Genomic and dental investigations of individuals and families with non-syndromic cleft lip and/ or cleft palate, Van der Woude and popliteal pterygium syndromes

Abstract: Van der Woude syndrome (VWS) is the most common oral cleft syndrome, accounting for two percent of all cleft lip and/or cleft palate (CL/P) cases. The main characteristics of VWS are lip pits (LP), cleft lip (CL), cleft lip and palate (CLP), cleft palate (CP) and/or hypodontia (H). Popliteal pterygium syndrome (PPS) has the same orofacial characteristics as VWS, combined with systemic anomalies. In 2002, the gene responsible for VWS/ PPS was found to be the interferon regulatory factor 6 gene (IRF6), located on chromosome 1, regio q32.2 The gene encodes for a transcription factor containing both a DNA binding domain and a protein binding domain. Non-syndromic (NS) cleft lip with or without cleft palate (CL±P) occurs without associated malformations in any other organs in approximately 70% of the affected subjects. The inheritance pattern for NSCL/P is complex, with a probable co-segregation of several genes and environmental factors. The IRF6 gene has been proposed to be part of the cause of NSCL/P. The aim of this thesis was to investigate individuals and families, mostly of Swedish and Finnish origin, with NSCL/P, VWS and PPS, with regard to their phenotypes, including dental anomalies, to detect mutations of the IRF6 gene in the syndromic cases and to investigate whether the IRF6 gene is responsible for the cleft phenotype in the nonsyndromic individuals. In Study I, 129 individuals affected with NS unilateral (U) CL±P were analysed for dental characteristics. Malformed lateral incisors were common in NSUCL, while hypodontia was more common in the NSUCLP phenotype. In the total material, hypodontia was found in 29.5% inside and in 15.5% outside the region of the cleft. Most of the existing lateral incisors were positioned distal to the cleft in both the primary and the permanent dentition. Study II revealed IRF6 gene mutation in 59% of the 17 VWS/ PPS families studied using direct sequencing of all exons of the gene. In Study III, the IRF6 gene was investigated in 17 Swedish NSCL/P families, using direct sequencing of the gene, in one affected and one healthy individual of each family. We could not detect any mutation in the protein-coding region of the gene. However, two noncoding SNPs – rs861019, a non-coding polymorphism in exon 2, and rs7552506, located in intron 3 – showed an association with the NSCLP phenotype. In Study IV, we tested two SNPs of IRF6, rs642961 in the promoter and rs2235371 in exon 7 (Val274Ile), for association with our entire sample set of NSCL/P, VWS and PPS families (119 families). In all but the Finnish VWS/ PPS families, the “A” allele of rs642961 was identified as a risk allele; transmission to an affected child occurred in a large majority on the same chromosome as the detected IRF6 mutation. The SNP rs642961, located in the AP-2a binding site in the promoter of the IRF6 gene, has previously been shown to be associated with NSCL±P but our results do not support this. However, we did find a significant risk (p=0.013) for transmission of the G-C haplotype (rs642961-rs2235371) to affected individuals in the NSCP subgroup of Swedish families. Of the 16 VWS/ PPS families found to have a mutation in IRF6 (Studies II and IV), 31% had a de novo mutation, that is, a mutation occurring in the proband only and not in the healthy parents. To conclude, NSCL/P is a complex anomaly, where disturbed dental development is a frequent finding. NSCL/P is not dependent on a single gene, as in Mendelian inherited VWS. Dividing our material into sub-phenotypes resulted in rather small groups, but we did find a significant risk with a haplotype of IRF6 in the NSCP group, and also an association for two SNPs of the IRF6 gene with NSCLP. Our results on NSCL/P emphasize the need for additional evaluation of the IRF6 gene and other genes/modifiers, to further clarify their roles in the development of the NSCL/P phenotype.