Functional tests of non-coding DNA variants associated with risk for orofacial cleft

About This Grant

Orofacial cleft (OFC, primarily cleft lip and/or cleft palate) is a relatively common structural birth defect with environmental and genetic contributions to etiology. Genome wide association studies (GWAS) and linkage studies have identified many gene variants, most in non-coding DNA, that are associated with elevated risk for isolated OFC. However, our understanding of the pathogenic mechanisms underlying this disease remains poor because, one, only a fraction of the heritable risk lies is derived from common variants, two, we have yet to distinguish the non- coding variants that directly influence risk for OFC (i.e., causal or functional variants) from those that are merely in linkage disequilibrium with them, three, it has never been directly shown that a common variant can affect the gross phenotype of an embryo. In Aim 1 we will conduct statistical analyses to identify de novo non-coding mutations that are likely to be functional. In Aim 2 we propose to identify the OFC-associated SNPs that are functional by filtering them against enhancer marks, testing them for allele-specific effects in reporter assays in vitro, and finally by engineering them singly or in combination into induced pluriopotent stem cells, differentiating the cells in to embryonic oral epithelium, and assessing allele-specific effects on gene expression and transcription factor binding. In Aim 3, we will engineer the genome of mouse strains that are genetically predisposed to cleft lip, cleft plate, or both, to be homozygous for risk or non-risk alleles of proven functional SNPs that are conserved in mice and humans, expecting the risk allele to increase the penetrance or expressivity of the cleft phenotype. The expected outcome of the proposed experiments is identification of the mechanisms by which genetic risk variants cause a common birth defect.