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Novel IRF6 Mutations in Chinese Patients with Van der Woude SyndromeDepartment of Oral & Maxillofacial Surgery, West China College of Stomatology, Sichuan University, Chengdu 610041, P.R. China Correspondence: * corresponding author, drtwd{at}sina.com
Van der Woude syndrome (VWS) (OMIM 119300) is a dominantly inherited, developmental disorder that is characterized by pits and/or sinuses of the lower lip and a cleft lip and/or cleft palate. Mutations in the interferon regulatory factor 6 gene (IRF6) have been recently identified in patients with VWS, with more than 60 mutations reported. However, the VWS phenotype, IRF6 mutation genotypes, and their interrelationships in Chinese VWS patients have not been studied. Here, we report 11 Chinese families with variable clinical phenotypes of VWS and identified mutations in all patients. Of the 11 mutations, 8 appeared to be novel: CC5.6GT, T342A, 566delA, C748T, C756A, C989A, C1209G, and 1316delT. Seven mutations caused a change or loss of the IRF6 domain. The marked phenotypic variation may be caused by the action of certain modifier genes on IRF6 function. Abbreviations: VWS, Van der Woude syndrome; IRF6, interferon regulatory factor 6; CL/P, cleft lip and/or cleft palate; DBD, DNA-binding domain; SMIR, Smad-interferon regulatory factor-binding domain; Kb, kilobase; PCR, polymerase chain-reaction.
Key Words: cleft lip/palate • lip pits • Van der Woude syndrome • IRF6 gene
Van der Woude syndrome (VWS) (OMIM 119300) is a dominantly inherited, developmental disorder that is characterized by pits and/or sinuses of the lower lip and a cleft lip and/or cleft palate (Van der Woude, 1954). It is the most common syndromic form of cleft lip or cleft palate, accounting for approximately 2% of cleft lip and/or cleft palate cases. VWS pedigrees suggest an autosomal-dominant inheritance pattern, yet mutations appear de novo in 30–50% of VWS cases (Conners, 2002). Localization of the gene locus responsible for VWS was assigned through chromosomal abnormalities involving 1q32–q41 (Bocian and Walker, 1987). Following linkage analysis, the VWS region was defined as a 4.1-cM area (Sander et al., 1995). To identify putative disease-causing mutations, investigators carried out direct sequence analyses of genes and their transcripts in a critical, 350-kilobase (kb) region (Schutte et al., 2000). Finally, a nonsense mutation in the interferon regulatory factor 6 gene (IRF6) was found in the affected twin of two monozygotic twins discordant for VWS, suggesting IRF6 as a candidate for VWS (Kondo et al., 2002). This hypothesis was confirmed in the same study by the detection of different IRF6 mutations in 45 unrelated VWS families. Since then, IRF6 mutations have been found in at least 20 cases of VWS from Thailand (Shotelersuk et al., 2003), China (Wang et al., 2003), Korea (Kim et al., 2003), Japan (Kayano et al., 2003; Matsuzawa et al., 2004), Italy (Gatta et al., 2004), Belgium (Ghassibe et al., 2004), and Turkey (Item et al., 2005). IRF6 belongs to a family of 9 transcription factors that share a highly conserved helix-turn-helix DNA-binding domain (DBD, amino acids 13–113) and a less-conserved, protein-binding domain called Smad-interferon regulatory factor-binding domain (SMIR, amino acids 226–394) (Eroshkin and Musheegian, 1999). The IRF6 protein presumably forms a dimer prior to binding DNA (Mamane et al., 1999). Of the 65 families examined, 59 different mutations and a 17-kb deletion in IRF6 were identified. Forty of the 59 mutations localized to regions encoding the DNA-binding domain and Smad-interferon regulatory factor-binding domain, both critical for IRF6 function. There were an additional 12 mutations, including frameshift and nonsense mutations, that caused protein domain loss. In previous reports on IRF6 gene mutations in VWS patients, the VWS phenotype, IRF6 mutation genotypes, and their interrelationships in Chinese VWS patients have not been studied. Here, we report on 11 Chinese families with variable clinical phenotypes of VWS. We screened these families to determine the prevalence of IRF6 as a disease-causing gene and analyzed the interrelationships between patient phenotype and mutation.
