Missense mutations that cause Van der Woude syndrome and popliteal pterygium syndrome affect the DNA-binding and transcriptional activation functions of IRF6.

Cleft lip and cleft palate (CLP) are common disorders that occur either as part of a syndrome, where structures other than the lip and palate are affected, or in the absence of other anomalies. Van der Woude syndrome (VWS) and popliteal pterygium syndrome (PPS) are autosomal dominant disorders characterized by combinations of cleft lip, CLP, lip pits, skin-folds, syndactyly and oral adhesions which arise as the result of mutations in interferon regulatory factor 6 (IRF6). IRF6 belongs to a family of transcription factors that share a highly conserved N-terminal, DNA-binding domain and a less well-conserved protein-binding domain. To date, mutation analyses have suggested a broad genotype-phenotype correlation in which missense and nonsense mutations occurring throughout IRF6 may cause VWS; in contrast, PPS-causing mutations are highly associated with the DNA-binding domain, and appear to preferentially affect residues that are predicted to interact directly with the DNA. Nevertheless, this genotype-phenotype correlation is based on the analysis of structural models rather than on the investigation of the DNA-binding properties of IRF6. Moreover, the effects of mutations in the protein interaction domain have not been analysed. In the current investigation, we have determined the sequence to which IRF6 binds and used this sequence to analyse the effect of VWS- and PPS-associated mutations in the DNA-binding domain of IRF6. In addition, we have demonstrated that IRF6 functions as a co-operative transcriptional activator and that mutations in the protein interaction domain of IRF6 disrupt this activity.

Genomic strategy identifies a missense mutation in WD-repeat domain 65 (WDR65) in an individual with Van der Woude syndrome.

Genetic variation in the transcription factor interferon regulatory factor 6 (IRF6) causes and contributes risk for oral clefting disorders. We hypothesized that genes regulated by IRF6 are also involved in oral clefting disorders. We used five criteria to identify potential IRF6 target genes; differential gene expression in skin taken from wild-type and Irf6-deficient murine embryos, localization to the Van der Woude syndrome 2 (VWS2) locus at 1p36-1p32, overlapping expression with Irf6, presence of a conserved predicted-binding site in the promoter region, and a mutant murine phenotype that was similar to the Irf6 mutant mouse. Previously, we observed altered expression for 573 genes; 13 were located in the murine region syntenic to the VWS2 locus. Two of these genes, Wdr65 and Stratifin, met 4 of 5 criteria. Wdr65 was a novel gene that encoded a predicted protein of 1,250 amino acids with two WD domains. As potential targets for Irf6 regulation, we hypothesized that disease-causing mutations will be found in WDR65 and Stratifin in individuals with VWS or VWS-like syndromes. We identified a potentially etiologic missense mutation in WDR65 in a person with VWS who does not have an exonic mutation in IRF6. The expression and mutation data were consistent with the hypothesis that WDR65 was a novel gene involved in oral clefting.

Prevalence and nonrandom distribution of exonic mutations in interferon regulatory factor 6 in 307 families with Van der Woude syndrome and 37 families with popliteal pterygium syndrome.

PURPOSE: Interferon regulatory factor 6 encodes a member of the IRF family of transcription factors. Mutations in interferon regulatory factor 6 cause Van der Woude and popliteal pterygium syndrome, two related orofacial clefting disorders. Here, we compared and contrasted the frequency and distribution of exonic mutations in interferon regulatory factor 6 between two large geographically distinct collections of families with Van der Woude and between one collection of families with popliteal pterygium syndrome. METHODS: We performed direct sequence analysis of interferon regulatory factor 6 exons on samples from three collections, two with Van der Woude and one with popliteal pterygium syndrome. RESULTS: We identified mutations in interferon regulatory factor 6 exons in 68% of families in both Van der Woude collections and in 97% of families with popliteal pterygium syndrome. In sum, 106 novel disease-causing variants were found. The distribution of mutations in the interferon regulatory factor 6 exons in each collection was not random; exons 3, 4, 7, and 9 accounted for 80%. In the Van der Woude collections, the mutations were evenly divided between protein truncation and missense, whereas most mutations identified in the popliteal pterygium syndrome collection were missense. Further, the missense mutations associated with popliteal pterygium syndrome were localized significantly to exon 4, at residues that are predicted to bind directly to DNA. CONCLUSION: The nonrandom distribution of mutations in the interferon regulatory factor 6 exons suggests a two-tier approach for efficient mutation screens for interferon regulatory factor 6. The type and distribution of mutations are consistent with the hypothesis that Van der Woude is caused by haploinsufficiency of interferon regulatory factor 6. On the other hand, the distribution of popliteal pterygium syndrome-associated mutations suggests a different, though not mutually exclusive, effect on interferon regulatory factor 6 function.