Our strategy allowed us to identify four novel LOF variants in three genes previously associated with NTD (MTHFR, DLC1, and ITGB1) and notably a significant mutation burden in a novel gene MYO1E. A replication MIP resequencing study in a larger NTD cohort identified an enrichment in this novel gene that approached statistical significance. This evidence concerns the gene DLC1 and neural tube defect.