RET基因新发突变和体细胞突变在先天性巨结肠的致病作用及机制研究

先天性巨结肠（HSCR）属于肠神经系统（ENS）发育异常疾病，遗传因素占主导地位。RET原癌基因功能异常是疾病发生的必要条件，但多数患者的病因和风险累积模式不清。我们的前期研究通过多样本高通量测序发现HSCR家系RET基因突变中新发突变和体细胞突变高达89%，为了明确其致病作用及机制，本项目拟利用高通量测序、快速基因型分析、基因表达定量、免疫组化染色等方法明确这两类突变在中国HSCR患儿中的发生频率和突变类别，阐明RET亚效等位基因的修饰作用和修饰强度，观察突变携带者肠组织中重要基因表达水平的变化情况和ENS的发育成熟度，同时辅以体外细胞功能实验、转基因斑马鱼模型分析突变对蛋白质功能和RET信号转导通路活性的干扰机制及其剂量依赖效应并在模式生物水平对突变干扰ENS发育进行进一步验证，为更好地解释本病临床表现多样、家族内部遗传规律不明确等现象提供新的线索和理论依据。

Hirschsprung disease (HSCR) is a congenital malformation of the enteric nervous system (ENS) standing as a model for genetic disorders with complex patterns of inheritance. The tyrosine kinase receptor RET is the major gene but clear heritability for most patients and familial cases are still missing. Our previous study detected a high frequency (8 out of 9, 89%) of de novo and somatic mutations in RET by the combination of targeted next-generation sequencing and amplicon-based deep sequencing on multiple DNA from the same person. With the aim of delineating the mechanism and consequences of these mutations, we here plan to explore the concrete frequency and type of the de novo and somatic mutations in RET in a larger Chinese HSCR cohort, analyze the contribution of hypomorphic allele (3 enhancer SNPs) to the disease susceptibility, assess expression level of the RET gene regulatory network-related genes and evaluate developmental maturity of the ENS in colons of the mutation carriers. In addition, we will also use in vitro cultured HEK293, Neuro-2a and SK-N-SH cells to test the effects of identified RET-coding variants on RET phosphorylation and activation via ERK. Finally, transgenic zebrafish model will be utilized to further demonstrate their role in inducing ENS defects.