开展耳聋遗传家系致聋基因克隆和功能研究有助于阐明遗传性耳聋发病机制，有助于该类疾病的诊治和预防。本课题组已收集到三个颇具研究价值的非综合征性耳聋遗传家系，一个为近亲家系，另外两个为常染色体显性家系。使用定向捕获联合二代测序技术，我们发现近亲家系的致病突变是KCNQ4新突变位点G228D，纯合和杂合突变均致聋；另外两家系已排除138个耳聋相关基因突变致聋的可能，为新基因致病的可能性较大。我们通过全基因组连锁分析，已将其中一个家系的致聋基因定位于染色体14q11.1区域（最大LOD值3.2），此区域不包含已报道的耳聋基因；拟使用全基因组外显子测序联合连锁分析的方法，鉴定所收集的两常染色体显性家系的致聋基因及突变位点；进一步通过构建基因敲入、敲除的小鼠和斑马鱼模型，结合生物化学、分子生物学、电生理、活体成像等技术手段对所克隆的致病基因进行功能研究，以揭示其致聋的分子机制。

Identification and functional studies of hereditary deafness genes are beneficial to understand both the complexity of the auditory system and impressive genetic heterogeneity of deafness, and are also helpful to elucidate the molecular pathogenesis of hearing impairment, which may offer opportunities for prevention and therapy of these diseases. Currently, we collected three Chinese nonsyndromic hearing impairment families segregated with deafness. Using targeted next-generation sequencing, we identified one novel missense mutation of KCNQ4, c.683G>A (p.G228D), co-segregating with the phenotype in one consanguineous family. Surprisingly, both the homozygous and heterozygous mutation can cause hearing loss, but the degree is different. Using targeted next-generation sequencing of 138 deafness-related genes, we excluded the possibility of causative mutations in known deafness genes in other two autosomal dominant nonsyndromic families. Genome-wide linkage analysis in one family showed linkage to chromosome 14q11.1 (maximum LOD score of 3.2). In this study, we proposed to identify the causative genes and their corresponding mutations by whole-exome sequencing and Genome-wide linkage analysis. Knockout, knock-in mouse and genetic-manipulated zebrafish model will be generated for functional studies of the causative genes of nonsyndromic deafness. In addition, several type of technologies, including: biochemical analysis, molecular biological analysis, electrophysiological analysis, live imaging, etc., will be employed to elucidate the complex mechanism of these hereditary deafness caused by identified gene mutations. The studies in this project will increase our knowledge of the molecular bases of nonsyndromic hereditary hearing loss, and provide new opportunities for the diagnosis and treatment of this disease in the hearing loss population.

耳聋是一种严重影响患者的言语、智力发展和社会交往的常见疾病，给家庭及社会带来沉重的精神及经济负担。耳聋发病率高，从刚出生的新生儿一直到老年人，均可发病，耳聋的发病原因50%以上是由遗传因素所导致，即遗传性耳聋；同时其具有很高的遗传异质性。本课题组对前期收集到的三个非综合征性耳聋遗传家系，进行了深入的研究，取较大的研究进展，并明确了下一步研究方向：1. 对于近亲家系HM，我们已成功构建了KCNQ4G228D基因敲入小鼠模型，该小鼠模型的听力表型与该耳聋家系患者的听力表现高度一致，纯合和杂合突变均致耳聋，免疫组化已证实该小鼠的外毛细胞、内毛细胞及螺旋神经节出现不同程度的变性；下一步研究是利用合适的小分子物质进行有效干预，修复突变体钾离子的完整性恢复其功能，改善突变小鼠的听力。2. 对于常染色体显性耳聋家系ZP，我们发现家系的致聋基因定位于染色体18p11.3区域（最大LOD值4.93），并联合全基因组外显子测序结果，成功鉴定了致聋基因及突变位点：THOC1 (c.547C>G, p.L183V)。研究还发现THOC1在小鼠内耳的外毛细胞和内毛细胞，及斑马鱼的otic vesicle都有表达, 并利用斑马鱼模型的毛细胞活体成像技术，证实了THOC1表达于毛细胞及听觉相关系统；我们进一步构建了基因敲入及敲除的斑马鱼模型，发现THOC1敲除斑马鱼的毛细胞出现了细胞凋亡。下一步研究方向是THOC1基因引起毛细胞凋亡的具体分子机制及探讨其有效的干预。3. 对于常染色体显性耳聋家系ZC，通过全基因组连锁分析，已将该家系的致聋基因定位于染色体15q15.1区域（最大LOD值3.2），此区域不包含已报道的耳聋基因；目前正在对该家系3个患病成员+2个正常成员进行使用全基因组外显子测序，以期发现和证实该耳聋家系致病基因和突变位点，并进一步完成致聋基因的功能实验。开展耳聋新基因的克隆与功能研究可为未来耳聋的分子诊断、预防、新药物开发和基因治疗提供重要的理论依据和指导方向，有助于该类疾病的精准诊断、治疗及预防，造福广大的耳聋患者。