先天性巨结肠相关Hedgehog及Notch信号通路的分子遗传机制研究

Hischsprung's disease (HSCR) is a complex genetic disorder attributed to abnormality of enteric nervous system development. Up to date, more than 10 genes have been found to be involved in HSCR development. However, molecular and genetic mechanisms of HSCR are still an unsolved puzzle. According to recent studies, PTCH1 and DLL3 as key factors of Hedgehog and Notch signaling pathways may confer higher risk to HSCR. We have recently explored the potential causes of HSCR and have analyzed the tissue-specific expression of the candidate genes. Thus, we will further testify the genetic links between PTCH1 & DLL3 genes and HSCR by linkage disequilibrium analysis, and apply the generalized multifactor dimensionality reduction (GMDR) method to detect Gene-by-Gene interactions in HSCR. Positive cSNPs or UTR SNPs will be investigated through allele-specific expression (ASE) analysis, somatic cell fusion, and monochromosomal hybrid clones. As for positive intron SNPs, chromatin immunoprecipitation will be included to find the allele with transcriptional activity. To assess the effect of positive SNPs on gene expression and neuronal proliferation and differentiation, functional studies will be performed taking advantage of Luciferase Reporter and RNA interference. Collectively, we will introduce a bran-new strategy of genetic analysis to interrogate gene-by-gene interactions in HSCR and provide a fresh clue in clinical therapeutics of HSCR.

先天性巨结肠（HSCR）是以肠神经系统发育异常为特征的一类多基因复杂遗传疾病，至今其分子遗传机制仍是未解之谜。研究发现Hedgehog和Notch信号通路及其关键组分PTCH1及DLL3会增加HSCR的患病风险。本课题组前期已完成对HSCR病因学机制的初步探索以及对候选基因的组织表达特异性分析。以此为基础，本研究引入连锁不平衡分析以证实PTCH1及DLL3等候选基因与HSCR发生之间的相关性，采用GMDR方法探索HSCR的基因互作网络。进而对编码区及UTR区的阳性SNP位点进行等位基因特异性表达检测，并结合单倍体杂交克隆进行体内验证；对内含子区阳性SNP位点进行ChIP分析；借助RNAi等技术分析突变位点对基因的表达调控作用及其对神经细胞增殖和分化的影响。本研究以新的遗传分析策略揭示HSCR的基因互作网络，进一步阐明HSCR的分子遗传机制，从而为HSCR的临床治疗提供新的线索。

先天性巨结肠（Hischsprung’s disease, HSCR）是一类主要由遗传因素导致的功能性肠梗阻，属于小儿常见的消化道畸形，其发病率约为1/5000。近期研究表明PTCH1、GABRG2和NRG3的特定遗传变异与HSCR增加的患病风险相关。本课题研究目标是从中国汉族人群出发，分析PTCH1、GABRG2和NRG3基因的遗传变异对HSCR遗传易感性的影响。我们引入104例散发HSCR病例和151例正常对照，首次采用Sequenom MassArray平台对上述基因的遗传多态性位点（SNP）进行连锁不平衡分析。结果显示其中某些遗传标记物与先天性巨结肠显著相关（P < 0.05），同时一些单倍型在HSCR病例组中出现的频率显著高于对照组。此外，等位基因特异性表达（ASE）分析表明在HSCR病例组和正常对照组之间，PTCH1基因的ASE程度存在显著性差异，并且在HSCR病例中ASE的出现频率要显著高于正常对照。本研究第一次在中国汉族人群中证实PTCH1、GABRG2和NRG3基因的常见遗传多态性可能与先天性巨结肠改变的患病风险相关，另在PTCH1中证实的ASE失衡可为阐明HSCR发病机制相关的顺/反调控体系提供新的线索，从而可进一步为先天性巨结肠早期临床诊断提供潜在的分子标记物。