Interferon Regulatory Factor 6 Gene Research Articles

Sequencing of the interferon regulatory factor 6 (IRF6) gene and correlation to its phenotypes in familial non-syndromic cleft lip and palate in North Indian population

The incidence of orofacial clefting varies depending upon geographical location, ethnicity, sex and even socioeconomic status. The study aimed to test the contribution of interferon regulatory factor 6 (IRF6) gene allelic variation to the incidence of non-syndromic cleft lip and/or cleft palate (CL/P) from the population of North India. Patients from 12 North Indian families with non-syndromic cleft lip with or without cleft palate (NSCL/P) with at least two members affected were clinically evaluated and tested. DNA was extracted from the blood, and three exonic part of the IRF6 gene (exons 2, 5 and 7) were amplified by PCR. Direct sequencing was performed by PCR product, and sequencing results were compared with the reference DNA sequence of the IRF6 gene. Three SNPs (rs861019, rs2013162 and rs2235372) in 4 out of 12 families were observed. SNP rs861019 was detected in family no. 4 (F4) around exon 2, SNP 2013162 was detected in family no. 10 (F10) and SNP 2235372 was detected in family nos. 5 and 9 (F5 and F9). This study demonstrates that the three SNPs within the IRF6 gene are associated with NSCL/P, suggesting IRF6 gene sequence variability and variation in this gene, providing first genetic clue to the NSCL/P phenotype in North Indian families. This is our initial step to demonstrate the involvement of the IRF6 gene in NSCL/P patients in North India. Further extensive study and statistical analysis are needed to validate our result. Level of Evidence: Level IV, diagnostic study.

Association of IRF6 (G820A) Gene Variant with Nonsyndromic Cleft Lip and Palate in South Indian Population

Introduction: Cleft lip and palate (CLP) is a major congenital structural anomaly. Cleft lip and palate has significant complex etiology involving both genetic and environmental factors and lifelong morbidity. Mutations in various genes like TGFB3, RARA, IRF6 have been responsible for causing CLP in various populations.IRF6 gene provides instructions for making a protein interferon regulatory factor, which is active in cells that give rise to tissues in the craniofacial region. The mutations in IRF6 gene can lead to NSCLP, tooth agenesis, etc. Aims and objectives: This study was undertaken to evaluate the relationship and determine the degree of association of Interferon Regulatory Factor 6 (IRF6) (G820A) gene variant with nonsyndromic CLP (NSCLP) in South Indian population. IRF6 gene provides instructions for making a protein interferon regulatory factor, which is active in cells that give rise to tissues in the craniofacial region. The mutations in IRF6 gene can lead to NSCLP, tooth agenesis, etc. Materials and methods: The study group consisted of 50 samples who were patients at the department of orthodontics. The samples were divided into Group A with 25 cases having NSCLP and Group B with 25 controls. Two milliliter blood sample was collected from cases and controls each, for DNA isolation. Polymerase chain reaction (PCR) was carried for amplification followed by restriction fragment length polymorphism (RFLP) to assess the genotype of all individuals. z-test was applied for statistical analysis. Results: The study revealed significant differences in the frequency distributions of genotypes of IRF6 (G820A) variant when cases were compared with controls, with high GG genotype frequency in cases whereas GA and AA genotype of IRF6 (G820A) gene variant was found more in control samples. Conclusion: The results of this study further support the role of IRF6 (G820A) gene variants in NSCLP in South Indian population.

A Novel Heterozygous Mutation (F252Y) in Exon 7 of the IRF6 Gene is Associated with Oral Squamous Cell Carcinomas

Interferon regulatory factor 6 (IRF6) is a transcription factor with distinct and conserved DNA and protein binding domains. Mutations within the protein binding domain have been significantly observed in subjects with orofacial cleft relative to healthy controls. In addition, recent studies have identified loss of expression of IRF6 due to promoter hypermethylation in cutaneous squamous cell carcinomas. Since mutational events occurring within the conserved domains are likely to affect the function of a protein, we investigated whether regions within the IRF6 gene that encodes for the conserved protein binding domain carried mutations in oral squamous cell carcinoma (OSCC). Total chromosomal DNA extracted from 32 post surgical OSCC tissue samples were amplified using intronic primers flanking the exon 7 of IRF6 gene, which encodes for the major region of protein binding domain. The PCR amplicons from all the samples were subsequently resolved in a 1.2% agarose gel, purified and subjected to direct sequencing to screen for mutations. Sequencing analysis resulted in the identification of a mutation within exon 7 of IRF6 that occurred in heterozygous condition in 9% (3/32) of OSCC samples. The wild type codon TTC at position 252 coding for phenylalanine was found to be mutated to TAC that coded for tyrosine (F252Y). The present study identified for the first time a novel mutation within the conserved protein binding domain of IRF6 gene in tissue samples of subjects with OSCC.

