ROBO1基因调控区突变鉴定及其导致人类半侧颜面短小畸形的功能研究

Craniofacial microsomia is the second most common craniofacial malformation that significantly affects the physical and mental health of the patients. Our team has performed the first genome-wide association study on craniofacial microsomia in Chinese and found 8 significant (P < 1E-8) associated loci in 1373 cases and 3681 controls. Within these loci, the most significant associated locus, located at the regulatory region of ROBO1, has a P-value of 2.15E-120 and an odds ratio of 5.18, suggesting that the common causal mutation(s) of craniofacial microsomia may be harbored in this locus. In this proposal, firstly, we will detect the causal mutation(s) by target-capture resequencing 40 patients and 8 controls. To investigate how a mutation frustrates the regulatory element and subsequently results in abnormal expression pattern of ROBO1, we will furtherperform chromosome conformation capture sequencing on the candidate mutation in neural crest stem cells. Finally, we will construct gene editing mice to reveal the genotype-phenotype relationship between the mutation and craniofacial development using CRISPR-Cas9 technology. Our study will establish the causal pattern of a mutation within the regulatory region of ROBO1 in the pathogenesis of craniofacial microsomia; and more importantly, provide genetic clues for diagnosis, therapy, and prevention of craniofacial microsomia.

半侧颜面短小畸形是第二大先天性颅面部发育畸形，严重影响患者身心健康。本项目研究团队前期对半侧颜面短小开展了首个全基因组关联研究，共找到了8个显著(P < 1E-8 )关联的染色体位点。其中的一个极显著关联位点位于ROBO1基因调控区，P值可达2.15E-120，比值比为5.18，这提示该关联位点内势必存在高频的可导致颅面部畸形的致病突变。本研究将采用目标区域捕获测序的手段对该调控区进行捕获测序以及突变位点探查，然后采用染色体构象捕获测序技术在细胞水平解析突变如何影响调控元件对ROBO1基因的调控，最后通过CRISPR-Cas9基因编辑技术构建突变体小鼠模型，探索突变和颅面部发育之间的关系。本研究可揭示存在于ROBO1基因调控区的致病突变导致半侧颜面短小畸形发病机制，为该病的诊断、治疗以及预防提供遗传学基础和病因学解释。

半侧颜面短小畸形是第二大先天性颅颌面部畸形，严重影响患者身心健康，但目前还未建立相关致病模型。本项目首先依据前期全基因组关联分析结果，选取连锁不平衡区间进行目标区域捕获测序；然后结合人类胚胎颌面部和神经嵴细胞的表观遗传学数据筛选致病突变位点；最后利用CRISPR/Cas9技术构建致病位点相关的神经嵴细胞模型，同时检测目标基因ROBO1的表达量。结果表明半侧颜面短小病人的稀有变异位点rs191424547位于一个神经嵴细胞特异的增强子内部。双荧光素酶实验表明此增强子在神经嵴细胞发育阶段具有调控活性，而其突变会显著降低增强子的调控活性。构建的细胞模型表明突变基因型中ROBO1的表达量显著升高。基于此，我们提出一个致病模型：rs191424547的突变会改变增强子的调控活性，进而改变ROBO1基因的表达量，从而导致神经嵴细胞的异常发育，最终诱使半侧颜面短小的发生。本项目的研究首次发现导致半侧颜面短小发生的调控区突变，初步揭示了其导致先天性畸形发生的致病机制，并提出致病模型，这对于该病的诊断以及预防提供了遗传学基础和病因学解释。