Identification of functional non-coding variants associated with orofacial cleft

Oral facial cleft (OFC) comprises cleft lip with or without cleft palate (CL/P) or cleft palate only. Genome wide association studies (GWAS) of isolated OFC have identified common single nucleotide polymorphisms (SNPs) in many genomic loci where the presumed effector gene (for example, IRF6 in the 1q32 locus) is expressed in embryonic oral epithelium. To identify candidates for functional SNPs at eight such loci we conduct a massively parallel reporter assay in a fetal oral epithelial cell line, revealing SNPs with allele-specific effects on enhancer activity. We filter these SNPs against chromatin-mark evidence of enhancers and test a subset in traditional reporter assays, which support the candidacy of SNPs at loci containing FOXE1, IRF6, MAFB, TFAP2A, and TP63. For two SNPs near IRF6 and one near FOXE1, we engineer the genome of induced pluripotent stem cells, differentiate the cells into embryonic oral epithelium, and discover allele-specific effects on the levels of effector gene expression, and, in two cases, the binding affinity of transcription factors FOXE1 or ETS2. Conditional analyses of GWAS data suggest the two functional SNPs near IRF6 account for the majority of risk for CL/P at this locus. This study connects genetic variation associated with OFC to mechanisms of pathogenesis.