Lack of association between IRF6 polymorphisms and nonsyndromic oral clefts in South Indian population

Objective: This present study is aimed to investigate the association between interferon regulatory factor 6 (IRF6), single nucleotide polymorphisms (SNPs), and nonsyndromic cleft lip without without cleft palate (NSCLP) in the South Indian population. Subject and Methods: For this study, 190 unrelated NSCLP patients and 189 controls without clefts were genotyped with rs2235371 (V2741) and rs642961 SNPs using PCR-RFLP. The associations between NSCLP groups and IRF6 gene polymorphisms, as well as haplotypes, were analyzed using chi-squared test and 95% confidence interval (95%CI) of the odds ratios were calculated with the control groups as reference. Results: For controls, the minor allele frequencies of both variants, V2741 and rs642961, were 7.1% and 21.1%, respectively. Genotype data for both variants in control and cleft groups follow the Hardy Weinberg Equilibrium. Between cases with NSCLP and controls, the two SNPs showed no differences in frequencies of the genotypes or alleles. The pairwise linkage disequilibrium (LD) values (D’=1 and r2=0.027) between V2741 and rs642961 revealed that these two SNPs are not in strong LD. Haplotype G-T showed a significantly reduced risk for oral clefts (p<0.001) and haplotype A-T increased the risk for oral clefts (p=0.043). Gene-gene interaction showed that the higher risk group contains more GG-CC combination of cases that the controls, but this model was not significantly associated with cleft status (p=0.136) Conclusion: In conclusion, while IRF6 is strongly associated in other populations, this study demonstrated that variants in IRF6 may play a role in NSCLP in a South Indian population, but other genes are expected to play a role in this population as well.

SNPs and interaction analyses of IRF6, MSX1 and PAX9 genes in patients with non-syndromic cleft lip with or without palate

Non‑syndromic cleft lip with or without cleft palate (NSCL/P) is a common congenital deformity, often associated with missing or deformed teeth. The genes interferon regulatory factor 6 (IRF6), muscle segment homeobox 1 (MSX1) and paired box gene 9 (PAX9) are important for the development of the maxillofacial region and dentition. The aim of this study was to explore how genetic variations in IRF6, MSX1 and PAX9, as well as gene‑gene interactions, are associated with NSCL/P. We investigated 9 IRF6 tag single nucleotide polymorphisms (SNPs), 2 MSX1 tag SNPs and 8 PAX9 tag SNPs selected from HapMap data from the Chinese population. The SNPs were examined for associations with NSCL/P in 204 patients and 226 controls. The results demonstrated a significant association between NSCL/P and rs2073485, rs2235371, rs2236909 and rs861020 in the IRF6 gene, and haplotype analysis supported these findings. A marginally significant difference was observed in the NSCL/P group for rs17176643 in the PAX9 gene compared to the control group. The results of gene‑gene interaction analyses also indicated that the combination of rs2073485, rs2235371 or rs2236909 in IRF6 and rs17176643 in PAX9, increased the risk of NSCL/P. Thus, our study provided additional understanding of IRF6 variations in patients with NSCL/P and suggested that interactions between the IRF6 and PAX9 genes are potentially important for susceptibility to NSCL/P.

