Two missense mutations in the IRF6 gene in two Japanese families with Van der Woude syndrome

Van der Woude syndrome (VWS) is a common autosomal dominant disorder with cleft lip and/or palate and lower lip pits. Its prevalence is estimated to be 1/33,600 in the Finnish Population, and 1/47,813 in the Japanese. We performed mutation analysis of the IRF6 gene by direct sequencing in 2 unrelated Japanese families that consist of a total of 3 affected members with cleft lip and palate associated with lower lip pits. Consequently, we found novel base substitutions, 25C>T, in IRF6-exon 3 in a boy, his mother, and his phenotypically normal maternal grandmother in one of the families. A known mutation, 250C>T, was identified in exon 4 of a girl and her unaffected father in the other family. The same mutations were never observed among 190 healthy Japanese. The results indicate incomplete penetrance and variable expressivity in the families. Because 25C>T and 250C>T predict to lead to R9W and R84C substitutions, respectively, at the most conserved DNA binding domain of IRF6, and because arginine at positions 9 and 84 is highly conserved among IRFs, the 2 mutations may lead to abolish the DNA binding activity in the developing craniofacial region. To our knowledge, this is the first report of IRF6 mutations observed in Japanese VWS patients.     

1个河南van Der Woude综合征家系IRF6基因的突变检测

目的:对1个河南省vanDerWoude综合征(VWS)家系进行IRF6基因的突变检测。方法:在IRF6(interferonregulatoryfactor6)基因内设计引物,经分段PCR(聚合酶链反应)和DNA测序,进行突变检测,运用PIX-ProteinIdentification软件对检测结果进行蛋白二级结构分析。结果:在该家系所有患者IRF6基因的第6密码子,均发现与表型一致的CGC>TGC(r.279c→t)突变,该突变引起IRF6蛋白二级预测结构的改变。结论:vanDerWoude综合征由IRF6基因突变引起,IRF6基因与唇、腭、牙的发育密切相关。     

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.     

一个范德伍德综合征家系的IRF6基因突变检测

目的:对收集的1个湖北Van der Woude综合征(VWS)家系进行临床和遗传特点分析,并进行IRF6基因的突变检测。方法:通过先证者及现场家系调查、临床检查和系谱分析收集VWS家系。在IRF6基因的外显子-内含子接头及9个外显子编码区分别设计引物,经聚合酶链式反应扩增并纯化后直接测序。结果:收集的VWS家系符合常染色体显性遗传特征,家系受累患者共3名(1名男性和2名女性),患者表现为典型的下唇瘘管或凹陷,且合并有唇腭裂和先天缺牙。患者表型在同一家系内有明显差异,且呈逐代加重趋势。在所有患者IRF6基因第412位密码子发现与表型一致的CGA>TGA(c.1234C>T)改变,经查证为一个已知的无义突变。结论:该VWS家系疾病表现度极不一致,是由IRF6基因的1个已知无义突变导致,IRF6是参与颌面部发育的重要基因。     

干扰素调节因子6基因致病的Van der Woude综合征的家系调查和遗传特点分析

目的 探讨中国人Van der Woude综合征（VWS）的临床表型及遗传学特点。方法 先证者法收集14个VWS家系并进行口腔专科检查、家系调查及基因突变分析,分析不同VWS家系个体或同一家系不同个体的临床表型,绘制家系图谱,明确遗传方式及致病基因,计算表型分布频率和表型基因频率。结果 VWS家系基本符合常染色体显性遗传特征,患者多数表现为典型的VWS,致病基因为干扰素调节因子6（IRF6）。VWS表型分布频率为：唇瘘91.9%,唇腭裂73.0%,牙畸形8.1%。不同家系个体和同一家系的不同个体临床表型存在明显差异。结论 收集的家系均为常染色体显性遗传,表现度变异大。中国人群VWS致病基因为IRF6,为Ⅰ型VWS。     

中国人群非综合征性唇腭裂患者IRF6基因突变检测

目的 探讨干扰素调节因子6(interferon regulatory factor 6, IRF6) 在非综合征性唇腭裂(non-sydromic cleft lip and/or cleft palate,NSCL/P)患者中的突变情况。方法：收集119例NSCL/P患者及288名健康人对照样本的外周血血样并提取DNA。在IRF6基因的全部外显子分别设计引物,PCR扩增其序列,通过测序找出IRF6基因突变,并将这些突变在对照样本中进行验证。结果：共发现5种在正常人中没有的突变,其中4种是新发现的突变。结论：IRF6基因突变在中国人群中参与了非综合征唇腭裂疾病的发生。     

