伴发色素异常的综合征型遗传性耳聋小型猪新模型建立及特性研究

A mutant minipig family of syndromic heredity hearing loss associated with pigmentary abnormalities was obtained in the pre-study. In the present study, we will conduct an analysis of the inner morphology, electrophysiology in vivo, and other clinical features of the family for identifying the systematic clinical otology and other characteristics. We will also identify the genetic characteristic by mapping the diseases causing gene and the mutant locus by genome-wide linkage/association analysis combined with the candidate gene method. To confirm the function of the discovered minipig mutation, we plan to introduce the mutation into the genomes of wild-type pigs and revert the mutation in the mutant individuals back to wild-type sequence via CRISPR/Cas9-mediated gene knock-in. To validate the similarity between the minipig model and human, we will confirm whether the human orthologue of the discovered minipig mutation and other mutations of the human orthologous gene can cause human heredity hearing loss by screening the clinic human samples derived from hearing loss patients and make the comparison of clinical characteristics between the mutant minipigs and hearing loss patients. Besides, to identify the characteristic and feasibility of the minipig model as a new animal model for medical device evaluation, suitability of the minipig model for electrical cochlea implant will be investigated. Accordingly, new large animal model with genetic stability and definite biological characteristic for human hereditary hearing loss will be created as important research tools in pathogenesis, clinical treatment and evaluation of drug and medical devices of human genetic hearing loss.

本课题在前期研究获得伴发色素异常的综合征型遗传性耳聋小型猪突变家系基础上，对该家系进行内耳形态学分析、活体电生理分析等系统临床特性鉴定，系统鉴定其耳科临床特点及其它临床特征；对该家系利用高密度SNP芯片进行全基因组关联/连锁分析，结合候选基因方法，确定该模型的致病基因和突变位点，鉴定该模型遗传特性；利用CRISPR/Cas9技术制备定点突变和回复突变小型猪模型并进行表型分析，确认该突变位点的致病功能；利用人耳聋病例资源库进行致病基因检测和临床特性比较，验证该模型与人的临床相似性；利用上述模型猪，进行人工电子耳蜗植入评价的动物试验，鉴定其作为医疗器械评价模型的应用特性。本研究将创建新的遗传稳定、生物学特性明确的耳聋大动物疾病模型，为人耳聋致病机理研究、临床治疗及药物和医疗器械评价等提供重要工具和材料。

猪耳聋动物模型与人在遗传及疾病特性上更相似，适于外科操作和临床转化研究，其作为大动物模型对于遗传性耳聋的致病机理研究、临床治疗及药物器械评价等具有独特优势。本项目建立及鉴定了2个新的伴发色素异常的、遗传背景清楚、生物学特性明确的遗传性耳聋猪疾病模型，对其进行了生物学特性、耳聋病理特性、遗传特性、基因定位、发病机制研究。SOX10 (R109W)突变猪是第一个遗传模式明确的人内耳Mondini畸形动物模型，KIT（D806E）突变猪模型首次发现KIT可能是Waardenburg综合征相关的新基因。上述模型和发现的新致病基因突变将为人耳聋致病机理研究、分子诊断、临床治疗及医疗器械评价等提供重要大动物模型及推动相关研究。