A combined targeted mutation analysis of IRF6 gene would be useful in the first screening of oral facial clefts

BackgroundInterferon Regulatory Factor 6 (IRF6) is a member of the IRF family of transcription factors. It has been suggested to be an important contributor to orofacial development since mutations of the IRF6 gene has been found in Van der Woude (VWS) and popliteal pterygium syndromes (PPS), two disorders that can present with isolated cleft lip and palate. The association between IRF6 gene and cleft lip and palate has also been independently replicated in many populations.MethodsWe screened a total of 155 Taiwanese patients with cleft lip with or without cleft palate (CL/P); 31 syndromic (including 19 VWS families), 44 non-syndromic families with at least two affected members, and 80 non-syndromic patients through a combined targeted, polymerase chain reaction (PCR)-based mutation analysis for the entire coding regions of IRF6 gene.ResultsWe found 11 mutations in 57.89% (11/19) of the VWS patients and no IRF6 mutation in 44 of the non-syndromic multiplex families and 80 non-syndromic oral cleft patients. In this IRF6 gene screening, five of these mutations (c.290 A>G, p.Tyr97Cys; c.360-375 16 bp deletion, p.Gln120HisfsX24; c.411_412 insA, p.Glu136fsX3; c.871 A>C, p.Thr291Pro; c.969 G>A, and p.Trp323X) have not been reported in the literature previously. Exon deletion was not detected in this series of IRF6 gene screening.ConclusionsOur results confirm the crucial role of IRF6 in the VWS patients and further work is needed to explore for its function in the non-syndromic oral cleft with vary clinical features.

De novo 2.3 Mb microdeletion of 1q32.2 involving the Van der Woude Syndrome locus

BackgroundVan der Woude syndrome is the most common among syndromes which include cleft lip and/or cleft palate as one of the presentations. It is usually caused by mutations in the interferon regulatory factor 6 (IRF6) gene.Case presentationWe previously reported on a patient with suspected deletion of the IRF6 gene. Using the Affymetrix Human SNP 6.0 Array, the interstitial deletion has been confirmed and found to be approximately 2.327–2.334 Mb within the 1q32.2 region. Although several known genes were deleted, the patient has no other phenotype apart from the orofacial presentations typical of VWS. The same deletion was not present in either parent and his two siblings were also phenotypically normal.ConclusionsOther than IRF6, the genes which are deleted in this patient appear to be insensitive to copy number and haploinsufficiency. We compared the deletion in this patient with another case which was also mapped by high resolution array but had additional phenotypic features.

Association between polymorphism of IRF6 rs2235371 locus and nonsyndromic cleft lip with or without cleft palate

To assess the association between polymorphism of interferon regulatory factor 6 (IRF6) gene rs2235371 locus and nonsyndromic cleft lip with or without cleft palate in Chinese population. Blood samples from 106 patients and their parents and 129 controls and their parents were collected. The polymorphism of IRF6 rs2235371 locus was determined with PCR-restriction fragment length polymorphism (PCR-RFLP) method. Case-control analysis, transmission disequilibrium test(TDT), haplotype-based haplotype relative risk analysis (HHRR) and family-based association test (FBAT) were carried out. By case-control analysis, no significant difference was found in the frequencies of GG, GA and AA genotypes of rs2235371 locus between the patient group and control group (P> 0.05), but there was a significant difference in allelic frequencies (P< 0.05). There was also a significant difference in genotype and gene frequencies of rs2235371 variant between family members from cleft lip only group and control group. However, in cleft lip with cleft palate group, no such difference was observed. TDT analysis suggested a linkage in the presence of disequilibrium (chi-square=5.56, P=0.024). Results of HHRR analysis (chi-square=5.115, P=0.024) and FBAT (Z=2.218, P=0.027) also indicated an association between IRF6 rs2235371 variant and the risk of NSCL with or without cleft palate. Genetic polymorphism of IRF6 gene rs2235371 locus is associated with NSCL with or without cleft palate.

Developmental factor IRF6 exhibits tumor suppressor activity in squamous cell carcinomas.

