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Strong Evidence of Linkage Disequilibrium between Polymorphisms at the IRF6 Locus and Nonsyndromic Cleft Lip With or Without Cleft Palate, in an Italian Population

2004; Elsevier BV; Volume: 76; Issue: 1 Linguagem: Inglês

10.1086/427344

1537-6605

Luca Scapoli, Annalisa Palmieri, Marcella Martinelli, Furio Pezzetti, Paolo Carinci, Mauro Tognon, Francesco Carinci,

dental development and anomalies

Cleft lip with or without cleft palate (CL/P) is one of the most common birth defects, but its etiology is largely unknown. It is very likely that both genetic and environmental factors contribute to this malformation. Mutations in the gene for interferon regulatory factor 6 (IRF6) have been shown to be the cause of Van der Woude syndrome, a dominant disorder that has CL/P as a common feature. Recently, it has been reported that genetic polymorphisms at the IRF6 locus are associated with nonsyndromic CL/P, with stronger association in Asian and South American populations. We investigated four markers spanning the IRF6 locus, using the transmission/disequilibrium test. A sample of 219 Italian triads of patients and their parents were enrolled in the study. Strong evidence of linkage disequilibrium was found between markers and disease in both single-allele (P=.002 at marker rs2235375) and haplotype (P=.0005) analyses. These findings confirm the contribution of IRF6 in the etiology of nonsyndromic CL/P and strongly support its involvement in populations of European ancestry. Cleft lip with or without cleft palate (CL/P) is one of the most common birth defects, but its etiology is largely unknown. It is very likely that both genetic and environmental factors contribute to this malformation. Mutations in the gene for interferon regulatory factor 6 (IRF6) have been shown to be the cause of Van der Woude syndrome, a dominant disorder that has CL/P as a common feature. Recently, it has been reported that genetic polymorphisms at the IRF6 locus are associated with nonsyndromic CL/P, with stronger association in Asian and South American populations. We investigated four markers spanning the IRF6 locus, using the transmission/disequilibrium test. A sample of 219 Italian triads of patients and their parents were enrolled in the study. Strong evidence of linkage disequilibrium was found between markers and disease in both single-allele (P=.002 at marker rs2235375) and haplotype (P=.0005) analyses. These findings confirm the contribution of IRF6 in the etiology of nonsyndromic CL/P and strongly support its involvement in populations of European ancestry. Orofacial development is a complex process that involves many genes and signaling pathways (Murray and Schutte Murray and Schutte, 2004Murray JC Schutte BC Cleft palate: players, pathways, and pursuits.J Clin Invest. 2004; 113: 1676-1678Crossref PubMed Scopus (139) Google Scholar). Alterations in one or more of these genes could cause one of the most common malformations in humans: cleft lip with or without cleft palate (CL/P). Although CL/P and isolated cleft palate are features of >300 syndromes, >70% of cases present with cleft as the sole anomaly. Nonsyndromic CL/P (MIM 119530) is a complex multifactorial disease that affects >1/1,000 live births in Europe. Genetic linkage and association analyses have provided evidence to support the involvement of several genes and chromosomal regions, although difficulties have been encountered in replicating previous findings (Carinci et al. Carinci et al., 2003Carinci F Pezzetti F Scapoli L Martinelli M Avantaggiato A Carinci P Padula E Baciliero U Gombos F Laino G Rullo R Cenzi R Carls F Tognon M Recent developments in orofacial cleft genetics.J Craniofac Surg. 2003; 14: 130-143Crossref PubMed Scopus (71) Google Scholar). Recent advances in gene mapping have resulted inthe identification of genes responsible for Mendelian-inherited clefting syndromes (Celli et al. Celli et al., 1999Celli J Duijf P Hamel BC Bamshad M Kramer B Smits AP Newbury-Ecob R Hennekam RC Van Buggenhout G van Haeringen A Woods CG van Essen AJ de Waal R Vriend G Haber DA Yang A McKeon F Brunner HG van Bokhoven H Heterozygous germline mutations in the p53 homolog p63 are the cause of EEC syndrome.Cell. 1999; 99: 143-153Abstract Full Text Full Text PDF PubMed Scopus (552) Google Scholar; Suzuki et al. Suzuki et al., 2000Suzuki K Hu D Bustos T Zlotogora J Richieri-Costa A Helms JA Spritz RA Mutations of PVRL1, encoding a cell-cell adhesion molecule/herpesvirus receptor, in cleft lip/palate-ectodermal dysplasia.Nat Genet. 