gms | German Medical Science

Van der Woude syndrome (VWS) is the most common type of syndromic orofacial cleft, which accounts for approximately 2% of all cleft lip and palate cases. It is characterised by variable association of lower lip pits, cleft lip and cleft palate, and hypodontia. VWS arises as the result of mutations in the gene encoding interferon regulatory factor 6 (IRF6). The disorder is transmitted in an autosomal dominant manner, with high penetrance and variable expressivity. Very recently, mutations of the (IRF6) gene on exons 2-9 have been found in VWS patients, suggesting that this gene plays an important role in the orofacial development [Ref. 1], [Ref. 4].

A German family with five first-degree relatives affected with CLP and one simplex case were investigated. After informed consent, a sample of buccal cells was obtained from all these five family members. DNA was extracted using the Puregene DNA Isolation Kit (Gentra Systems) according to the manufacturer's instructions. Exons 2-9, containing the coding region of IRF6, were amplified with primers already published before [Ref. 5]. The sequences were compared with GenBank No. NT_021877 using the CEQ 8000 software (Beckman Coulter).

A nine month old baby girl with unilateral cleft lip and palate and bilateral lower lip fistulas was referred for phoniatric assessment. Her mother also had VWS with similar fistulas, but with the clefts confined to the right side. Within the family, a sister of the mother had isolated lower lip pit, and a brother and a nephew bilateral lip and palate clefts with lower lip pits. The simplex case was a 2 year old boy with VWS (bilateral CLP and bilateral lower lip pits).

We sequenced exons 2-9 including all coding exons of the IRF6 gene as well as the flanking intronic regions in the simplex case and one member of the family. This approach led to the identification of one primarily non-described missense mutation, one nonsense mutation and two single nucleotide polymorphisms (SNP). In the sporadic case a heterozygous missense mutation in exon 4 could be identified. This mutation c.251G>A (p.R84H) has already been described [Ref. 6]. Furthermore, this patient carried a c.459G>T transition at codon 153 in exon 5 not leading to an amino acid change. This sequence variant has also been reported before (refSNP ID: rs2013162). All 5 patients with the familial context were heterozygous for a base substitution c.201C>A changing the Tyrosine codon at amino acid position 67 into a stop codon (p.Y67X) in exon 4. One of our patients also carried the c.459G>T transition in a homozygous state and moreover, we found a homozygous change from C to G in intron 6 (refSNP ID: rs11811534). In all other affected members of the family we only analyzed exon 4. We found that all of them carried the p.Y67X disease causing mutation in a heterozygous state.

We described a sporadic case and a new family with autosomal dominantly inherited VWS. The sporadic patient carried the p.R84H mutation in exon 4 of the IRF6 gene, leading to an amino acid change from Arginine to Histidine at amino acid position 84. This mutation was formerly described to cause popliteal pterygium syndrome (PPS) [Ref. 7]. Due to the fact that this is the first case of VWS in the family, de novo mutation has to be assumed. We also found a novel mutation in the IRF6 gene. All of the available affected members of the family carried the p.Y67X mutation in exon 4. This mutation changes a Tyrosine codon into a stop codon at amino acid position 67. The premature stop codon is responsible for a truncated protein lacking parts of the well conserved DNA binding domain and the complete SMIR domain probably essential for interactions with the Smad transcription factors. The mutation produced different phenotypes within our family. This is a strong hint for at least one modifying gene [Ref. 7].

We can conclude that the present data confirm the association between IRF6 and VWS proving the pathogenetic role of IRF6 mutations in this syndrome and the important role of the IRF6 gene in the orofacial development. We suggest that molecular analysis of VWS and PPS patients should be performed on the complete coding region of the IRF6 gene by direct sequencing because mutations can be distributed all over the whole gene.