福建范德伍兹综合征1例家系IRF6基因突变的检测

目的检测福建省一范德伍兹综合征(VWS)家系IRF6基因的突变。方法在IRF6(interferonregulatoryfactor6)基因内设计引物,经分段聚合酶链反应(PCR)和DNA测序进行突变检测,运用PIX-ProteinIdentification软件对检测结果进行蛋白二级结构分析。结果在所有患者IRF6基因的第379密码子发现TGG>TGA(r.1400g→a)的碱基变化,该突变引入终止码,引起IRF6蛋白转录提前终止。结论范德伍兹综合征由IRF6基因突变引起,IRF6基因与唇腭、牙齿发育密切相关。     

Van der Woude syndrome: dentofacial features and implications for clinical practice

Background:  Van der Woude syndrome (VWS) is the most common clefting syndrome in humans. It is characterized by the association of congenital lower lip fistulae with cleft lip and/or cleft palate. VWS individuals have a high prevalence of hypodontia. Although caused by a single gene mutation, VWS has variable phenotypic expression. This study aimed to describe the range of clinical presentations in 22 individuals with VWS to facilitate its diagnosis.
Methods:  A retrospective study of 22 patients with a diagnosis of VWS was undertaken at the Australian Craniofacial Unit (ACFU) in Adelaide. Three extended families with affected members were included in the study cohort.
Results:  The overall prevalence of lip pits in this study cohort was 86%. Cleft phenotypes included bilateral cleft lip and palate (32%); unilateral cleft lip and palate (32%); submucous cleft palate (23%); and isolated cleft hard and soft palate (9%). Missing permanent teeth were reported in 86% of affected individuals.
Conclusions:  Submucous cleft palate in VWS may go undiagnosed if the lower lip pits are not detected. Associated hypodontia and resultant malocclusions will also require management by a dental team.     

Clinical and genetic studies of Van der Woude syndrome in Sweden.

Van der Woude syndrome (VWS) is an autosomal dominant craniofacial disorder characterized by pits of the lower lip, hypodontia and cleft lip and/or cleft palate. It has been reported as the most common form of syndromic orofacial clefting with very high penetrance and varied expressivity. The disease locus for VWS has been mapped to chomosome 1q32, but the gene is yet to be cloned. Here we report a total of 11 Swedish VWS patients: 9 familial cases from two families and two isolated cases. Clinical examination of these patients showed phenotypic variability, even between patients from the same family. Genetic studies were performed using four microsatellite markers from chromosome 1q32. Constitutional deletion in this region was not demonstrated in any of the familial or isolated cases. However, in the two VWS families, linkage analysis using these markers showed positive LOD (logarithm of the odds) scores ranging from 2.56 to 2.88 to all individual markers. The highest LOD score of 3.75 was obtained with the combined haplotypes of D1S491 and D1S205, thus confirming linkage of VWS in these two families to 1q32. We conclude that there is varied expressivity but no evidence of genetic heterogeneity in VWS.     

Van der Woude综合征家系先天缺牙患者MSX1基因突变的检测

目的探讨MSX1基因与Van der Woude综合征(VWS)家系中缺牙的关系。方法从VWS家系9中伴发缺牙患者2人及家系正常成员2人、60个牙列完整的健康者共64人的静脉血中提取DNA,设计MSX1基因引物,采用PCR方法扩增MSX1基因外显子1、2的编码区,而后对外显子1、2的PCR纯化产物测序,进行序列比对分析。结果 VWS家系9两个缺牙患者MSX1基因中有ivs2+68 C>T多态;伴IRF6基因突变的VWS患者缺牙较多。结论 VWS家系9中先天缺牙患者的牙先天缺失与MSX1基因的ivs2+68 C>T多态可能相关。     

A novel missense mutation in Van der Woude syndrome: usefulness of fingernail DNA for genetic analysis

Van der Woude syndrome (VWS) is an autosomal-dominant oral facial disorder. To find a gene mutation in a Japanese family using fingernail DNA samples, we performed this study. We hypothesized that a gene mutation in IRF6 might be involved in VWS, and that fingernail DNA samples may be valuable for detecting such mutations. Linkage and haplotype analyses of the family mapped the disease locus to the 1q32-q41 region. Mutation analysis with an improved extraction method for fingernail DNA detected a novel missense mutation (1046A>T, E349V) in exon 7 of IRF6 in all the affected members of the family. Since the E349V change may disturb the hydrophobic core and affect regulatory activity of IRF6, it is most likely that the mutation is causative for VWS in this family. Fingernail DNA is thus useful for linkage and mutation analyses, since the fingernail can be easily obtained non-invasively, sent through the mail, and stored for a long period. We emphasize here the usefulness of fingernail DNA for the genetic analysis of a disease.     

