Interferon regulatory factor 6 (IRF6) belongs to a family of nine transcription factors that share a highly conserved helix-turn-helix DNA-binding domain and a less conserved protein-binding domain. Most IRFs regulate the expression of interferon-alpha and -beta after viral infection, but the function of IRF6 is unknown. The gene encoding IRF6 is located in the critical region for the Van der Woude syndrome (VWS; OMIM 119300) locus at chromosome 1q32-q41 (refs 2,3). The disorder is an autosomal dominant form of cleft lip and palate with lip pits, and is the most common syndromic form of cleft lip or palate. Popliteal pterygium syndrome (PPS; OMIM 119500) is a disorder with a similar orofacial phenotype that also includes skin and genital anomalies. Phenotypic overlap and linkage data suggest that these two disorders are allelic. We found a nonsense mutation in IRF6 in the affected twin of a pair of monozygotic twins who were discordant for VWS. Subsequently, we identified mutations in IRF6 in 45 additional unrelated families affected with VWS and distinct mutations in 13 families affected with PPS. Expression analyses showed high levels of Irf6 mRNA along the medial edge of the fusing palate, tooth buds, hair follicles, genitalia and skin. Our observations demonstrate that haploinsufficiency of IRF6 disrupts orofacial development and are consistent with dominant-negative mutations disturbing development of the skin and genitalia.

Case-parent trios were used in a genome wide association study of cleft lip with/without cleft palate (CL/P). SNPs near two genes not previously associated with CL/P [MAFB: most significant SNP rs13041247, with odds ratio per minor allele OR=0.704; 95%CI=0.635,0.778; p=2.05*10 −11 ; and ABCA4: most significant SNP rs560426, with OR=1.432; 95%CI=1.292,1.587; p=5.70*10 −12 ] and two previously identified regions (chr. 8q24 and IRF6) attained genome wide significance. Stratifying trios into European and Asian ancestry groups revealed differences in statistical significance, although estimated effect sizes were similar. Replication studies from several populations showed confirming evidence, with families of European ancestry giving stronger evidence for markers in 8q24 while Asian families showed stronger evidence for MAFB and ABCA4. Expression studies support a role for MAFB in palate development.Corresponding author: THB (tbeaty@jhsph.edu). NIH Public Access Author ManuscriptNat Genet. Author manuscript; available in PMC 2010 September 17. Published in final edited form as:Nat Genet. 2010 June ; 42(6): 525-529. doi:10.1038/ng.580. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptCleft lip with or without cleft palate (CL/P) is a common human birth defect with documented genetic and environmental risk factors 1 . While CL/P can occur in many Mendelian malformation syndromes, the isolated, non-syndromic form constitutes 70% of all cases2. Evidence for genetic control of CL/P is compelling: recurrence risks are 20-30 times greater than population prevalences3 , 4 and both twin and family studies 5 suggest a major role for genes, Mutations in IRF6 cause VanderWoude syndrome, the most common Mendelian syndrome including CL/P, and markers in IRF6 have repeatedly shown evidence of association with isolated, non-syndromic CL/P 6-9 . An allele disrupting an AP2 binding site near IRF6 showed particularly strong evidence among European CL families, although multiple risk alleles are likely 10 .Birnbaum et al. 11 conducted a case-control genome wide association study (GWAS) in Germany and found significant evidence of association with markers in 8q24.21, and a US case-control GWAS confirmed this region 12 , with rs987525 being the most significant marker in both studies. Here we present a GWAS using a case-parent trio design in a consortium drawing cases from Europe, the US, China, Taiwan, Singapore, Korea and the Philippines. This design has the advantage of being robust to confounding due to population stratification, which is important when cases from diverse populations are combined. ResultsBecause these case-parent trios came from different populations (Table 1), we conducted a principal components analysis (PCA) on all parents to document genetic variation in our consortium (Supplementary Figure 1). Approximately 50% of parents could be classified as Asian and 45% as European, with remaining parents being of African or "other" ancestry (including mixed). Transmission disequilibrium tests...

DNA-sequence variants associated with IRF6 are major contributors to cleft lip, with or without cleft palate. The contribution of variants in single genes to cleft lip or palate is an important consideration in genetic counseling.

Orofacial clefts (OFCs), which include non-syndromic cleft lip with or without cleft palate (CL/P), are among the most common birth defects in humans, affecting approximately 1 in 700 newborns. CL/P is phenotypically heterogeneous and has a complex etiology caused by genetic and environmental factors. Previous genome-wide association studies (GWASs) have identified at least 15 risk loci for CL/P. As these loci do not account for all of the genetic variance of CL/P, we hypothesized the existence of additional risk loci. We conducted a multiethnic GWAS in 6480 participants (823 unrelated cases, 1700 unrelated controls and 1319 case-parent trios) with European, Asian, African and Central and South American ancestry. Our GWAS revealed novel associations on 2p24 near FAM49A, a gene of unknown function (P = 4.22 × 10), and 19q13 near RHPN2, a gene involved in organizing the actin cytoskeleton (P = 4.17 × 10). Other regions reaching genome-wide significance were 1p36 (PAX7), 1p22 (ARHGAP29), 1q32 (IRF6), 8q24 and 17p13 (NTN1), all reported in previous GWASs. Stratification by ancestry group revealed a novel association with a region on 17q23 (P = 2.92 × 10) among individuals with European ancestry. This region included several promising candidates including TANC2, an oncogene required for development, and DCAF7, a scaffolding protein required for craniofacial development. In the Central and South American ancestry group, significant associations with loci previously identified in Asian or European ancestry groups reflected their admixed ancestry. In summary, we have identified novel CL/P risk loci and suggest new genes involved in craniofacial development, confirming the highly heterogeneous etiology of OFCs.

Nonsyndromic orofacial clefts (OFCs) are a heterogeneous group of common craniofacial birth defects with complex etiologies that include genetic and environmental risk factors. OFCs are commonly categorized as cleft lip with or without cleft palate (CL/P) and cleft palate alone (CP), which have historically been analyzed as distinct entities. Genes for both CL/P and CP have been identified via multiple genome-wide linkage and association studies (GWAS), however, altogether, known variants account for a minority of the estimated heritability in risk to these craniofacial birth defects. We performed genome-wide meta-analyses of CL/P, CP, and all OFCs across two large, multiethnic studies. We then performed population specific meta-analyses in sub-samples of Asian and European ancestry. In addition to observing associations with known variants, we identified a novel genome-wide significant association between SNPs located in an intronic TP63 enhancer and CL/P (p = 1.16 × 10−8). Several novel loci with compelling candidate genes approached genome-wide significance on 4q21.1 (SHROOM3), 12q13.13 (KRT18), and 8p21 (NRG1). In the analysis of all OFCs combined, SNPs near FOXE1 reached genome-wide significance (p = 1.33 × 10−9). Our results support the highly heterogeneous nature of OFCs and illustrate the utility of meta-analysis for discovering new genetic risk factors.