胎儿中枢神经组织体细胞突变与神经管缺陷的关系研究

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.

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。

【背景】神经管缺陷(NTDs)是胚胎早期发育神经管闭合不全导致的一组严重先天畸形。平面细胞极化(PCP)信号通路基因突变与NTDs关系密切。然而既往研究仅限于基于种系遗传，且绝大多数报道的突变来源于胎儿血液或唾液DNA。研究表明，体细胞突变在神经系统疾病的发生中起重要作用。因此，本研究旨在探讨NTDs患儿的中枢神经系统病变处组织中体细胞突变与NTDs的关系。.【内容及方法】.(1)人群研究：对48例NTDs神经系统病变处和脐带组织进行PGM二代测序，筛选体细胞突变；用Sanger测序验证并纳入同一患儿不同胚层的正常器官组织测序，探讨体细胞突变在不同胚层的器官的分布。.(2)细胞实验：验证目标突变对细胞功能的影响。用免疫荧光染色法检测突变位点对蛋白定位的影响，用蛋白质印迹法检测目标蛋白的表达，用荧光素酶报告系统检测突变对WNT 信号通路影响，用活细胞成像技术检测突变对细胞迁移的影响。.(3)小鼠模型：用CRISPR/Cas9技术构建小鼠点突变模型。在胚胎发育第12.5天解剖小鼠，记录神经管缺陷表型。.【重要结果及关键数据】.第一部分：PCP通路基因CNVs与NTDs关联研究.(1) 人群研究：PGM测筛选到位于15个基因上共27个体细胞突变，10个经过了.Sanger验证，其在不同胚层的组织中分布不同。生物信息学分析后纳入FZD6 p.Gln88Glu，VANGL1 p.Arg374His，MED12 p.Arg1782Cys进行后续研究。.(2) 细胞实验：FZD6 p.Gln88Glu影响FZD6定位、与CELSR共定位到细胞-细胞接触点和FZD6对于非经典WNT信号通路的调控。VANGL1 p.Arg374His影响VANGL1和CELSR共定位到细胞-细胞接触点、上调VANGL1蛋白的表达以及导致细胞迁移速度上调。而MED12 p.Arg1782Cys导致MED12蛋白水平下降，升高WNT信号通路表达。.(3) 小鼠模型：Med12 p.Arg17842Cys突变小鼠均表现出现严重的露脑、脊柱裂和卷尾表型，表明该突变可导致小鼠NTDs表型。.【结论及意义】.PCP通路基因VANGL1、FZD6、MED12神经系统病变处的体细胞突变与人类NTD的发生有关，为NTDs的遗传机制提供了新的线索。