2000; 25: 427-430Crossref PubMed Scopus (250) Google Scholar; Kondo et al. Kondo et al., 2002Kondo S Schutte BC Richardson RJ Bjork BC Knight AS Watanabe Y Howard E et al.Mutations in IRF6 cause Van der Woude and popliteal pterygium syndromes.Nat Genet. 2002; 32: 285-289Crossref PubMed Scopus (602) Google Scholar). It is likely that mutations having a mild effect on the function of genes causing the syndromes could cause phenotypes not distinct from nonsyndromic CL/P, which, in turn, means that those genes could be involved in the etiology of nonsyndromic CL/P. An increasing amount of evidence seems to support this hypothesis. Van der Woude syndrome (VWS [MIM 119300]) is an autosomal dominant disorder in which lower-lip pits and occasional hypodontia are the only features distinguishing the disorder from nonsyndromic CL/P. Lip pits are variable findings in VWS and may not be present at all in some affected individuals belonging to kindred with VWS. It has been shown that deletions involving the gene coding for interferon regulatory factor 6 (IRF6) or mutations causing haploinsufficiency are responsible for VWS (Kondo et al. Kondo et al., 2002Kondo S Schutte BC Richardson RJ Bjork BC Knight AS Watanabe Y Howard E et al.Mutations in IRF6 cause Van der Woude and popliteal pterygium syndromes.Nat Genet. 2002; 32: 285-289Crossref PubMed Scopus (602) Google Scholar). Recently, some original evidence of a significant association between nonsyndromic CL/P and SNPs at the IRF6 locus was reported (Zucchero et al. Zucchero et al., 2004Zucchero TM Cooper ME Maher BS Daack-Hirsch S Nepomuceno B Ribeiro L Caprau D et al.Interferon regulatory factor 6 (IRF6) gene variants and the risk of isolated cleft lip or palate.N Engl J Med. 2004; 351: 769-780Crossref PubMed Scopus (444) Google Scholar). In that study, strong evidence of overtransmission of the ancestral allele of the V274I polymorphism to patients with nonsyndromic CL/P was observed in Asians but not in groups of European descent. However, analysis of additional SNPs showed that linkage disequilibrium with CL/P extended from 40 kbp in the 5′ portion of IRF6 to 135 kbp in the 3′ portion of IRF6 and was also detectable in the Iowan and Danish populations. The complexity of CL/P genetics has been evident in the last decade. Several genes and loci have been claimed to be etiologically involved, although difficulties have been encountered in replicating positive findings, and different results have been obtained for the different populations investigated. In the present investigation, we examined a sample of patients with nonsyndromic CL/P recruited in Italy, to verify the involvement of IRF6 in cases of CL/P in southern Europe. Our study group consisted of 219 unrelated patients with nonsyndromic CL/P and their parents. All of them were white and of Italian ancestry. Among them, 132 were sporadic, whereas 87, who had relatives affected with CL/P, were classified as familial cases. CL/P was the sole disorder affecting these patients. To assess the nonsyndromic status and to exclude known teratogenic influences, the patients and their relatives were asked specific questions about the presence in the family of any other somatic and neurological disorders and the use of clefting drugs—such as phenytoin, warfarin, and ethanol—during pregnancy. After informed consent was obtained, peripheral blood samples were drawn from each individual. Clinical analyses, blood collection, and DNA extraction were performed using a consolidated protocol described elsewhere (Pezzetti et al. Pezzetti et al., 2004Pezzetti F Martinelli M Scapoli L Carinci F Palmieri A Marchesini J Carinci P Caramelli E Rullo R Gombos F Tognon M Maternal MTHFR variant forms increase the risk in offspring of isolated nonsyndromic cleft lip with or without cleft palate.Hum Mutat. 