携带m.1555A>G突变的聋病特异性诱导多能干细胞模型的建立及分子机制研究

Deafness is one of the major public health problems, affecting 360 million persons worldwide. Deafness can result from hereditary factors (nuclear and/or mitochondrial genes), environmental factors such as aminoglycoside antibiotics, additionally from the interaction of hereditary and environmental factors. The m.1555A>G mutation is the primary factor for the aminoglycoside induced and non-syndronmic deafness. However, the tissue-specific pathogenesis of the maternally inherited deafness with m.1555A>G mutation remains poorly understood. Patient-specific iPS cells can act as a new disease cell model, which keep the pathogenic gene unchanged, and have the potential of amplication and differentiation. During previous work, we confirmed a maternally transmitted non-syndronmic deafness pedigree carrying m.1555A>G mutation. We generated the primary cells and iPS cells of the proband, and performed some basic experiments on the mitochondrial function assays and hair cell differentiation. In this project we will establish the deaf-specific primary cells, iPS cells and differentiated hair cells from the affected and non-affected of maternal family members, and normal control individuals. We will study the aminoglycoside sensitivity, mitochondrial structure and functions, and their effects on autophagy and apoptosis. These studies will provide a new insight into the molecular pathogenesis of aminoglycoside induced and non-syndronmic deafness associated with m.1555A>G mutation.

耳聋是重大公共卫生问题，全球约有3.6亿耳聋人口。耳聋主要由遗传因素、环境因素或两者共同作用所致。m.1555A>G突变是造成氨基糖苷类抗生素诱导的非综合征聋的主要分子基础，但是其组织特异性致病机制尚不清楚。患者特异性iPS细胞保持患者致病基因不变、具大量扩增和定向分化潜能，是一类新的良好的疾病细胞模型。我们前期工作采集了一个确诊的携带m.1555A>G突变的母系遗传性非综合征聋家系，构建了先证者的原代细胞系和iPS细胞系，进行了初步的线粒体功能分析和毛细胞分化实验。在此基础上，本项目拟系统地构建家系母系成员聋病患者、非聋病患者和正常对照个体的原代细胞系、iPS细胞系及其定向分化内耳毛细胞模型，研究氨基糖苷类抗生素敏感性差异、和线粒体结构功能变化，以及对细胞自噬和凋亡的影响，揭示m.1555A>G突变相关氨基糖苷类抗生素诱导的非综合征聋的致病分子机制，为母系遗传性聋病的早期预警提供理论基础

耳聋是重大公共卫生问题，全球约有3.6亿耳聋人口。耳聋主要由遗传因素、环境因素或两者共同作用所致。MT-RNR1 m.1555A>G突变是造成氨基糖苷类抗生素诱导的非综合征聋的主要分子基础。但是，由于缺乏合适的细胞和动物模型，有关m.1555A>G突变相关聋病的分子致病机制尚不清楚。我们前期工作采集了一个确诊的携带m.1555A>G突变的母系遗传性非综合征聋家系，本项目进一步构建携带m.1555A>G突变的聋病特异性诱导多能干细胞（iPSCs）及其定向分化神经干细胞、听神经样细胞等，结果显示m.1555A>G突变不影响细胞重编程，同时重编程过程没有产生新的突变或导致原有突变消失；发现m.1555A>G突变导致iPSCs-听神经样细胞的线粒体超微结构异常，电子传递链复合体亚基的蛋白质表达降低，造成线粒体膜电势和ATP产量降低等线粒体功能障碍，同时ROS水平显著升高可能引起iPSCs-听神经样细胞的突触可塑性障碍，影响谷氨酸信号传递；听神经样细胞出现去极化电流阈值升高，动作电位幅度下降、上升时间延长、时程延长等动作电位异常，导致聋病发生。线粒体药物处理可拯救线粒体功能，并改善听神经样细胞的神经突长度和动作电位，部分恢复听神经样细胞的电生理功能。本项目研究初步揭示了聋病相关m.1555A>G突变的致病分子机制，为母系遗传性聋病的早期预警和治疗提供理论和实验基础。