? Project I: Human genetics of meningomyelocele and risk mitigation by folic acid This project focuses on the characterization of genomic variation in human patients with Meningomyelocele (MM), the most common CNS birth defect, with heritability estimated at 70-75% 1,2, and a cumulative incidence of 3.72/10,000 live US births. MM is a debilitating structural birth defect, the most common form of NTD compatible with life, and with substantial associated morbidity and mortality. National folic acid (FA) supplementation has reduced incidence >3-fold, but there is little understanding of the mechanism of this Gene-Environment interaction (GXE). Here we propose to study the molecular basis of human MM through a world-wide recruitment of trios with narrowly defined inclusion/exclusion criteria, stratified by prenatal FA exposure. We hypothesize that de novo mutations (DNMs) make a critical contribution to the risk of MM, and that FA increases the mutational burden required for phenotypic expressivity. MM shares features with other severe childhood diseases that show strong DNM contributions such as congenital structural disorders and autism. Our preliminary data point to a strong DNM contribution to MM, but like autism, these DNM increase risk but likely act with other factors to determine risk. We propose to ascertain a total of 2000 carefully phenotyped MM trios, recruited worldwide, stratified based upon national dietary FA supplementation status at the time of conception (+FA:fortified vs -FA:nonfortified). Trios will undergo whole genome sequencing (WGS), then analyzed for de novo and inherited mutations as risk factors, compared with control trios. Results from Project I will be incorporated into workflow of Project II and III to model mutations, and results from Project II and III will be used to refine WGS analysis in Project I. Project I will rely on Core B to identify candidate FA-responsive genes from changes in epigenetic signatures, and on Core C for bioinformatic analysis. Project I has already: 1] Founded the Spina Bifida Sequencing Consortium and enrolled a cohort of >1500 MM trios using social media, and historic cohorts, stratified as +FA or -FA. 2] Extracted and QC?d DNA from >700 of these trios. 3] Competed successfully for NICHDs Gabriella Miller-Kids First program access for 1000 WGS samples. 4] Performed sequencing on 600 trios, as well as optimized algorithms to achieve uniform mutation calling. 5] Identified 12 MM candidate genes, including 3 recurrently mutated genes, and one recurrent copy number variant (CNV). 6] Found that +FA trios but not -FA trios demonstrate a striking accumulation of damaging DNMs compared with controls. We will test the model that de novo and inherited mutations interact with FA to determine risk. The application proposes to complete recruitment, identify de novo and inherited gene mutations in MM, correlate with maternal FA exposure, and uncover mechanisms of disease within a clinical context.
