基于遗传性耳聋猪新模型的内耳Mondini畸形发育分子机制研究

Deafness is the second disabling disease in China. Clinically, Mondini inner ear malformation is of the highest occurrence in inner ear malformations causing sensorineural hearing loss. However, the underlying molecule(s) causing Mondini malformation and molecular mechanism for Mondini malformation development remain to be investigated due to the lack of animal models. Based on our previously generated novel pig model with hereditary hearing loss exhibiting simple Mondini inner ear malformation similar to human, we found that the SOX10 p.R109W mutation was completely genetically linked to Mondini malformation, and besides, this mutant SOX10 was confirmed to be the Mondini malformation-causing molecule by introducing SOX10 p.R109W mutation precisely into wild type pig genome. In this project, using the fetal inner ear tissues of different developmental stages collected from the pig models within the key time window of Mondini inner ear malformation development, we are planning to perform intensive molecular mechanism researches for Mondini inner ear malformation via SOX10-based chromatin immunoprecipitation-deep sequencing (ChIP-Seq), transcriptomic and proteomic analysis, and thereby construct dynamic and integrated omics databases for Mondini inner ear malformation development. Based on the established omics databases, the molecules differentially expressed and(or) regulated between the malformational and healthy inner ear tissues will be found out, and the molecular-interacting network and molecular pathway(s) regulating Mondini inner ear malformation development will be elucidated. The key molecule(s) in the pathway(s) will be further functionally validated via expression in cultured porcine cells, generation and analysis of gene-modified animals, and genetic and expression analysis in clinic human samples. Based on the work mentioned above, we are to systematically decipher the molecular mechanism for Mondini inner ear malformation development, and find out molecular targets for the diagnosis and therapy of Mondini inner ear malformation.

Mondini畸形是最常见的人内耳发育畸形，引起感音性耳聋，但发病机制不清、未发现致病基因、缺乏动物模型。基于前期首创的遗传性Mondini畸形耳聋猪新模型，首次发现SOX10 p.R109W突变与Mondini畸形完全连锁，并确证该突变分子可导致Mondini畸形，完成了首个Mondini畸形致畸分子的功能鉴定。在此基础之上，本项目拟在Mondini畸形发育关键时间窗内，通过基于突变分子的全基因组ChIP-Seq深度测序与转录组、蛋白组学技术，分析突变分子在不同发育时期的内耳组织基因组中的结合位点及其导致的转录组和蛋白组学变化，构建动态、完整、准确的Mondino畸形发育基因表达和调控的组学数据库，寻找表达、调控差异分子，建立畸形发育分子通路，并对分子通路的关键分子在内耳发育中的表达、功能及其在临床样本中的突变与表达模式进行综合分析，阐明Mondini畸形发育分子机制，探寻诊断、防治靶点

耳聋是一种常见的严重影响人类生活质量的感觉障碍疾病。由于人体临床样本存在表型差异大、遗传背景复杂、样本数量少以及研究材料（内耳组织等）获取困难等问题，动物模型在耳聋相关基因发现、功能验证、发病机理研究、诊断预防治疗方法评价等方面具有不可替代的作用。尽管啮齿类动物在耳聋基因发现及研究中发挥了重要作用，但啮齿类动物与人听力表型差异较大，且并不适宜于临床耳聋治疗与矫正评价研究。非人灵长类动物与人类进化关系最接近，但由于其繁殖周期长、繁殖力低下、成本高以及动物福利伦理的限制，不利于科学研究的进行。除灵长类以外，猪和人类进化关系最近，其与人在听觉器官的形态、结构和听觉功能方面具有极高的相似性，是创制耳聋动物模型的理想材料。本项目前期培育了首例遗传性内耳Mondini畸形耳聋猪模型，并基于该模型鉴定了首个遗传性内耳Mondini畸形致病突变基因SOX10 p.R109W。依照项目研究计划，本项目基于遗传性耳聋猪模型，开展了内耳发育畸形分子机制研究。首先，系统解析了SOX10 p.R109W突变猪的病理变化以及突变分子致聋的分子机制，系统分析了SOX10 p.R109W突变分子的生物学活性、表达剂量、遗传补偿效应以及分子结构变化，发现SOX10 p.R109W突变分子是完全的单倍剂量不足的模式发挥致病效应，其致病机制和病理改变与Sox10基因完全呈单倍剂量表达的Sox10 c.321dupC突变猪一致；通过对SOX10分子的n-NLS区域进行了模拟临床的饱和点突变，系统分析了该区域单个氨基酸的改变所致的蛋白转录活性变化，并通过与临床病例表型结合分析，初步揭示了该区域基因型与表型的相关性。其次，开展了SOX10 p.R109W突变猪模型内耳发育的组学研究，发现DNA表观修饰酶TET2启动子活性受到SOX10转录因子蛋白的直接调控，而SOX10突变导致TET2异常表达进而影响全基因组5hmC修饰水平，并以表观遗传学这一全新视角预测了TET2异常表达对耳蜗基因组5hmC修饰的影响以及这些变化如何通过临近或远端作用方式调控内耳发育关键基因的表达，为大动物耳蜗及听神经发育乃至瓦氏综合征疾病机制提供了新的线索。

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