2004; 24: 104-105Crossref PubMed Scopus (67) Google Scholar). To facilitate comparisons between studies, we selected polymorphisms that had been used before to investigate other populations (Zucchero et al. Zucchero et al., 2004Zucchero TM Cooper ME Maher BS Daack-Hirsch S Nepomuceno B Ribeiro L Caprau D et al.Interferon regulatory factor 6 (IRF6) gene variants and the risk of isolated cleft lip or palate.N Engl J Med. 2004; 351: 769-780Crossref PubMed Scopus (444) Google Scholar). Four SNPs at the IRF6 locus, with high heterozygosity in whites, were chosen because they showed a very strong association with the disorder (Zucchero et al. Zucchero et al., 2004Zucchero TM Cooper ME Maher BS Daack-Hirsch S Nepomuceno B Ribeiro L Caprau D et al.Interferon regulatory factor 6 (IRF6) gene variants and the risk of isolated cleft lip or palate.N Engl J Med. 2004; 351: 769-780Crossref PubMed Scopus (444) Google Scholar). Marker information is included in table 1. Genotypes were obtained using an ABI PRISM 7700 Sequence Detection System and TaqMan chemistry. Reagents and SNP genotyping assays were supplied by Applied Biosystems.Table 1Marker Information and TDT ResultsPforfGlobal P value for haplotype analyses. In each case, the P value is placed on the line of the first SNP of the haplotype.dbSNPAllelesaBase change in coding strand, with major allele listed first.Physical LocationbPhysical locations are from the UCSC Human Genome Browser, July 2003 assembly.Genetic LocationAllele FrequencycMajor-allele frequency was calculated using all parental chromosomes.T/NTdTransmission/nontransmission (T/NT) counts from heterozygous parents are given for the common allele.PeP value for TDT.2 SNPs3 SNPs4 SNPsrs1319435G/A20712700770 kb 5′ of IRF6.8543/61.077.0004.0014.0005rs2013162G/T207048073In IRF6.67118/78.004.0098.0087…rs2235375C/G207044976In IRF6.67111/80.002.0376……rs2235543A/G206940057100 kb 3′ of IRF6.8753/47.55………a Base change in coding strand, with major allele listed first.b Physical locations are from the UCSC Human Genome Browser, July 2003 assembly.c Major-allele frequency was calculated using all parental chromosomes.d Transmission/nontransmission (T/NT) counts from heterozygous parents are given for the common allele.e P value for TDT.f Global P value for haplotype analyses. In each case, the P value is placed on the line of the first SNP of the haplotype. Open table in a new tab Hardy-Weinberg equilibrium was observed for all polymorphisms in both patients and parents. Single-marker results were analyzed by the transmission/disequilibrium test (TDT), which examines the transmission of alleles from heterozygous parents (Spielman et al. Spielman et al., 1993Spielman RS McGinnis RE Ewens WJ Transmission test for linkage disequilibrium: the insulin gene region and insulin-dependent diabetes mellitus (IDDM).Am J Hum Genet. 1993; 52: 506-516PubMed Google Scholar). Significant linkage disequilibrium with the disease locus was apparent for markers rs2013162 and rs2235375 (P=.004 and .002, respectively); in both cases, the frequent allele was significantly overtransmitted to the affected individuals. Haplotype analyses were performed, because they enhance the power of linkage disequilibrium studies by increasing the number of heterozygous parents that are processed by TDT statistics. Haplotype analysis was performed using the Web-based interface GLUE and the program TDTPHASE (Dudbridge Dudbridge, 2003Dudbridge F Pedigree disequilibrium tests for multilocus haplotypes.Genet Epidemiol. 