Mutations in IRF6 cause Van der Woude and popliteal pterygium syndromes

Abstract

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.

Fig. 1

Mutations in IRF6 cause VWS and PPS. a, Family number and mutation found for two VWS pedigrees and one PPS pedigree. The gender of each individual was randomly assigned to preserve the anonymity of the pedigrees; the actual pedigrees are available on request. Unaffected individuals (open), probands (arrow) and individuals with VWS (blue) or PPS (red) are indicated. Symbols representing specific phenotypes are shown below the pedigree for family VWS25. The sequence chromatogram derived from the affected proband is shown below the pedigrees for families VWS14 and PPS6. Above is an image of an agarose gel that shows the restriction-fragment length polymorphism (RFLP) assay used to confirm these mutations. Numbers on the side of each gel represent the size of the RFLP products. The mutation in family VWS14 abolishes an EcoRI restriction site, whereas the mutation in family PPS6 abolishes an HhaI site. Consequently, individuals with either mutation exhibit the large undigested DNA fragment in addition to two smaller digested products. Below the pedigree for VWS25 is an image of an agarose gel used to detect the 18-bp deletion mutation (132-bp fragment) or the wildtype allele (150-bp fragment). b, The structure of the IRF6 gene. Exons (rectangles) are drawn to scale except for exon 9, which is longer than shown. The brackets connecting the exons represent spliced introns, and the break between exons 9 and 10 represents an unspliced intron of 1,621 nt that is present in the most common 4.4-kb IRF6 transcript. The untranslated portions are in gray. The predicted IRF6 protein contains a winged-helix DNA-binding domain (yellow) and a SMIR/IAD protein-binding domain (green). The DNA-binding domain includes a pentatryptophan (w) motif. The arrowheads indicate the relative position of protein-truncation (above exons) and missense mutations (below exons) that cause VWS (blue) or PPS (red) or that are polymorphisms (green). The arrow above exon 4 represents the Glu92X nonsense mutation identified in the affected twin of family VWS14. The amino-acid change for each missense mutation is shown and an asterisk indicates mutations affecting residues that contact the DNA.

Fig. 2

Protein modeling of IRF6. The predicted IRF6 protein structure was aligned with the crystalline structure of the DNA-binding domain of IRF1. In the wildtype protein (green), the Arg84 residue (red) binds to the guanine (yellow) in the consensus sequence GAAA (blue), found in the IFN-β promoter, by means of three interactions. A bidentate hydrogen bond forms between two amine groups in the guanine base and the two amine groups in the basic side chain of the arginine, measuring a distance of 2.6 Å. An electrostatic 2.2-Å salt link also forms between the positively charged amine group of the arginine and the negatively charged 5′ phosphate group that precedes the guanine base. In the Arg84Cys mutant, the gap between the cysteine side chain and guanine base is greater than 3.10 Å, and is thus too great to support a hydrogen bond. Cysteine cannot physically form hydrogen or electrostatic bonds with the DNA, and this results in a disrupted DNA–protein interaction. In the Arg84His mutant, the aromatic ring of the histidine side chain is predicted to be oriented perpendicular to the DNA groove. This position would reduce the flexibility of the protein, impeding its ability to hydrogen bond.

Fig. 3

Expression of mouse Irf6. a, RT–PCR analysis of mouse tissues. Irf6 is expressed throughout a range of embryonic and adult tissues, although at low levels in brain, heart and spleen. Greater Irf6 expression seems to occur in secondary palates dissected from day 14.5–15 mouse embryos and in adult skin. PCR reactions carried out for 25 (not shown), 30 (shown) and 35 (not shown) cycles yielded similar results. Control RT–PCR experiments were done using the ubiquitously expressed gene Tcof1 (ref. 28). b, Northern-blot analysis of total RNA derived from whole mouse embryos at the day indicated. The Irf6 probe detects a transcript of approximately 4 kb and a larger transcript (arrow) whose size could not be determined. The amount of total RNA loaded into each lane was verified by ethidium bromide staining of the 28S rRNA transcript. c–h, Whole-mount in situ hybridization of day 14.5 mouse embryos. High Irf6 expression is observed in the hair follicles (d, white arrow), palatal rugae (d, black arrow), medial edge of the secondary palate immediately before and during fusion (d, arrowhead), mandibular molar tooth germs (f, arrow), thyroglossal duct (f, arrowhead) and penis (h). c,e,g, Embryos from the same litter hybridized with the sense probe are presented for comparison.