Study Members This study included members of 11 families of Chinese descent. The probands of six families were referred to the West China College of Stomatology at Sichuan University, and the others were referred to Nanyang Stomatology Hospital in the Henan Province for treatment of cleft lip and/or cleft palate. All probands were Han Chinese. Each family had one proband, each of whom had lower lip pits or had one or more relatives with lip pits. Pedigrees were constructed by clinical examination of available family members and by interviews. From one to nine affected individuals in each family were identified (Fig  .). We obtained orthopantomographic radiographs to verify tooth agenesis in patients with hypodontia. In addition to these families, 96 unrelated individuals with no family history of VWS and cleft lip and/or cleft palate were recruited as controls. We gained approval for all protocols from the Ethics Committee of Sichuan University.
Mutational Analysis of IRF6 Peripheral blood samples were collected for genomic DNA extraction by means of the QIAmp Blood Mini Kit (Qiagen, Cologne, Germany). We amplified exons 1–8 and their flanking splice junctions and part of exon 9 of the IRF6 gene from genomic DNA samples by polymerase chain-reaction (PCR). Primer sequences were obtained from Drs. Murray and Schutte (University of Iowa), except for the forward primer of exon 7 (5'-ATGGCCTTGACCTCCTCCAGAC-3'). After PCR, we directly sequenced all the probands’ products using an ABI-377 sequencer (Applied Biosystems, Dalian, China). Each PCR product was sequenced on both the sense and antisense strands. Sequencing results were compared with the IRF6 mRNA sequence (NM_006147). We confirmed detected mutations by repeating the sequencing analysis on a new PCR product and testing it in other patients, with unaffected individuals from the same family and 96 unrelated individuals as controls.
Clinical Examination A summary of the relevant phenotype of each patient from the 11 VWS families is shown in the pedigrees (Fig  .); in total, 32 VWS patients were identified. The male/female ratio was 1.46 (19/13); 29 (90.6%) affected individuals had lip pits, and 22 (68.75%) had a cleft lip and/or cleft palate. These results are, to some extent, different from data which reported that 80% of VWS patients had lip pits and 50% of VWS patients had a cleft lip and/or cleft palate (Rintala and Ranta, 1981). In VWS4, five patients had only cleft palate, but one had cleft lip and palate. In VWS10, one patient had only cleft palate, but three had cleft lip and palate. There were only three patients who had hypodontia outside the cleft region: II:7 in VWS4, I:1 and II:1 in VWS9. There were three patients with one lip pit each, and 26 patients with two lip pits each. In family VWS8, there were two cleft lip patients (individuals III:1 and III:5). III:5 was identified as a VWS patient with lip pits. III:1 was not a VWS patient and, according to his parents, lacked lip pits. Sequencing was not performed on this patient, because he died at eight months of age.
Mutational Analysis
All 11 mutations were located in or proximal to the critical regions of IRF6. The mutation Pro76Ser affected the DNA-binding domain, which is involved in DNA interactions. Three mutations (i.e., Phe252Leu, Arg250X, and Ser330X) were found in the Smad-interferon regulatory factor-binding domain that is critical for both homo- and hetero-dimerization of IRF6 (Au et al., 2001). Two nonsense mutations (i.e., Cys114X and Gln118X) and the Gly189fs frameshift mutation mapped to an area downstream of the DNA-binding domain and led to a truncated protein that lacked the Smad-interferon regulatory factor-binding domain. There was one missense mutation (i.e., Ala2Gly) that was localized upstream of the DNA-binding domain, one frameshift mutation (i.e., Ala439fs), and two nonsense mutations (i.e., Arg412X and Tyr403X) that mapped downstream of the Smad-interferon regulatory factor-binding domain, creating non-functional proteins. Mutational analysis showed complete segregation of IRF6 mutations with phenotype. These mutations were not observed in any unaffected family members or in the 96 controls.
