Risk Genes and Environmental Interactions in Neural Tube Defects

NTDS中的风险基因和环境相互作用 神经管缺陷（NTDs）是由多个基因和环境因素的复杂相互作用引起的。 暴露。在人群中，叶酸（FA）补充剂可以预防高达70%的NTD发生- - 包括无脑畸形和脊柱裂-其机制尚不清楚。然而，FA未能受益 至少三分之一的家庭和最近的数据表明，在某些特定的遗传背景下，FA可能是 对发育中的胚胎有害。显然，如果家庭的个人风险能够 准确评估，包括确定FA代谢途径的哪个方面-或 完全涉及另一种途径的补充剂-将为他们提供最大的益处， 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类知识， 基因组学与人类和小鼠的生物范例，以确定关键的基因-环境相互作用。 项目1（罗斯PI与Finnell & Gross）已经积累了200个全基因组序列（WGS）， 病例和200例对照，并确定了罕见的无义，移码和非编码变异与 脊柱裂在更新中，我们将采用一种强大的高通量方法， 探针（MIP）对超过2，000例NTD病例的复制队列进行重新测序。最前沿的CRISPR-Cas9 hESC和小鼠中的依赖性基因组编辑将探测所鉴定的变体的功能影响， 神经上皮细胞极性、增殖和反应性氧化/亚硝化物质（RONS）的产生。 项目2（罗斯和芬内尔的总PI）将检验叶酸保护的主要作用 抑制NTD的方法是抑制RONS的产生。他们将使用一种新的非靶向稳定同位素 方法追踪NTD易感小鼠模型中叶酸介导的1-C运输。此外，他们还将雇用 一种新的氧化还原酶体平台，用于定量NTD易感小鼠中氧化修饰的小分子。与项目 1&3，他们将研究确定的NTD相关的人类变异对细胞氧化还原状态和1- C运输和补充小分子可以调节这些作用的程度。 项目3（Finnell PI与Gross & Ross）将研究遗传变异和RONS的相互作用， 破坏信号通路并在NT关闭期间引起细胞损伤。他们将测试人类的能力 NO合酶NOS 3中的NTD相关变异体，增加细胞中的ROS过氧亚硝酸盐， N 0 S3在Ser 633上的磷酸化。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、营养和免疫学之间的相互作用。 利用广泛的人类基因组学、蛋白质组学/代谢组学， hESC、患者干细胞（iPSC）和小鼠中的CRISPR-Cas9依赖性基因组编辑。

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.