Risk Genes and Environmental Interactions in Neural Tube Defects

NTDS中的危险基因与环境互作 神经管缺陷是多种基因和环境相互作用的结果。 曝光。在人类群体中，补充叶酸(FA)可以防止高达70%的NTD发生- -包括无脑儿和脊柱裂-机制尚不清楚(S)。然而，英足总并没有从中受益 至少三分之一的家系和最近的数据表明，在某些特定的遗传背景下，FA可能是 对发育中的胚胎有害的。显然，如果家庭的个人风险能够得到更好的服务 准确评估，包括确定FA代谢途径的哪一方面--或哪一方面 完全涉及另一条途径的补充--会给他们提供最大的好处，所以NTD 预防策略可根据个体遗传危险因素进行优化。 该计划旨在通过整合先进的人力资源来改进NTD风险评估和预防 基因组学与人类和小鼠的生物学范式，以确定关键的基因-环境相互作用。 项目1(Finnell&Gross的Ross Pi)已经积累了200个全基因组序列 病例和200个对照，并发现了罕见的无意义、移码和非编码变体 脊柱裂。在更新中，我们将使用一种强大的高通量方法，使用分子反转 探针(MIP)对2,000多例NTD病例的复制队列进行重新排序。尖端CRISPR-CAS9 人类胚胎干细胞和小鼠的依赖基因组编辑将探索已识别的变异对基因功能的影响 神经上皮细胞的极性、增殖和反应性氧化/亚硝化物种(RON)的产生。 项目2(Ross&Finnell的Gross Pi)将检验叶酸保护的主要作用这一假设 针对NTD的是抑制RON的产生。他们将使用一种新的非靶向稳定同位素 方法在NTD易感小鼠模型中追踪叶酸介导的1-C转运。此外，他们还将雇用 一种新的氧化还原体平台，用于定量易患NTD的小鼠体内氧化修饰的小分子。通过项目 1和3，他们将研究已发现的与NTD相关的人类变异对细胞氧化还原状态的影响，以及1- C贩运以及补充小分子可以在多大程度上调节这些活动。 项目3(Gross&Ross的Finnell Pi)将研究遗传变异和启动子之间的相互作用 在NT关闭期间，扰乱信号通路并造成细胞损伤。他们将测试人类的能力 NTD相关的一氧化氮合酶变异体NOS3，增加细胞中的ROS过氧化亚硝酸盐，这是由于 Ser633上NOS3的磷酸化。它将测试线粒体是否是Rons的主要来源 神经形成。项目1、2和3将共同帮助定义母体/胚胎遗传学、营养 使用广泛的人类基因组学、蛋白质组学/代谢组学，以及 依赖CRISPR-Cas9的hESCs、患者干细胞(IPSCs)和小鼠基因组编辑。

RISK GENES AND ENVIRONMENT INTERACTIONS IN NTDS Neural tube defects (NTDs) arise from a complex interplay of multiple genes and environmental exposures. In human populations, folic acid (FA) supplementation can prevent up to 70% of NTD occurrences- -including anencephaly and spina bifida—by as yet unknown mechanism(s). Nevertheless, FA fails to benefit at least a third of families and recent data suggest that in some specific genetic contexts, FA may be deleterious to the developing embryo. Clearly, families would be far better served if their individual risks could be accurately assessed, including identification of which aspect of the FA metabolic pathway--or which supplement involving another pathway entirely--would provide the most benefit to them, so that NTD prevention strategies could be optimized according to individual genetic risk factors. This program aims to improve NTD risk assessment and prevention by integrating advanced human genomics with biological paradigms in humans and mice for identifying key gene-environment interactions. Project 1 (Ross PI with Finnell & Gross) has accumulated 200 whole genome sequences (WGS) from cases and 200 controls and has identified rare nonsense, frameshift and non-coding variants associated with spina bifida. In the renewal, we will employ a powerful high throughput method using molecular inversion probes (MIPs) to resequence a replication cohort of over 2,000 NTD cases. Cutting edge CRISPR-Cas9 dependent genome editing in hESCs and mice will probe the functional impact of identified variants on neuroepithelial cell polarity, proliferation, and the generation of reactive oxidative/nitrosative species (RONS). Project 2 (Gross PI with Ross & Finnell) will test the hypothesis that a major role for folate protection against NTD is to suppress the generation of RONS. They will employ a novel untargeted stable isotope method to trace folate-mediated 1-C trafficking in NTD-susceptible mouse models. In addition, they will employ a novel redoxome platform to quantify oxidatively-modified small molecules in NTD prone mice. With Projects 1&3, they will examine the impact of identified NTD associated human variants on cellular redox status and 1- C trafficking and the extent to which supplementation with small molecules can modulate these actions. Project 3 (Finnell PI with Gross & Ross) will examine the interaction of genetic variants and RONS to disrupt signaling pathways and cause cell damage during NT closure. They will test the ability of a human NTD-associated variant in NO synthase, NOS3, to increase ROS peroxynitrite in cells due to the phosphorylation of NOS3 on Ser633. It will test whether mitochondria are a major source of RONS during neurulation. Together, Projects 1, 2, & 3 will help define interactions of maternal/embryonic genetics, nutritional status and 1-C metabolism with NTD risk, using extensive human genomics, proteomics/metabolomics, and CRISPR-Cas9-dependent genome editing in hESCs, patient stem cells (iPSCs) and mice.