Identification of functional non-coding variants associated with orofacial cleft

Oral facial cleft (OFC) is a multifactorial disorder that can present as a cleft lip with or without cleft palate (CL/P) or a cleft palate only. Genome wide association studies (GWAS) of isolated OFC have identified common single nucleotide polymorphisms (SNPs) at the 1q32/ IRF6 locus and many other loci where, like IRF6 , the presumed OFC-relevant gene is expressed in embryonic oral epithelium. To identify the functional subset of SNPs at eight such loci we conducted a massively parallel reporter assay in a cell line derived from fetal oral epithelium, revealing SNPs with allele-specific effects on enhancer activity. We filtered these against chromatin-mark evidence of enhancers in relevant cell types or tissues, and then tested a subset in traditional reporter assays, yielding six candidates for functional SNPs in five loci (1q32/ IRF6 , 3q28/ TP63 , 6p24.3/ TFAP2A , 20q12/ MAFB , and 9q22.33/ FOXE1 ). We further tested two SNPs near IRF6 and one near FOXE1 by engineering the genome of induced pluripotent stem cells, differentiating the cells into embryonic oral epithelium, and measuring expression of IRF6 or FOXE1 and binding of transcription factors; the results strongly supported their candidacy. Conditional analyses of a meta-analysis of GWAS suggest that the two functional SNPs near IRF6 account for the majority of risk for CL/P associated with variation at this locus. This study connects genetic variation associated with orofacial cleft to mechanisms of pathogenesis.