Nonsyndromic cleft lip with or without cleft palate has been associated with SNPs in the IRF6 gene. Now Jeff Murray and colleagues report the identification of a common variant in a previously unknown IRF6 enhancer that shows strong evidence of association with cleft lip only and disrupts the binding site of transcription factor AP-2α. Previously we have shown that nonsyndromic cleft lip with or without cleft palate (NSCL/P)1 is strongly associated with SNPs in IRF6 (interferon regulatory factor 6)2. Here, we use multispecies sequence comparisons to identify a common SNP (rs642961, G>A) in a newly identified IRF6 enhancer. The A allele is significantly overtransmitted (P = 1 × 10−11) in families with NSCL/P, in particular those with cleft lip but not cleft palate. Further, there is a dosage effect of the A allele, with a relative risk for cleft lip of 1.68 for the AG genotype and 2.40 for the AA genotype. EMSA and ChIP assays demonstrate that the risk allele disrupts the binding site of transcription factor AP-2α and expression analysis in the mouse localizes the enhancer activity to craniofacial and limb structures. Our findings place IRF6 and AP-2α in the same developmental pathway and identify a high-frequency variant in a regulatory element contributing substantially to a common, complex disorder.

Orofacial clefts are congenital structural anomalies of the lip and/or palate that affect ∼1/1000 live births. Their frequent occurrence as well as their extensive psychological, surgical, speech and dental involvement emphasize the importance of understanding the underlying causes. The etiology of orofacial clefts is complex, including multiple genetic and environmental factors. Rare forms, where they occur as one component of multiple congenital anomaly syndromes, have Mendelian or teratogenic origins; the non-syndromic forms of orofacial clefts are more common and are likely due to secondary gene—environment interactions. Recent advances in both molecular and quantitative approaches have begun to identify the genes responsible for the rare syndromic forms of cleft and have also identified both candidate genes and loci for the more common and complex non-syndromic variants. Animal models, in particular the mouse, have also contributed greatly to an understanding of these disorders. This review describes genes that are involved in orofacial clefts in humans and animal models and explores genetic approaches to identifying additional genes and gene—environment interactions that constitute the many factors of orofacial clefts.

Nonsyndromic cleft lip and palate (NS CLP) is a complex birth defect resulting from a combination of genetic and environmental factors. Several members of the FGF and FGFR families are expressed during craniofacial development and can rarely harbor mutations that result in human clefting syndromes. We hypothesized that disruptions in this pathway might also contribute to NS CLP. We sequenced the coding regions and performed association testing on 12 genes (FGFR1,FGFR2,FGFR3,FGF2,FGF3,FGF4,FGF7,FGF8,FGF9,FGF10,FGF18, andNUDT6) and used protein structure analyses to predict the function of amino acid variants. Seven likely disease-causing mutations were identified, including: one nonsense mutation (R609X) in FGFR1, ade novomissense mutation (D73H) in FGF8, and other missense variants in FGFR1, FGFR2, and FGFR3. Structural analysis of FGFR1, FGFR2, and FGF8 variants suggests that these mutations would impair the function of the proteins, albeit through different mechanisms. Genotyping of SNPs in the genes found associations between NS CLP and SNPs inFGF3,FGF7,FGF10,FGF18, andFGFR1. The data suggest that the FGF signaling pathway may contribute to as much as 3–5% of NS CLP and will be a consideration in the clinical management of CLP.