X-连锁遗传综合征型听神经病（AUNX1）的分子病理机制研究

The genetics basis, the lesion site and the underlying molecular pathology of auditory neuropathy (AN) have been the research hotspots in recent years, especially for the syndromic type. AUNX1 (auditory and peripheral sensory neuropathy, X-linked locus 1) was the first reported X-linked recessive syndromic AN locus which was mapped in a large Chinese pedigree in 2004. In our previous study, we identified the specific missense mutations (c.778A>G, c.1030C>T, c.1352G>A, ect.) of AIFM1 gene as the causative variants of AUNX1 phenotype. AIFM1 gene encodes an inner-membrane-anchored protein in mitochondrion. Its mutations are also known to be associated with mitochondrial encephalomyopathy, prenatal ventriculomegaly and Cowchock syndrome. Our work expands the spectrum of AIFM1-associated phenotypes, but the related pathological mechanism remains unknown. In the present study, we’ll focus on the AIFM1 specific causal mutations (c.778A>G, c.1030C>T, c.1352G>A, ect.). The mitochondrial complex I-V enzyme activity and oxygen consumption rate in AUNX1 patients will be analyzed to identify the markers for early molecular diagnosis (clinical genetics part). The transgenic mouse and cell line model with the specific causal mutations (c.778A>G, c.1030C>T, c.1352G>A, ect.) will be created using CRISPR/Cas9 system to explore the effect target and mechanism of AIFM1 gene defect, by investigating the function of hearing and peripheral nervous system in animal body, AIFM1 expression in inner ear tissue, and redox reaction of cell metabolism in specific mutant cell (lines) (molecular genetics part). The results of this study would provide experimental evidence and theoretical basis for early diagnosis and new therapeutic approach to AN.

听神经病相关的病因、病变部位和病理机制一直是国际研究的热点和难点，而累及多系统的综合征型听神经病更为复杂。AUNX1基因座是2004年报道的X染色体连锁遗传性听神经病伴周围感觉神经病新位点，我们的前期研究首次证实凋亡诱导因子基因AIFM1多种特异性点突变（c.778A>G、c.1030C>T、c.1352G>A等）与该型听神经病相关。AIFM1表达在线粒体内膜，其不同突变还可导致线粒体肌脑病、腓骨肌萎缩症等表型。同一基因导致多种疾病，其相关病理机制尚不明确。本研究以特异性致病点突变（c.778A>G、c.1030C>T、c.1352G>A等）为切入点：检测突变对AUNX1患者线粒体呼吸链复合物活性、氧耗速率及酶等代谢指标的影响，为寻找可能的病理代谢通路提供线索（临床遗传学）；同时利用CRISPR/Cas9技术构建点突变小鼠或细胞系模型，观察动物水平听觉及周围神经电生理特征改变，组织水平基因内耳分布，细胞水平突变蛋白表达差异、氧化还原代谢变化，探索基因缺陷的作用靶点和分子机制（分子遗传学），为研发治疗新途径提供理论基础

凋亡诱导因子基因AIFM1突变可导致X-连锁隐性遗传性听神经病，但是相关的病理生理机制仍不清楚。本课题从细胞和模式动物水平进行研究，一方面分离培养大鼠的螺旋神经节细胞(SNGs)，通过siRNA沉默AIFM1表达，观察SNGs线粒体功能、膜电势等病理生理改变，以及对诱导细胞凋亡这一过程的作用。同时利用CRISPR/Cas9技术，构建AFIM1致病点突变的小鼠模型，观察模式动物的临床表型、内耳解剖及基因表达变化。结果发现：在细胞水平，siRNA干扰AIF表达可导致大鼠SNGs氧化应激水平明显增高，线粒体膜电位降低、呼吸链功能明显下降，同时抗凋亡、抗氧化蛋白及线粒体呼吸链复合酶I的水平明显减低，提示AIF表达异常可引起线粒体氧化呼吸功能受损，氧化应激损伤增加，从而引发SNGs细胞功能障碍，可能导致遗传性听神经病。但是，在动物水平，AIFM1突变型小鼠的听力水平（ABR阈值）正常，大体及内耳组织形态正常，AIF蛋白表达较野生型未见明显差异，说明点突变小鼠模型不能模拟人类AIFM1相关的X-连锁遗传听神经病表型，提示可能需要构建与人类更相似的大型哺乳动物模型以进一步研究相关的分子机制。综合上述结果，本研究为探讨AIFM1突变导致遗传性听神经病的分子病理机制提供了实验依据和重要线索，也为人类疾病动物模型的构建提供了一定的借鉴经验。