PROJECT 1: Gene Variants and their Interactions Defining Human NTD Risk Neural tube defects (NTDs), primarily spina bifida and anencephaly, arise from a complex interplay of multiple gene interactions and environmental exposures. After 30 years of clinical and basic research, the field remains unable to accurately predict the risk for an individual couple of having a child affected by NTD, how folic acid (FA) works to prevent NTDs, whether or what dose of FA is likely provide effective prevention for them, or whether there is another nutrient/supplement or intervention that would provide greater benefit. The recent confluence of advances in genomics and computational genetics, enhanced by information from genetic mouse models, capabilities of molecular biological and biochemical detection in embryonic systems, now provide outstanding opportunity to address this complex genetic disorder. In the initial funding period, Project 1 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. This has thus far generated over 100 candidate gene and transcription factor binding sites associated in both expected and novel molecular interaction pathways in humans that are enriched for rare sequence variants in NTD cases compared to non-malformed controls and public databases. Among the most striking findings are that pathways modulating or affected by oxidative stress are heavily represented among the rare variants in our NTD cohort. 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 and validate the most highly significant genes and non-coding, regulatory regions of the genome associated with NTD risk. Cutting edge CRISPR-Cas9 dependent genome editing in hESCs and mice will probe the functional impact of identified variants on neuroepithelial cell polarity, proliferation, differentiation, survival and the generation of reactive oxidative/nitrosative species (RONS). With Projects 2 and 3 we will explore the impact of these variants on oxidative stress in in vitro hESC models bearing patient variants, and the ability of compounds that manipulate RONS in these mutant cells to exacerbate or reverse aberrant cell morphology, differentiation and, in genome edited mouse models, promote successful neural tube closure (NTC). Building upon the results obtained in our original funding period, Project 1, in collaboration with Projects 2 and 3, rigorously applies next generation DNA sequencing, genome editing, animal modeling, protein chemistry, and transcriptomic approaches to illuminate the causes of human NTDs, with a goal to ultimately reduce their prevalence.
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