Mutation Research 547 (2004) 49–53

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

Valentina Gattaa, Oronzo Scarciollaa, Massimo Cupaiolib, Chiara Palkaa,Pierluigi Lelli Chiesab, Liborio Stuppiaa,c,∗

a Dipartimento di Scienze Biomediche, Università “G. D’Annunzio”, Via dei Vestini 35, 66013 Chieti-Pescara, Italyb Cattedra di Chirurgia Pediatrica, Università “G. D’Annunzio”, Chieti-Pescara, Italy

c ITOI-CNR, Unit of Bologna c/o IOR, Bologna, Italy

Received 24 June 2003; received in revised form 29 October 2003; accepted 28 November 2003

Abstract

Van der Woude syndrome (VWS) is the most common type of syndromic orofacial cleft, being characterised by variableassociation of lower lip pits, cleft lip and cleft palate. VWS is transmitted in an autosomal dominant manner, with highpenetrance and variable expressivity, and a gene for this disease has been mapped in 1q32-q41. Very recently, mutations ofthe interferon regulatory factor 6 (IRF6) gene have been found in VWS patients, suggesting that this gene plays an importantrole in the orofacial development. We report a novel mutation of the IRF6 in an Italian family with six members affected byVWS with different expression. This mutation, the W217X, produces a stop codon within exon 6 of the IRF6 gene, with lossof the SMIR domain of the IRF6 protein.© 2004 Elsevier B.V. All rights reserved.

Keywords: IRF6 gene; Orofacial cleft; Van der Woude syndrome

1. Introduction

Up to 1 in 500 infants at the birth presents orofacialclefts which can be subdivided in two categories: syn-dromic and not-syndromic[1]. The most common ofthe syndromic orofacial clefts is the Van der Woudesyndrome (VWS) (OMIM #119300), characterisedby a random association between lower lip pits (withor without sinuses), cleft lip (CL), cleft lip/palate(CL/CP) or isolated cleft palate (CP). Natal teeth, ab-sence of second premolars, ankyloglossia, syndactylyand equinovarous foot deformity, although reported

∗ Corresponding author. Tel.:+39-08713554137;fax: +39-08713554135.E-mail address: stuppia@unich.it (L. Stuppia).

in literature, seem to be incidental[2–4]. VWS isinherited as an autosomal dominant trait with highpenetrance (96.7%) and variable expressivity rangingfrom a single barely evident depression to bilateralfistulae of the lower lip and from a bifid uvula toa complete cleft palate and lip, determining variousdegrees of clinical relevance[5–7]. These signs mayeven belong to the popliteal pterygium syndrome(PPS) (OMIM #119500), a more complex diseasewhose other stigmata are bilateral popliteal webs,syndactyly, genital anomalies, ankyloblepharon andnail abnormalities[8].

VWS gene has been mapped by linkage analysis tochromosome 1q32-q41[9–12]. The critical region forthe VWS has been subsequently restricted to a 350 Kbgenomic region containing more than 20 candidate

0027-5107/$ – see front matter © 2004 Elsevier B.V. All rights reserved.doi:10.1016/j.mrfmmm.2003.11.011

50 V. Gatta et al. / Mutation Research 547 (2004) 49–53

genes[13]. Finally, Kondo et al.[14], investigatingtwo monozigotic twins discordant for VWS, found anonsense mutation in the interferon regulatory factor6 (IRF6) gene of the affected twin, suggesting IRF6 asa candidate for VWS. This hypothesis was confirmedin the same study by the detection of different IRF6mutations in 45 unrelated VWS families and in 13families with PPS, suggesting that VWS and PPS areallelic [14]. Very recently, a novel mutation of IRF6associated with VWS has been reported[15].

To confirm the association between IRF6 and VWS,we report on a molecular analysis of the IRF6 genecarried out in a family with six members affected byVWS.

