Mutations in interferon regulatory factor 6 (IRF6) account for ∼70% of cases of Van der Woude syndrome (VWS), the most common syndromic form of cleft lip and palate. In 8 of 45 VWS-affected families lacking a mutation in IRF6, we found coding mutations in grainyhead-like 3 (GRHL3). According to a zebrafish-based assay, the disease-associated GRHL3 mutations abrogated periderm development and were consistent with a dominant-negative effect, in contrast to haploinsufficiency seen in most VWS cases caused by IRF6 mutations. In mouse, all embryos lacking Grhl3 exhibited abnormal oral periderm and 17% developed a cleft palate. Analysis of the oral phenotype of double heterozygote (Irf6(+/-);Grhl3(+/-)) murine embryos failed to detect epistasis between the two genes, suggesting that they function in separate but convergent pathways during palatogenesis. Taken together, our data demonstrated that mutations in two genes, IRF6 and GRHL3, can lead to nearly identical phenotypes of orofacial cleft. They supported the hypotheses that both genes are essential for the presence of a functional oral periderm and that failure of this process contributes to VWS.

Although genome-wide association studies (GWASs) for nonsyndromic orofacial clefts have identified multiple strongly associated regions, the causal variants are unknown. To address this, we selected 13 regions from GWASs and other studies, performed targeted sequencing in 1,409 Asian and European trios, and carried out a series of statistical and functional analyses. Within a cluster of strongly associated common variants near NOG, we found that one, rs227727, disrupts enhancer activity. We furthermore identified significant clusters of non-coding rare variants near NTN1 and NOG and found several rare coding variants likely to affect protein function, including four nonsense variants in ARHGAP29. We confirmed 48 de novo mutations and, based on best biological evidence available, chose two of these for functional assays. One mutation in PAX7 disrupted the DNA binding of the encoded transcription factor in an in vitro assay. The second, a non-coding mutation, disrupted the activity of a neural crest enhancer downstream of FGFR2 both in vitro and in vivo. This targeted sequencing study provides strong functional evidence implicating several specific variants as primary contributory risk alleles for nonsyndromic clefting in humans.

Interferon Regulatory Factor 6 (IRF6) is a transcription factor that, in mammals, is required for the differentiation of skin, breast epithelium, and oral epithelium. However, the transcriptional targets that mediate these effects are currently unknown. In zebrafish and frog embryos Irf6 is necessary for differentiation of the embryonic superficial epithelium, or periderm. Here we use microarrays to identify genes that are expressed in the zebrafish periderm and whose expression is inhibited by a dominant-negative variant of Irf6 (dnIrf6). These methods identify Grhl3, an ancient regulator of the epidermal permeability barrier, as acting downstream of Irf6. In human keratinocytes, IRF6 binds conserved elements near the GHRL3 promoter. We show that one of these elements has enhancer activity in human keratinocytes and zebrafish periderm, suggesting that Irf6 directly stimulates Grhl3 expression in these tissues. Simultaneous inhibition of grhl1 and grhl3 disrupts periderm differentiation in zebrafish, and, intriguingly, forced grhl3 expression restores periderm markers in both zebrafish injected with dnIrf6 and frog embryos depleted of Irf6. Finally, in Irf6 deficient mouse embryos, Grhl3 expression in the periderm and oral epithelium is virtually absent. These results indicate that Grhl3 is a key effector of Irf6 in periderm differentiation.

DNA variation in Interferon Regulatory Factor 6 (IRF6) causes Van der Woude syndrome (VWS), the most common syndromic form of cleft lip and palate (CLP). However, an etiologic variant in IRF6 has been found in only 70% of VWS families. To test whether DNA variants in regulatory elements cause VWS, we sequenced three conserved elements near IRF6 in 70 VWS families that lack an etiologic mutation within IRF6 exons. A rare mutation (350dupA) was found in a conserved IRF6 enhancer element (MCS9.7) in a Brazilian family. The 350dupA mutation abrogated the binding of p63 and E47 transcription factors to cis-overlapping motifs, and significantly disrupted enhancer activity in human cell cultures. Moreover, using a transgenic assay in mice, the 350dupA mutation disrupted the activation of MCS9.7 enhancer element and led to failure of lacZ expression in all head and neck pharyngeal arches. Interestingly, disruption of the p63 Motif1 and/or E47 binding sites by nucleotide substitution did not fully recapitulate the effect of the 350dupA mutation. Rather, we recognized that the 350dupA created a CAAAGT motif, a binding site for Lef1 protein. We showed that Lef1 binds to the mutated site and that overexpression of Lef1/β-Catenin chimeric protein repressed MCS9.7-350dupA enhancer activity. In conclusion, our data strongly suggest that 350dupA variant is an etiologic mutation in VWS patients and disrupts enhancer activity by a loss- and gain-of-function mechanism, and thus support the rationale for additional screening for regulatory mutations in patients with CLP.

No abstract