目标区域捕获测序鉴定“眼-耳-脊柱综合征”的致病基因的分子遗传学研究

Oculo-auriculo-vertebral spectrum (OAVS) is a congenital genetic disease in Asian race,characterized byeyes, ears, face and spinal deformities. Since the pathogenesis of OAVS is complicated, traditional genetic testing is difficult to effectively capture its pathogenic genes. In this project, we investigated the Chinese family history of Chinese with positive OAVS, and confirmed that OAVS is highly heterogeneous and non-chromosomal aberrant. With the repeated detection of FOXI3 mutation by exome sequencing,we supposedthat FOXI3 mutation was responsable for OAVS. In order to locate the OAVS pathogenic genes accurately and select the genes to be tested, we first take advantage of the efficiency and accuracy of gene-targeted capture and sequencing. By detecting large OAVS sample, we comprehensively analyze the functional mutations of candidate genes and screened the pathogenic genes. Then we obtain the disease mutation spectrum by correlatively analyzing the relavancy between the type of mutation and phenotype. Finally, we study the function of pathogenic genes and elucidate the underlying mechanism with cytology and knockout mouse. We hope that with the implementation of this project, the pathogenicity of FOXI3 can be verified,the pathogenic genes and their mutation profiles of OAVS can be found. Meanwhile, we aim at building a screening platform for mutation of OAVS gene, which will not only provide the basis for more in-depth study on the pathogenic mechanism of OAVS in the future, but also establish the foundation of rapid detection of disease diagnosis and final gene therapy.

眼-耳-脊柱综合征（OAVS）是以眼、耳及颜面、脊柱畸形为主要症状，好发于东方人群的遗传性疾病；其发病机制复杂，传统基因检测手段难以有效捕获致病基因，其致病的遗传学机制目前尚不明确。前期研究分析所建立的有阳性家族史的汉族OAVS家系，证实OAVS有高度遗传异质性且非染色体畸变所致；结合对FOXI3突变的重复检出，推测存在致病基因引起OAVS。为准确定位OAVS致病基因，先利用基因靶向捕获测序检测大样本OAVS病例，全面分析候选基因功能性突变并筛选出致病基因；再关联性分析突变类型与表型之间关系获得疾病突变谱；最后利用细胞学实验和基因敲除小鼠模型，研究致病基因功能并探讨其致病机制。希望通过本研究项目，验证FOXI3的致病性，锁定OAVS的致病基因及其突变谱，同时构建OAVS基因突变筛查平台，不但为今后更深入地研究OAVS的发病机制提供依据，而且为OAVS的快速诊断和基因治疗奠定理论基础。

眼-耳-脊柱综合征（OAVS）是以眼、耳及颜面、脊柱畸形为主要症状，好发于东方人群的遗传性疾病；其发病机制复杂，致病的遗传学机制目前尚不明确。目前已知的OAVS致病基因的数量有限，对其中的病因机制仍缺乏了解，加之既往的遗传学研究多局限于少数家系中，研究发现多不能在其他家系中得到重现与证实，难以推广到临床基因检测和遗传咨询，患者的实际获益十分有限。本课题通全外显子组测序技术，对37例散发OAVS患者以及1个OAVS的三口之家家系样本（共计40例）的基因编码序列进行检测，鉴定其基因突变谱，分析和挑选可能与OAVS发病相关的候选基因,通过CRISPR/Cas9技术，对C3H10T1/2与ATDC5细胞OAVS发病相关的候选基因进行编辑，并通过细胞增殖实验、迁移实验以及陈软骨分化模型，初步观察其敲除对细胞行为学和成软骨分化过程的影响，以从细胞表型上了解这些基因参与OAVS发病的潜力；利用上述体外细胞模型，通过WesternBlotting等手段对这些基因在OAVS发病中可能存在的上下游调节因子进行初步挖掘。最终鉴定了本研究OAVS队列的基因突变谱，结合基因功能富集分析结果和家系遗传模式分析结果，筛选了PRODH，TTN，TTC21B，BCR等4个候选致病基因进行功能机制研究。最终发现TTC21B基因的失活可通过Gli1-Wnt5a抑制Wnt/β-catenin通路进而抑制成软骨分化过程。同时，过表达该基因还可部分逆转RA的成软骨分化抑制作用。这些结果提示TTC21B可能是一个新的OAVS致病候选基因和潜在干预治疗靶点。后期将利用细胞学实验和基因敲除小鼠模型，研究致病基因功能并探讨其致病机制，为今后更深入地研究OAVS的发病机制提供依据，而且为OAVS的快速诊断和基因治疗奠定理论基础。