2. Materials and methods

2.1. Patients

Two sisters, ageing 6 years and 1 month, respec-tively, with a family history of lip pits, were referred

LIP PITS ONLY

LIP PITS AND BIFD UVULA

LIP PITS AND CLEFT PALATE

I

II

III

IV

Fig. 1. Pedigree with phenotypic features as indicated: blue, lip pits; green, bifid uvula; red, cleft palate. Arrows indicate the members ofthe family who underwent molecular analysis (for interpretation of the references to color in this figure legend, the reader is referred tothe web version of the article).

in 1997 to the Pediatric Surgical Unit of Spirito SantoHospital, “G. D’Annunzio” University, in Pescara. Theclinical presentation was different, since the older girlhad lower lip pits and bifid uvula, while her sistershowed lower lip pits and incomplete palatoschisis. Noother sign of VWS or PPS was present. Their fatherhad bifid uvula and had been operated in childhoodfor lower lip pits; his mother, uncle and grandmotherhad lower lip pits only (Figs. 1 and 2).

The older sister underwent surgical correction of theanomaly due to aesthetic and psychological discom-fort, that represents the only indication for surgical re-moval of the pits. The younger sister was operated forboth cleft palate and pits at the age of 1 year. Both sis-ters show good aesthetic and functional results after a5 and 6 years follow up.

2.2. Molecular analysis

Molecular analysis was performed on DNA sam-ples of the two probands, of their affected father, of

V. Gatta et al. / Mutation Research 547 (2004) 49–53 51

Fig. 2. Clinical signs of VWS in three affected members of thefamily: (a) father; (b) older sister; (c) younger sister.

their mother, of the healthy paternal aunt, of the af-fected grandmother and of her affected brother. Link-age analysis was carried out using PCR amplificationof specific markers for the 1q32-q41 region (D1S205,

Fig. 3. DNA sequences showing the G650A mutation of the IRF6 gene in the forward (a) and reverse (b) strand.

D1S245, D1S414, D1S471). PCR products were runon a 8% polyacrilamide gel for 12 h at 120 V. Gelswere stained with Ethidium Bromide and observed us-ing a Biorad Gel Doc 1000 Image Analyzer.

Screening of mutations of IRF6 gene was carriedout using PCR amplifications with specific primers forexon 1–8 and part of exon 9 followed by direct se-quencing with an ABI PRISM 310 Genetic Analyzer.Each PCR product was sequenced both in the forwardand in the reverse strand. Detected mutations wereconfirmed by repeating the sequencing analysis on anew PCR product. Sequence was analysed using theSequencing Analysis 3.4 software.

3. Results

Linkage analysis using specific markers for the1q32-q41 region identified an haplotype segregatingin the affected patients, supporting the hypothesisthat the disease in the investigated family was linkedto the VWS1 locus. Screening of mutations of theIRF6 gene detected a G650A transition in exon 6

52 V. Gatta et al. / Mutation Research 547 (2004) 49–53

causing the formation of a stop codon (W217X)(Fig. 3). The same mutation was detected in all theaffected members of the family, but not in theirhealthy relatives and in 50 unrelated healthy individ-uals from the same geographic origin investigated ascontrols.

4. Discussion

IRF6 is a member of a gene family of transcrip-tion factors characterised by the presence of an highlyconserved helix-turn-helix DNA-binding domain anda less conserved protein-binding domain termed SMIR(Smad-interferon regulatory factor binding domain),required to form homo- and heterodimers[14]. Thisgene lies within the critical region for the VWS, andhas been found mutated in families both with VWSand PPS[14]. Mutations of IFR6 so far reported inVWS families are both protein-truncation and mis-sense mutations, clustered within the DNA bindingand SMIR domains. In this study we analyzed theIFR6 gene in an Italian family with VWS, and wefound a nonsense mutation in exon 6 consisting ofa G650A transition with formation of a stop codonat the aminoacid residue 217 (W217X). The W217Xmutation was not present among those described inthe only two studies so far reported on IRF6 muta-tions in VWS[14,15], thus representing a novel mu-tation. In fact, the majority of mutations of the IRF6falls within exons 4 and 7, corresponding to the DNAbinding and the SMIR domain, and only two non-sense mutations involving exon 6 have been described,namely a C558A and a G576A. Although mutationsof the exon 6 are rare and do not fall neither withinthe DNA binding or the SMIR domains of the IRF6gene, nevertheless the formation of a stop codon inexon 6 produces a protein lacking the entire SMIRdomain, fitting well to the model of the VWS caus-ing mutations[14]. Moreover, the detection of a non-sense mutation of IRF6 in a VWS family confirm thatprotein-truncation mutations are frequent in VWS andsupport the hypothesis that the VWS phenotype iscaused by haploinsufficiency. Interestingly, also in thepresent family the mutation produced different phe-notypes within the family, confirming the variable ex-pressivity of the VWS. In conclusion, the present re-port confirms the association between IRF6 and VWS,

