应用全外显子组测序技术鉴定我国外中耳畸形的致病基因及功能研究

Microtia is a congenital deformity where the external ear is underdeveloped. In China, the prevalence rate of microtia is 5.18 per 10000 births. Greater than 90% of individuals with microtia experience conductive hearing loss on the affected side, which harms the patient's sound in both mind and health. Reconstruction of external ear and hearing cost lots of money and give a heavy burden for family and society. Previous studies on nonsyndromic microtia were mainly focused on plastic surgery and environment causes, while no confident genetic risk factors were reported so far. Thus, causal genes mapping is still the hotspot for investgating microtia. We collected 3 large nonsyndromic microtia pedigrees and more than 500 microtia trios, and solved the major problem of sample limited for exploring genetics basis of microtia. Whole exome sequencing is a new powerful technology and has been successfully used in mapping causal genes for several rare and common diseases. We have implemented this technology on one of three microtia pedigrees and found a potential causal mutation in DMP1 gene. However, only 8% of gentic risk was contributed by mutations within DMP1 gene suggested that many other gentic risks need to be revealed. Therefore, we will use whole-exome sequencing method to explore the majority genetic risks of microtia with our large pedigree and trio samples in this study. We will obtain the causal genes of microtia with methods of bioinfomatic analzing, GoldenGate genotyping, and functional experiment validating. Our results will not only improve the etiology knowledge of microtia, but also be helpful for genetic consultation and future gene therapy.

先天性外中耳畸形是我国第二大颅面发育异常疾患，发病率高达5.18/万人。约90%的患者患侧听力丧失，严重影响认知能力和身心健康。外耳和听力重建花费巨大，给家庭和社会带来沉重负担。查找外中耳畸形的致病基因仍是该病的研究热点，而大家系和散发样本的获取是研究该病的瓶颈。本项目组已拥有非综合征型外中耳畸形3个大家系及约500个核心家系。我们前期对其中一大家系研究后找到的致病突变对群体患病风险的贡献度仅8%，提示仍有大量遗传风险因素需要挖掘。全外显子组捕获结合二代测序技术是目前鉴定疾病遗传因素的最有效手段。我们将使用该方法，采用大家系结合核心家系的策略，深入开展先天性外中耳畸形的遗传定位研究。应用生物信息学方法，结合GoldenGate分型平台和细胞功能研究平台，确定影响我国外中耳畸形的主要基因。本研究将为阐释该病的分子病因和发病机制提供理论基础，为临床早期干预以及靶向治疗提供指导。

先天性外中耳畸形是我国第二大颅面发育异常疾患，发病率高达5.18人/万人。约90%的患者患侧听力丧失，严重影响认知能力和身心健康。本研究使用家系样本和散发样本挖掘我国外中耳畸形的致病突变。针对家系的致病突变挖掘，我们收集到了7个家系，包括3个20人以上的大家系，通过外显子测序、目标区域捕获测序手段，发现1个拷贝数（CNV）变异和7个家系完全共分离，提示为外中耳畸形的致病突变位点。我们前期芯片的研究同样也发现了该拷贝数变异只存在于患者（942例）中，而正常人群（3000余例）不存在该变异。针对散发样本的致病突变挖掘，我们使用全基因组测序的手段对21例外中耳畸形样本进行研究，找到了4个可引起功能变异的突变，该结果仍需开展更大样本量的验证工作才能确定这些变异是否为真正的致病突变。在挖掘到家系样本的致病突变后，我们采用CRISPR-Cas9的基因编辑技术构建了拷贝数变异核心保守序列的小鼠模型，包括敲除和敲入模型，通过传代后我们拿到了CNV=0，1，3，4以及CNV=2的片断性单亲二倍体鼠。目前已经明确CNV=0的小鼠耳朵部分结构缺失，而CNV=3和4的小鼠耳郭明显变大。