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.
PROGRAM NARRATIVE Neural tube defects (NTDs) are among the most common of all human structural malformations. They arise when the neural tube fails to close during early embryo development. Up to 70% NTDs in human populations are thought to be due to genetic factors, with intrauterine environmental factors tipping the balance toward NTD in at risk individuals. NTDs stand out as a preventable birth defect, as periconceptional use of folic acid (FA) prevents a significant percentage of the population burden of NTDs. The strength of this Program is in its translation of mechanistic hypotheses generated from human genetic studies, interpreted in light of mouse mutant data and tested in human stem cell-derived and ?humanized? mouse models of NTD-associated genetic polymorphisms and maternal-fetal conditions in the intrauterine environment. These studies are the necessary antecedent of more effective, and individually tailored therapies to ensure healthy birth outcomes.
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