In this study, we describe 11 Chinese families with VWS, all bearing mutations in IRF6. In five of the 11 families (i.e., VWS1, 2, 4, 5, and 10), these mutations affected from three to nine patients whose ages spanned multiple generations. Since the mutation in VWS1 was not found in either I:1 or I:2, it was likely to have appeared de novo. There was one patient in five of the VWS families (VWS6, 7, 8, 9, and 11) who exhibited de novo mutations. IRF6 mutations were found in approximately 75% of VWS cases in two large cohorts (de Lima, Murray, and Schutte, personal communication). In our study, mutations were revealed in all VWS patients. This finding may be due to our small sample size or to the fact that the families were phenotypically more homogeneous. In family VWS9, we found a missense CC5, 6GT mutation. The proband II:1 had a complete left cleft lip and palate, two lower lip pits, and was congenitally missing the left lower lateral incisor, canine, second premolar, and second molar. We did not find an IRF6 mutation in her father, who was congenitally missing the two lower incisors. However, he did not fit the inclusion criteria for a VWS patient. Furthermore, we did not find, in either this proband or her father, mutations of MSX1 and PAX9, responsible for most non-syndromic hypodontia and oligodontia (Klein et al., 2005). This observation may suggest that hypodontia in this family is due to some as-yet-unidentified factor. Thus, in our study, there was only one case of a VWS patient with hypodontia (II: 7 in family VWS4) that could be attributed to an IRF6 mutation. We found the ratio of hypodontia to be 3% in VWS patients, different from a previous report of 25% (Rintala and Ranta, 1981). Although VWS has been demonstrated to be caused by IRF6 mutations, the phenotypes in the same family varied. Of families with known mutations, we observed five families with multiple combinations of orofacial anomalies and two families with mixed clefting phenotypes (e.g., VWS4 and VWS10). These two families consisted of eight or nine patients. The marked phenotypic variation in large families strongly suggests that certain stochastic factors or modifier genes act on IRF6 function. If the VWS family is large enough, there will be mixed clefting phenotypes for different modifiers. In this context, we found the sequence variant, V274I, and other SNPs of IRF6. The V274I variant occurs at an absolutely conserved residue within the Smad-interferon regulatory factor-binding domain. Analysis of our data did not show different V274I distributions between the normal population and patients (p > 0.05). Furthermore, three genotypes were found in VWS patients with the same phenotype. The actions of either V274I or other SNPs on IRF6 function may be revealed with additional samples. Previous detection of deletions that encompass the VWS region had suggested that the VWS phenotype was caused by haploinsufficiency (Schutte et al., 1999). Haploinsufficiency was also underscored by the identification of several protein-truncating mutations in IRF6 that were common in VWS. In this study, we found protein-truncation mutations (e.g., nonsense and frameshift) in eight families. Our research further confirmed that mutations in IRF6 were pathogenic for VWS. Earlier observations supported a role for IRF6 in the transforming growth factor-β (TGF-β) signaling pathway. Additionally, MSX1 and IRF6 might be involved in a common genetic pathway (Kondo et al., 2002). Further work is needed to delineate and integrate the molecular pathways that underlie the morphogenesis of the lip and palate.
We thank the patients and their families for their participation in this study. We also thank Lisheng Sun and Pengcheng Chen from Nanyang Stomatology Hospital for sample collection. We appreciate the advice of Drs. Murray and Schutte from The University of Iowa, and that of Prof. Taylor B. Guo from Shanghai Second Medical University. This study was supported by the Outstanding Youth Teacher Foundation of the Education Ministry of China.
A supplemental appendix to this article is published electronically only at http://www.dentalresearch.org. Received for publication July 1, 2005. Revision received May 27, 2006. Accepted for publication June 7, 2006.
Journal of Dental Research, Vol. 85, No. 10,
937-940 (2006)
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