The transcription factor interferon regulatory factor 6 (IRF6) regulates craniofacial development and epidermal proliferation. We recently showed that IRF6 is a component of a regulatory feedback loop that controls the proliferative potential of epidermal cells. IRF6 is transcriptionally activated by p63 and induces its proteasome-mediated down-regulation, thereby limiting keratinocyte proliferative potential. We hypothesized that IRF6 may also be involved in skin carcinogenesis. Hence, we analyzed IRF6 expression in a large series of squamous cell carcinomas (SCCs) and found a strong down-regulation of IRF6 that correlated with tumor invasive and differentiation status. IRF6 down-regulation in SCC cell lines and primary tumors correlates with methylation on a CpG dinucleotide island located in its promoter region. To identify the molecular mechanisms regulating IRF6 potential tumor suppressive activity, we performed a genome-wide analysis by combining ChIP sequencing for IRF6 binding sites and gene expression profiling in primary human keratinocytes after siRNA-mediated IRF6 depletion. We observed dysregulation of cell cycle-related genes and genes involved in differentiation, cell adhesion, and cell-cell contact. Many of these genes were direct IRF6 targets. We also performed in vitro invasion assays showing that IRF6 down-regulation promotes invasive behavior and that reintroduction of IRF6 into SCC cells strongly inhibits cell growth. These results indicate a function for IRF6 in suppression of tumorigenesis in stratified epithelia.

Three novel mutations of the IRF6 gene with one associated with an unusual feature in Van der Woude syndrome

Van der Woude syndrome (VWS) is a dominantly inherited disorder characterized by cleft lip with or without cleft palate and lip pits. It remains the most common syndromic form of oral clefts. Mutations in the interferon regulatory factor 6 (IRF6) gene have been identified in patients with VWS. We reported three unrelated families with lower lip anomalies. Two had lower lip pits, a cardinal sign of VWS, but the other had a heart-shaped mass on lower lip without pits, oral clefts, or hypodontia. This isolated anomaly has not been previously observed in VWS. We performed mutation analysis by PCR-sequencing the entire coding region of the IRF6 gene. Three potentially pathogenic mutations, c.145C>T (p.Q49X), c.171T>G (p.F57L), and 1306C>G (p.L436V) were successfully identified. All the missense mutations were not detected in 100 unaffected ethnic-matched control chromosomes and have never been previously reported. The p.Q49X and p.F57L mutations were located in the highly conserved DNA binding domain while the p.L436V was located at the carboxy-terminal region. This study reported an undescribed clinical feature of VWS and three novel mutations, expanding the phenotypic spectrum of VWS and mutational spectrum of IRF6.

Association of Single Nucleotide Polymorphisms in IRF6 and TGFA Genes With Nonsyndromic Cleft Lip With Or Without Cleft Palate in Chinese Patients

Objective Nonsyndromic cleft lip with or without cleft palate(NSCL/P) is a common birth defect with unclear etiology. Both genetic and environmental factors may contribute to NSCL/P. Many genes have been identified as candidate genes associated with this disease. Interferon regulatory factor 6( IRF6) gene and transforming growth factor-a( TGFA) gene seem to be crucial in the predisposition of NSCL/P. Here we evaluated some single nucleotide polymorphisms(SNPs) loci of TGFA and IRF6 genes in Chinese nuclear families consisting of fathers, mothers and affected offspring with NSCL/P. Methods Fifty patients of NSCL/P were confirmed by the plastic surgeons. They and their parents were included in the study, all with the informed consents. SNPs loci of TGFA and IRF6 genes were analyzed by microarray technology. Some PCR products were randomly chosen and sequenced to check microarray results. The distribution of gene type and allele frequency between patient group and parents group were compared. Then a Haplotype Relative Risk(HRR) and Transmission Disequilibrium Test(TDT) were performed. Results The sequences of randomly selected PCR products were all consistent with the microarray results. All loci were in Hardy-Weinberg equilibrium. There were no significant differences in the distribution of genotypes and alleles between patients and their parents. Using HRR and TDT analyses the V274I of IRF6 was associated with NSCL/P, while another SNP locus of IRF6 was not. Strong evidence of linkage disequilibrium was found between the 2 SNP loci of TGFA and disease with the HRR analysis, but not with the TDT analysis. Conclusion Our study confirms the contribution of IRF6 in the etiology of NSCL/P in populations of Asian ancestry. The association of TGFA with NSCL/P requires further research.