2003; 25: 115-121Crossref PubMed Scopus (1044) Google Scholar), part of the UNPHASED package, available at the Human Genome Mapping Project Web site. The analysis was restricted to phase-certain haplotypes, in a conditional logistic regression model. Rare haplotypes with a frequency <0.03 were excluded from statistical analysis. Highly significant transmission distortion was observed in two-, three-, and four-marker haplotype analyses. Global P values, most of which are <.01, are reported in table 1. Haplotypes carrying the frequent alleles at markers rs2013162 and rs2235375 were always found to be overtransmitted to patients, whereas those carrying the rare alleles were negatively associated (table 2).Table 2Two-Marker and Three-Marker Haplotypes Showing Significant Transmission DistortionFrequency in HaplotypesHaplotypeT/NTTNTPTwo markers, with allele: G-G-X-X95/66.53.37.0420 G-T-X-X38/86.21.48.0001 X-G-C-X83/49.63.37.0098 X-T-G-X49/83.37.63.0098 X-X-G-A40/66.27.44.0227 X-X-C-A82/49.55.33.0120Three markers, with allele: G-T-G-X37/79.22.46.0004 X-G-C-A81/43.57.30.0029 X-T-G-A35/66.25.46.0050Four markers, with allele: G-G-C-A80/52.49.32.0261 G-T-G-A25/61.15.38.0003 A-G-C-A33/17.20.10.0277Note.—T = transmitted; NT = nontransmitted. Open table in a new tab Note.— T = transmitted; NT = nontransmitted. Linkage disequilibrium between markers was calculated by D′ and r2 statistics from parental haplotypes, by use of the program ldmax from the GOLD software package (Abecasis and Cookson Abecasis and Cookson, 2000Abecasis GR Cookson WO GOLD—graphical overview of linkage disequilibrium.Bioinformatics. 2000; 16: 182-183Crossref PubMed Scopus (673) Google Scholar). The results are shown in table 3, with D′ values above and r2 values below the diagonal. A comparison of these data with those regarding Filipinos, available in the report by Zucchero et al. (Zucchero et al., 2004Zucchero TM Cooper ME Maher BS Daack-Hirsch S Nepomuceno B Ribeiro L Caprau D et al.Interferon regulatory factor 6 (IRF6) gene variants and the risk of isolated cleft lip or palate.N Engl J Med. 2004; 351: 769-780Crossref PubMed Scopus (444) Google Scholar), shows that linkage disequilibrium between markers was stronger among Italians.Table 3Linkage Disequilibrium between Markers Genotyped in the StudyMarkerrs1319435rs2013162rs2235375rs2235543rs1319435….280.316.001rs2013162.007….995.866rs2235375.008.968….868rs2235543.000.237.245…Note.—D′ is above the diagonal; r2 is below the diagonal. Open table in a new tab Note.— D′ is above the diagonal; r2 is below the diagonal. In the report by Zucchero et al. (Zucchero et al., 2004Zucchero TM Cooper ME Maher BS Daack-Hirsch S Nepomuceno B Ribeiro L Caprau D et al.Interferon regulatory factor 6 (IRF6) gene variants and the risk of isolated cleft lip or palate.N Engl J Med. 2004; 351: 769-780Crossref PubMed Scopus (444) Google Scholar), a significant association was reported between nonsyndromic CL/P and the V274I polymorphism in IRF6. In particular, the V allele was overtransmitted to patients from Asian and South American populations. Moreover, it was reported that the I allele was very rare or absent among Africans and Europeans. Specifically, it was not detected in 76 chromosomes from Italy. On the strength of this result, our study did not investigate the V274I polymorphism. On the other hand, it is very unlikely that V274I, or the SNPs investigated in the present study, may be directly implicated in CL/P etiology. The genetic alterations increasing the susceptibility to the disease are as yet unknown, and further investigations are needed to address this important issue. Above all, in the present investigation, we found strong evidence supporting the involvement of IRF6 in nonsyndromic CL/P. This is the first study to confirm the original finding reported by Zucchero et al. (Zucchero et al., 2004Zucchero TM Cooper ME Maher BS Daack-Hirsch S Nepomuceno B Ribeiro L Caprau D et al.Interferon regulatory factor 6 (IRF6) gene variants and the risk of isolated cleft lip or palate.N Engl J Med. 2004; 351: 769-780Crossref PubMed Scopus (444) Google Scholar) and to report an IRF6 contribution to the etiology of CL/P in southern Europe. We wish to thank Jeff Murray and Theresa Zucchero, for help with the study design. We are also grateful to all the patients and families who participated in this investigation. This study was supported in part by grants from Telethon (E. 1147 to P.C.); Ministero dell' Istruzione, Universita, e Ricerca Cofinanziamento and Local (to M.T., F.C., and P.C.); and the Fondazione Cassa di Risparmio di Bologna (to P.C.).