（HARS2）基因，校正tRNAHis突变引起的细胞功能障碍。另一方面，构建患者特异性诱导多能性干细胞（iPSCs），分析m.12201T>C突变的组织特异性表型，评估线粒体遗传缺陷对患者特异性iPSCs诱导分化毛细胞的影响。我们筛选了稳定的HARS2过表达细胞株，详细分析HARS2过表达对m.12201T>C突变体tRNA代谢和线粒体功能的影响。研究发现，HARS2过表达不影响突变型tRNAHis的高级结构，但是提高了tRNAHis氨酰化比例，同时增加了tRNAHis、tRNAAla、tRNALys和tRNAMet等稳态水平，改变了TUFM、KARS等翻译相关蛋白的表达，并最终影响氧化磷酸化复合体亚基的表达水平以及三个由线粒体DNA参与编码的复合体的活性。在HARS2过表达的m.12201T>C转线粒体融合细胞中，细胞呼吸速率下降、ATP产生减少、膜电位破坏、ROS生成和细胞凋亡比例增加等细胞功能缺陷得到持续修复。我们构建并鉴定了携带m.12201T>C突变的iPSCs，初步实现了定向分化毛细胞，尝试向携带m.12201T>C突变的iPSCs中导入HARS2基因，探索内耳毛细胞功能重建的策略。本项目的实施将有助于进一步诠释耳聋的致病机制，为聋病及其他母系遗传性疾病细胞干预治疗提供科学依据。