Unraveling human cleft lip and palate research

The focus of this work is to highlight the most recent advances in the understanding of cleft lip and palate occurrence. Information regarding research on long-term outcomes, genes and their interactions with other genes, and gene-environment interactions is compiled to provide the reader with a critical and up-to-date overview on the current knowledge of the etiology of cleft lip and palate. Recent epidemiological evidence strongly suggests that individuals born with clefts have a shorter lifespan and may have a higher incidence of cancer and psychological disorders. IRF6 has been shown to be an important contributor to cleft lip and palate, but the functional variant leading to the defect has not yet been defined. Inactivation of MSX1 and genes in the FGF family has also been shown to lead to cleft lip and palate. In addition, missense mutations in several candidate genes may cause cleft lip and palate, but definitive evidence regarding the biological consequences of these mutations is yet to be unraveled. Maternal cigarette smoking increases the risk of a baby born with clefts, in particular when the mother carries the GSTT1-null variants. The latest approaches in cleft research include the analysis of several additional phenotypical features of the population, with the goal of increasing the statistical power of genetics studies.     

Alveolar bone grafting in the treatment of midline alveolar cleft and diastema in incomplete median cleft lip

Median cleft lip is a rare congenital anomaly. The wide diastema with mesial tipping observed in these patients has been largely overlooked. A midline submucosal alveolar cleft prevents adequate treatment. The purpose of this article is to describe an alveolar bone grafting (ABG) technique used in the combined surgical-orthodontic approach to diastema treatment in patients presenting with incomplete median cleft lip. Patients treated for incomplete median cleft lip and diastema were identified in the clinic registry from 1981 to 2007. Six patients were identified; 4 underwent ABG before permanent maxillary incisor eruption, the other 2 were seen later when they were 11 years old. All 6 ABGs were successful. The incisors erupted through the graft or were successfully moved into it with lasting results. Follow-up ranged from 8 to 21 years. The existence of a midline submucosal alveolar cleft and subsequent diastema should be recognized and addressed in all patients who present with incomplete median cleft lip repair. This includes taking maxillary occlusal view X-rays before the age of 5 years to detect the cleft, and proceed to ABG if necessary, generally before permanent maxillary incisor eruption.     

Congenital lower lip pits (Van der Woude syndrome): report of a case

Van der Woude syndrome (VWS) is a rare autosomal dominant disorder that is characterized by a cleft lip and palate with congenital lip pits. This is a report of a case of VWS with sinuses in the lower lip, a cleft in the upper lip, and a supernumerary tooth in the maxilla. The main characteristics of this disorder are discussed. Dental treatment of the patient was performed, but the surgical removal of the sinus was rejected by the parents. This case report brings this condition to the attention of dentists and surgeons and emphasizes lip pits may not always be identical in appearance.     

Haplotype‐based gene–gene interaction of bone morphogenetic protein 4 and interferon regulatory factor 6 in the etiology of non‐syndromic cleft lip with or without cleft palate in a Chilean population

Non‐syndromic cleft lip with or without cleft palate (NSCL/P) is the most common craniofacial birth defect in humans, the etiology of which can be dependent on the interactions of multiple genes. We previously reported haplotype associations for polymorphic variants of interferon regulatory factor 6 (IRF6), msh homeobox 1 (MSX1), bone morphogenetic protein 4 (BMP4), and transforming growth factor beta 3 (TGFB3) in Chile. Here, we analyzed the haplotype‐based gene–gene interaction for markers of these genes and NSCL/P risk in the Chilean population. We genotyped 15 single nucleoptide polymorphisms (SNPs) in 152 Chilean patients and 164 controls. Linkage disequilibrium (LD) blocks were determined using the Haploview software, and phase reconstruction was performed by the Phase program. Haplotype‐based interactions were evaluated using the multifactor dimensionality reduction (MDR) method. We detected two LD blocks composed of two SNPs from BMP4 (Block 1) and three SNPs from IRF6 (Block 2). Although MDR showed no statistical significance for the global interaction model involving these blocks, we found four combinations conferring a statistically significantly increased NSCL/P risk (Block 1–Block 2): T‐T/T‐G C‐G‐T/G‐A‐T; T‐T/T‐G C‐G‐C/C‐G‐C; T‐T/T‐G G‐A‐T/G‐A‐T; and T‐T/C‐G G‐A‐T/G‐A‐T. These findings may reflect the presence of a genomic region containing potential causal variants interacting in the etiology of NSCL/P and may contribute to disentangling the complex etiology of this birth defect.     