Identification of IRF6 gene variants in three families with Van der Woude syndrome

Van der Woude syndrome is the most common cause of syndromic orofacial clefting. It is characterised by the presence of lip pits, cleft lip and/or cleft palate. It is transmitted in an autosomal dominant manner, with high penetrance and variable expressivity. Several mutations in the interferon regulatory factor 6 (IRF6) gene have been found in VWS families, suggesting that this gene is the primary locus. We screened for mutations in this gene in three families in our population. There was a recurrent nonsense mutation within exon 9 of the gene for a Malay family consisting of five affected members with different presentations. We also found a co-segregating rare polymorphism which would result in a non-synonymous change 23 bases downstream of the nonsense mutation. This polymorphism was present in <1% of the Malay subjects screened, but was not found among the Chinese and Indians in our population. For another family, a 396C-->T mutation (R45W in the DNA-binding domain) was found in the proband, although the possibility of a genetic defect elsewhere could not be excluded because his mother and twin sister (both unaffected) also had this variant. In the third case with complete absence of family history, a de novo deletion spanning the whole IRF6 gene was detected in the child with VWS. This case of haploinsufficiency caused disruption of orofacial development but not other organ systems as the child has no other medical or developmental abnormalities despite the deletion of at least five other genes.

Familial non-syndromic cleft lip and palate--analysis of the IRF6 gene and clinical phenotypes

The aim of this study was to characterize Swedish families with non-syndromic cleft lip and/or palate (NSCL/P) for mutations or other sequence variants in the interferon regulatory factor 6 (IRF6) gene, as well as to describe their cleft phenotypes and hypodontia. Seventeen Swedish families with at least two family members with NSCL/P were identified and clinically evaluated. Extracted DNA from blood samples was used for IRF6 mutation screening. Exonic fragments of the IRF6 gene were sequenced and chromatograms were inspected. Statistical analysis was undertaken with marker- and haplotype association tests. No disease-associated IRF6 mutation could be determined in the families analyzed. One new and seven known single nucleotide polymorphisms (SNPs) were detected. The A allele of SNP rs861019 in exon 2 and the G allele of SNP rs7552506 in intron 3 showed association with cleft lip and palate (CLP; odds ratios of 3.1 and 5.45, respectively). Hypodontia was observed more commonly in individuals affected with CL/P as compared with family members without a cleft (P < 0.01). The hypodontia most often affected the cleft area, possibly representing a secondary effect. The distribution of cleft phenotypes in 15 of the 17 families with NSCL/P differed from the mixed cleft types seen in Van der Woude syndrome (VWS), in that CLP did not occur together with an isolated cleft palate within the same family. It was concluded that mutations of the IRF6 gene are not a common cause for cleft predisposition in Swedish NSCL/P families.

A novel IRF6 nonsense mutation (Y67X) in a German family with Van der Woude syndrome

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 in exons 2-9 have been found in VWS patients, suggesting that this gene plays an important role in orofacial development. We report a novel mutation of the IRF6 gene in a German family. Five out of the 12 persons affected were able to be investigated. The mutation produced a stop codon within exon 4 of the IRF6 gene. All 5 patients 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. The premature stop codon was responsible for a truncated protein lacking parts of the DNA- binding domain and the complete Smad-interferon regulatory factor-binding domain probably essential for interactions with the Smad transcription factors.

Interferon regulatory factor-6: a gene predisposing to isolated cleft lip with or without cleft palate in the Belgian population

Cleft lip with or without cleft palate is the most frequent craniofacial malformation in humans ( approximately 1/700). Its etiology is multifactorial; some are a result of a genetic mutation, while others may be due to environmental factors, with genetic predisposition playing an important role. The prevalence varies widely between populations and the mode of inheritance remains controversial. The interferon regulatory factor-6 (IRF6) gene has been shown to harbor mutations in patients with van der Woude syndrome, a dominant form of clefts associated with small pits of the lower lip. Moreover IRF6 has been associated with nonsyndromic cleft of the palate (CL/P) in two separate studies. We investigated the role of IRF6 in a set of 195 trios from Belgium. Cleft occurred as an isolated feature. We studied association of the IRF6 locus using two variants: one in the IRF6 gene and the other 100 kpb 3' of the gene. Our independent study group confirms that the IRF6 locus is associated with nonsyndromic cleft lip with or without palate. This result, with previous studies performed in the United States and Italy, shows for the first time the implication of IRF6 in isolated CL/P in northern Europe. It is likely that association to this locus can be identified in various populations and that the IRF6 locus thus represents an important genetic modifier for this multifactorial malformation.

