神经管畸形(NTDs)是一类高致死致残性先天缺陷，表现为胚胎发育早期神经管闭合障碍。其病因尚未完全清楚。研究显示遗传因素占全部病因的50%以上。但基于种系突变假设的遗传学研究至今一直未有突破性进展，只极少数基因突变被确认为与NTDs发生密切相关；而体细胞突变与NTDs的关系尚无报道。本课题组前期小样本研究提示，源于生殖细胞外的体细胞突变可能与NTDs发生有关。本项目采用两阶段病例对照研究，利用患者病变处中枢神经组织为实验材料，以脐带组织为参照，针对神经管发育的关键通路——细胞平面极化通路基因，运用深度测序技术，筛选与NTDs相关的体细胞突变位点，然后用另一组病例进行扩大样本量验证；对获得确认的突变位点，采用细胞定点诱变实验，观察变异对基因表达、亚细胞结构定位及蛋白质间相互作用的影响，同时分析病例对照神经组织mRNA及蛋白的表达差异；以期确证体细胞突变在NTDs发生中的作用并揭示其分子机制。

Neural tube defects (NTDs) are severe congenital malformations with high rates of disability and lethality that often lead to perinatal deaths. NTDs result from failure of the neural tube to close during early embryonic development. The etiology of NTDs remains poorly understood. Up to 50% of the variance in NTDs prevalence attributes to genetic factors. So far, little progress has been made in classical genetics based on germline variation and only a few gene variants are recognized as risk factors for NTDs. There are no reports on the somatic variation in central nervous tissue in relation to NTD development. Our preliminary study shows that somatic variation may be correlated to NTDs. In the proposed research project, we aim to examine the association of NTDs with somatic variation in planar cell polarity pathway genes, NTD key pathway genes, by performing deep DNA sequencing in NTD lesions and control tissues. The project will be conducted using case-control study design with two stages, mutation discovery and validation with expanded sample size. Then, functional analysis will be conducted by using in vitro site-specific mutagenesis study. The effects of somatic variation will be examined on gene expression, subcellular localization and interaction between protein-protein. In the meanwhile, the differential expression of mRNA and protein that a target gene encodes will be assayed in the neural tissues of cases and controls. The proposed research project will partly discover the molecular mechanism of somatic variation in PCP pathway contributing to the development of NTDs.