The complex genetics of cleft lip and palate

Clefts of the lip and palate are a common craniofacial anomaly, requiring complex multidisciplinary treatment and having lifelong implications for affected individuals. The aetiology of both cleft lip with or without cleft palate (CLP) and isolated cleft palate (CP) is thought to be multifactorial, with both genetic and environmental factors playing a role. In recent years, a number of significant breakthroughs have occurred with respect to the genetics of these conditions, in particular, characterization of the underlying gene defects associated with several important clefting syndromes. These include the identification of mutations in the interferon regulatory factor-6 (IRF6) gene as the cause of van der Woude syndrome and the poliovirus receptor related-1 (PVRL1) gene as being responsible for an autosomal recessive ectodermal dysplasia syndrome associated with clefting. While no specific disease-causing gene mutations have been identified in non-syndromic clefting, a number of candidate genes have been isolated through both linkage and association studies. However, it is clear that environmental factors also play a role and an important area of future research will be to unravel interactions that occur between candidate genes and environmental factors during early development of the embryo. Orthodontists are intimately involved in the therapeutic management of individuals affected by CLP and it is important that they keep abreast of current knowledge of the aetiology behind these conditions. This review aims to summarize some of the more significant advances in the genetics of CLP and highlight current thinking on the modes of inheritance and genetic loci that might be involved in this complex disorder.     

IRF6 gene variants in Central European patients with non‐syndromic cleft lip with or without cleft palate

Variants in the interferon regulatory factor 6 (IRF6) gene have repeatedly been associated with non‐syndromic cleft lip with or without cleft palate (NSCL/P). A recent study has suggested that the functionally relevant variant rs642961 is the underlying cause of the observed associations. We genotyped rs642961 in our Central European case–control sample of 460 NSCL/P patients and 952 controls. In order to investigate whether other IRF6 variants contribute independently to the etiology of NSCL/P, we also genotyped the non‐synonymous coding variant V274I (rs2235371) and five IRF6‐haplotype tagging single nucleotide polymorphisms (SNPs). A highly significant result was observed for rs642961 (P = 1.44 × 10^?6) in our sample. The odds ratio was 1.75 [95% confidence interval (CI): 1.38–2.22] for the heterozygous genotype and 1.94 (95% CI: 1.21–3.10) for the homozygous genotype, values that are similar to those reported in a previously published family‐based study. Our results thus confirm the involvement of the IRF6 variant, rs642961, in the etiology of NSCL/P in the Central European population. We also found evidence suggestive of an independent protective effect of the coding variant V274I. In order to understand fully the genetic architecture of the IRF6 locus, it will be necessary to conduct additional SNP‐based and resequencing studies using large samples of patients.     

C392T polymorphism of the Wnt10a gene in non-syndromic oral cleft in a northeastern Chinese population

Non-syndromic oral cleft is one of the most common congenital malformations, and more than 40 genes may be involved in its aetiology. Recent studies have shown that the Wnt10a gene may also contribute. We recruited 198 patients with non-syndromic oral clefts, comprising 96 elementary families (restricted to the patients and their parents) and 187 controls, to investigate their associations with the risk of such clefts and their subgroups in a Chinese Han population. The variant evaluated in this study was a single nucleotide polymorphism – specifically, a missense mutation C392T of Wnt10a. Polymerase chain reaction (PCR) restriction fragment length polymorphism (RFLP) was used to genotype the marker, and case–control and family-based associations were analysed. Although in the case-control study there were no significant differences in frequency distributions of genotypes or alleles between cases and controls in the groups with cleft palate and cleft lip and palate, the genotypic and allelic frequencies of C392T in the total groups and the group with cleft lip alone differed significantly from those in the controls (p = 0.04, and 0.01, respectively). A transmission disequilibrium test showed a transmitted disequilibrium in C392T. In conclusion, we found an association between the C392T variant and non-syndromic oral clefts.