核修饰基因调控母系遗传性耳聋发病机制及听觉功能重建的策略研究

Deafness is one of the major public health problems worldwide. In China, approximately 30 million subjects sufferred from severe to profound hearing loss. Deafness can be caused by genetic or environemtnal factors or interaction between two factors. The A1555G mutation in the mitochondrial 12S rRNA gene is the most common mutation for maternally transmitted deafness. Our previous data showed that modifier fators including nuclear modifier gene TRMU may modualte the phenotypic manifestation of the A1555G mutation. However, the 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 stragery for the regenation of hearing organ. The first aim will be to evaluate the effect of the TRMU A10S mutation on mitochondrial dysfunction associated with A1555G mutation. For this purpose, we will examine the effect of A10S mutation on the level of 2-thiouridine modification at position 34 in mitochondrial tRNALys, tRNAGlu and tRNAGln, the posttranscriptional modifications of mitochondrial tRNA, the aminoacylation of mitochondrial tRNA, and mitochondrial protein synthesis, oxygen consumption and ROS production. The second aim is to assess the effects of the knock-down TRMU on mitochondrial dysfunction associated with the A1555G mutation. The TRMU knock-down cell lines will be assayed for the levels of mitochondrial tRNAs, the rate of mitochondrial protein synthesis by 35S-methionine pulse-labeling experiments, the endogenous respiration rates by determining the O2 consumption rate in intact cells and the O2 consumption on digitonin-permeabilized cells using different substrates and inhibitors. The third aim is examine if the A1555G mutation and TRMU mutation affect the induction of iPSCs and iPSCs differentiating into inner ear hair cell like cells. The fourth aim is to examine if overexpression of TRMU gene in cells carrying both TRMU A10S and A1555G mutation or only A1555G mutation can correct the mitochodnrial dysfunction associated with deafness. Finally, we will test if the cell lines overexpressing TRMU can be induced into iPSCs and these iPSCs can be differentiated into inner ear hair cell like cells. Success of this project will define the role of nuclear genes in the pathogenesis of maternally inherited deafness. This, in turn, provides new insights into the molecular mechanism of maternally inherited deafness as well as other disease process. Also, this success will give rise to a deepen understanding the mechanism of the interaction between the nuclear and mitochondrial genome. In addition, the success of this research will provide the valuable information and technology 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。我国有近3000万耳聋患者。耳聋由遗传因素、环境因素或两者共同作用所致。线粒体12S rRNA A1555G突变是母系遗传性耳聋的重要原因，TRMU等核修饰基因则可能调控该突变的表型表达。本项目拟在我们前期研究成果基础上，探讨母系遗传性耳聋的分子致病机制及听觉功能重建的策略。首先，利用携带线粒体12S rRNA和TRMU基因突变的细胞模型，研究线粒体和核修饰基因互作导致母系遗传性耳聋发病的机制；其次，构建携带12S rRNA基因和核修饰基因TRMU突变的患者特异性诱导多能性干细胞，探讨线粒体遗传缺陷对iPSCs诱导分化毛细胞的影响；最后，通过向携带耳聋相关线粒体基因突变的iPSCs中导入核修饰基因，校正线粒体的功能缺陷，实现内耳毛细胞功能重建。本项目的实施将有利于全面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和技术支撑。

耳聋是全球性重大公共卫生问题。我国有近3000万耳聋患者。耳聋由遗传因素、环境因素或两者共同作用所致。线粒体12S rRNA A1555G突变是母系遗传性耳聋的重要原因，TRMU等核修饰基因则可能调控该突变的表型表达。本项目拟在我们前期研究成果基础上，探讨母系遗传性耳聋的分子致病机制及听觉功能重建的策略。首先，利用携带线粒体12S rRNA和TRMU基因突变的细胞模型，研究线粒体和核修饰基因互作导致母系遗传性耳聋发病的机制，结果表明TRMU突变使得线粒体1555A>G 相关线粒体功能失常的进一步恶化，最终超过耳聋表型表达的阈值，引发聋病。我们的研究为母系遗传耳聋的致病机制提供了新的思路，即通过线粒体突变与核基因突变相互作用引发聋症发生，丰富了聋病致病机制体系。其次，构建携带12S rRNA基因和核修饰基因TRMU突变的患者特异性诱导多能性干细胞，探讨线粒体遗传缺陷对iPSCs诱导分化毛细胞的影响，结果显示研究发现携带TRMU A10S纯合突变与线粒体1555A>G突变时，iPSC诱导分化为听祖细胞效率降低、毛细胞样细胞静纤毛形态功能异常；利用CRISPR/Cas9技术成功完成上述双突变iPSC模型的A10S突变的遗传矫正。该研究具有重要的临床意义，尽管目前很难通过技术手段来矫正线粒体突变，但通过对核修饰基因进行遗传矫正则可能实现表型修复。此外，研究中构建了核修饰基因TRMU及GTPBP3敲除及敲减的多种斑马鱼、小鼠疾病模型，在体水平研究核修饰基因缺陷引发聋病机制，充分阐释其分子致病机制；同时发现母系遗传性聋病致病新机制，多种线粒体tRNA突变tRNA修饰，进而影响线粒体及细胞功能进而引发聋病。本项目全面诠释修饰基因调控母系遗传性耳聋的致病机制，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和技术支撑。