supporting the pathogenetic role of IFR6 mutationsin this disease and the key role played by this genein the orofacial development. Moreover, we detecteda novel mutation producing a functional disruptionof the SMIR domain, confirming that IRF6 gene canbe affected by a number of different mutations. Thissuggests that the molecular analysis of VWS patientsshould be based on the screening of the entire geneby direct sequencing, since mutations can be scatteredalong the entire gene, also outside exons 4 and 7.More studies on Italian families with VWS will clar-ify if the W217X represents a specific Italian muta-tion.

References

[1] M. Muenke, The pit, the cleft and the web, Nat. Genet. 32(2002) 219–220.

[2] J.H. Hersh, G.D. Verdi, Natal teeth in monozygotic twinswith Van der Woude syndrome, Cleft Palate Craniofac. J. 29(1992) 279–281.

[3] E.L. Schneider, Lip pits and congenital absence of secondpremolars: varied expression of the lip pits syndrome, J. Med.Genet. 10 (1973) 346–349.

[4] W.B. Taylor, D.K. Lane, Congenital fistulas of the lowerlip. Associations with cleft lip-palate and anomalies of theextremities, Arch. Dermatol. 94 (1966) 421–424.

[5] J. Cervenka, R.J. Gorlin, V.E. Anderson, The syndromeof pits of the lower lip and cleft lip and/or palate.Genetic considerations, Am. J. Hum. Genet. 19 (1967) 416–432.

[6] P. Janku, M. Robinow, T. Kelly, R. Bralley, A. Baynes, M.T.Edgerton, The Van der Woude sindrome in a large kindred:variabilità, penetrance, genetic risks, Am. J. Med. Genet. 5(1980) 117–123.

[7] A. Velez, F.J. Alamillos, A. Dean, J.J. Ruiz-Masera,Congenital lower lip pits (Van der Woude syndrome), J. Am.Acad. Dermatol. 32 (1995) 520–521.

[8] D. Soekarman, J.M. Cobben, A. Vogels, P.H. Spauwen,J.P. Fryns, Variable expression of the popliteal pterygiumsyndrome in two 3-generation families, Clin. Genet. 47 (1995)169–174.

[9] T.F. Wienker, G. Hudek, S. Bissbort, A. Mayerova, G. Mauff,K. Bender, Linkage studies in a pedigree with Van der Woudesyndrome, J. Med. Genet. 24 (1987) 160–161.

[10] J.C. Murray, D.Y. Nishimura, K.H. Buetow, et al., Linkage ofan autosomal dominant clefting syndrome (Van der Woude)to loci on chromosome 1q, Am. J. Hum. Genet. 46 (1990)486–491.

[11] B.C. Schutte, A. Sander, M. Mailk, J.C. Murray, Refinementof the Van der Woude gene location and construction ofa 3.5-Mb YAC conting and STS map spanning the criticalregion in 1q32-q42, Genomics 36 (1996) 507–514.

V. Gatta et al. / Mutation Research 547 (2004) 49–53 53

[12] C. Houdayer, V. Soupre, M. Rosenberg-Bourgin, et al.,Linkage analysis of 5 novel Van der Woude syndromekindreds to 1q32-q41 markers further supports locus homo-geneity of the disease trait, Ann. Genet. 42 (1999) 69–74.

[13] B.C. Schutte, B.C. Bjork, K.B. Coppage, et al., A preliminarygene map for the Van der Woude syndrome critical regionderived from 900 kb of genomic sequence at 1q32-q41,Genome Res. 10 (2000) 81–94.

[14] S. Kondo, B.C. Schutte, R.J. Richardson, et al., Mutationsin IRF6 cause Van de Woude and popliteal pterygiumsyndromes, Nat. Genet. 32 (2002) 285–289.

[15] V. Shotelersuk, C. Srichomthong, K. Yoshiura, N. Niikawa,A novel mutation, 1234del(C), of the IRF6 in a Thai familywith Van der Woude syndrome, Int. J. Mol. Med. 11 (4)(2003) 505–507.