Genomic, cDNA and embryonic expression analysis of zebrafish IRF6, the gene mutated in the human oral clefting disorders Van der Woude and popliteal pterygium syndromes

Van der Woude syndrome (VWS) and popliteal pterygium syndrome (PPS) are autosomal dominant clefting disorders recently discovered to be caused by mutations in the IRF6 ( Interferon Regulatory Factor 6) gene. The IRF gene family consists of nine members encoding 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-α and -β after viral infection, but the function of IRF6 remains unknown. We have isolated a full-length zebrafish irf6 cDNA, which encodes a 492 amino acid protein that contains a Smad-IRF interaction motif and a DNA-binding domain. The zebrafish irf6 gene consists of eight exons and maps to linkage group 22 closest to marker unp1375. By in situ hybridization analysis of embryo whole-mounts and cryosections, we demonstrate that irf6 is first expressed as a maternal transcript. During gastrulation, irf6 expression was concentrated in the forerunner cells. From the bud stage to the 3-somite stage, irf6 expression was observed in the Kupffer's vesicle. No expression could be detected at the 6-somite and 10-somite stages. At the 14-somite stage, expression was detected in the otic placode. At the 17-somite stage, strong expression was also observed in the cloaca. During the pharyngula, hatch and larva periods up to 5 days post-fertilization, irf6 was expressed in the pharyngeal arches, olfactory and otic placodes, and in the epithelial cells of endoderm derived tissues. The latter tissues include the mouth, pharynx, esophagus, endodermal lining of swim bladder, liver, exocrine pancreas, and associated ducts. Overall, the zebrafish expression data are consistent with the observations of lip pits in VWS patients, as well as more recent reports of alae nasi, otitis media and sensorineural hearing loss documented in some patients.

A novel mutation of the IRF6 gene in an Italian family with Van der Woude syndrome

Van der Woude syndrome (VWS) is the most common type of syndromic orofacial cleft, being characterised by variable association of lower lip pits, cleft lip and cleft palate. VWS is transmitted in an autosomal dominant manner, with high penetrance and variable expressivity, and a gene for this disease has been mapped in 1q32-q41. Very recently, mutations of the interferon regulatory factor 6 (IRF6) gene have been found in VWS patients, suggesting that this gene plays an important role in the orofacial development. We report a novel mutation of the IRF6 in an Italian family with six members affected by VWS with different expression. This mutation, the W217X, produces a stop codon within exon 6 of the IRF6 gene, with loss of the SMIR domain of the IRF6 protein.

Novel mutations in the IRF6 gene for Van der Woude syndrome.

Van der Woude syndrome (VWS, OMIM 119300) is an autosomal dominant craniofacial disorder characterized by pits of the lower lip, hypodontia, and cleft lip and/or cleft palate. It is the most common form of syndromic orofacial clefting and has very high penetrance with varied expressivity. The disease locus for VWS has been mapped to a 1.6-cM region on 1q32-41 between D1S205 and D1S491. Recently, mutations have been found in the interferon regulatory factor 6 ( IRF6) gene in patients with VWS and popliteal pterygium syndrome. To identify novel mutations of IRF6 in VWS patients, we screened four Chinese VWS families in all nine exons and their flanking splice junctions by direct sequencing. We identified three missense mutations and one nonsense mutation in IRF6. Our study further confirmed that IRF6 is essential for craniofacial development.

Novel mutations in the IRF6 gene in Brazilian families with Van der Woude syndrome

Van der Woude Syndrome (VWS) is an autosomal craniofacial disorder characterized by lower lip pits and cleft lip and/or palate. Mutations in the interferon regulatory factor 6 (IRF6) gene have been identified in patients with VWS. To identify novel IRF6 mutations in patients affected by VWS, we screened 2 Brazilian families, sequencing the entire IRF6-coding region and flanking intronic boundaries. Two novel heterozygous mutations were identified: a frame shift mutation with deletion of G at the nucleotide position 520 in the exon 6 (520delG), and a missense single nucleotide substitution from T to A at nucleotide position 1135 in exon 8 (T1135A). By using restriction enzyme analysis, we were able to demonstrate the lack of similar mutations in unrelated healthy individuals and non-syndromic cleft lip and palate patients. Our results further confirmed that haploinsufficiency of the IRF6 gene results in VWS.