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Disruption of an AP-2α binding site in an IRF6 enhancer is associated with cleft lip

Nature Genetics volume 40, pages 1341–1347 (2008)Cite this article

Abstract

Previously we have shown that nonsyndromic cleft lip with or without cleft palate (NSCL/P)1 is strongly associated with SNPs in IRF6 (interferon regulatory factor 6)2. Here, we use multispecies sequence comparisons to identify a common SNP (rs642961, G>A) in a newly identified IRF6 enhancer. The A allele is significantly overtransmitted (P = 1 × 10−11) in families with NSCL/P, in particular those with cleft lip but not cleft palate. Further, there is a dosage effect of the A allele, with a relative risk for cleft lip of 1.68 for the AG genotype and 2.40 for the AA genotype. EMSA and ChIP assays demonstrate that the risk allele disrupts the binding site of transcription factor AP-2α and expression analysis in the mouse localizes the enhancer activity to craniofacial and limb structures. Our findings place IRF6 and AP-2α in the same developmental pathway and identify a high-frequency variant in a regulatory element contributing substantially to a common, complex disorder.

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Figure 1: Transcription factor AP-2α binds to MCS-9.7 and rs642961 disrupts its binding site.
Figure 2: MCS-9.7 shows IRF6 enhancer activity in transgenic mouse assay.

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References

  1. Jugessur, A. & Murray, J.C. Orofacial clefting: recent insights into a complex trait. Curr. Opin. Genet. Dev. 15, 270–278 (2005).

    Article  CAS  Google Scholar 

  2. Zucchero, T.M. et al. Interferon regulatory factor 6 (IRF6) gene variants and the risk of isolated cleft lip or palate. N. Engl. J. Med. 351, 769–780 (2004).

    Article  CAS  Google Scholar 

  3. Kondo, S. et al. Mutations in IRF6 cause Van der Woude and popliteal pterygium syndromes. Nat. Genet. 32, 285–289 (2002).

    Article  CAS  Google Scholar 

  4. Scapoli, L. et al. Strong evidence of linkage disequilibrium between polymorphisms at the IRF6 locus and nonsyndromic cleft lip with or without cleft palate, in an Italian population. Am. J. Hum. Genet. 76, 180–183 (2005).

    Article  CAS  Google Scholar 

  5. Blanton, S.H. et al. Variation in IRF6 contributes to nonsyndromic cleft lip and palate. Am. J. Med. Genet. A. 137, 259–262 (2005).

    Article  Google Scholar 

  6. Ghassibe, M. et al. Interferon regulatory factor-6: a gene predisposing to isolated cleft lip with or without cleft palate in the Belgian population. Eur. J. Hum. Genet. 13, 1239–1242 (2005).

    Article  CAS  Google Scholar 

  7. Park, J.W. et al. Association between IRF6 and nonsyndromic cleft lip with or without cleft palate in four populations. Genet. Med. 9, 219–227 (2007).

    Article  Google Scholar 

  8. Schorle, H., Meier, P., Buchert, M., Jaenisch, R. & Mitchell, P.J. Transcription factor AP-2 essential for cranial closure and craniofacial development. Nature 381, 235–238 (1996).

    Article  CAS  Google Scholar 

  9. Milunsky, J.M. et al. TFAP2A mutations result in branchio-oculo-facial syndrome. Am. J. Hum. Genet. 82, 1171–1177 (2008).

    Article  CAS  Google Scholar 

  10. Horvath, S., Xu, X. & Laird, N.M. The family based association test method: strategies for studying general genotype–phenotype associations. Eur. J. Hum. Genet. 9, 301–306 (2001).

    Article  CAS  Google Scholar 

  11. Graham, R.R. et al. Three functional variants of IFN regulatory factor 5 (IRF5) define risk and protective haplotypes for human lupus. Proc. Natl. Acad. Sci. USA 104, 6758–6763 (2007).

    Article  CAS  Google Scholar 

  12. Bille, C. et al. Oral clefts and life style factors–a case-cohort study based on prospective Danish data. Eur. J. Epidemiol. 22, 173–181 (2007).

    Article  Google Scholar 

  13. Nguyen, R.H., Wilcox, A.J., Moen, B.E., McConnaughey, D.R. & Lie, R.T. Parent's occupation and isolated orofacial clefts in Norway: a population-based case-control study. Ann. Epidemiol. 17, 763–771 (2007).

    Article  Google Scholar 

  14. Mitchell, L.E. et al. Guidelines for the design and analysis of studies on nonsyndromic cleft lip and cleft palate in humans: summary report from a Workshop of the International Consortium for Oral Clefts Genetics. Cleft Palate Craniofac. J. 39, 93–100 (2002).

    Article  Google Scholar 

  15. Harville, E.W., Wilcox, A.J., Lie, R.T., Vindenes, H. & Abyholm, F. Cleft lip and palate versus cleft lip only: are they distinct defects? Am. J. Epidemiol. 162, 448–453 (2005).

    Article  Google Scholar 

  16. Marazita, M.L. et al. Meta-analysis of 13 genome scans reveals multiple cleft lip/palate genes with novel loci on 9q21 and 2q32–35. Am. J. Hum. Genet. 75, 161–173 (2004).

    Article  CAS  Google Scholar 

  17. Mossey, P. Epidemiology underpinning research in the aetiology of orofacial clefts. Orthod. Craniofac. Res. 10, 114–120 (2007).

    Article  Google Scholar 

  18. Poulin, F. et al. In vivo characterization of a vertebrate ultraconserved enhancer. Genomics 85, 774–781 (2005).

    Article  CAS  Google Scholar 

  19. Knight, A.S., Schutte, B.C., Jiang, R. & Dixon, M.J. Developmental expression analysis of the mouse and chick orthologues of IRF6: the gene mutated in Van der Woude syndrome. Dev. Dyn. 235, 1441–1447 (2006).

    Article  CAS  Google Scholar 

  20. Sharpe, J. et al. Optical projection tomography as a tool for 3D microscopy and gene expression studies. Science 296, 541–545 (2002).

    Article  CAS  Google Scholar 

  21. Cirulli, E.T. & Goldstein, D.B. In vitro assays fail to predict in vivo effects of regulatory polymorphisms. Hum. Mol. Genet. 16, 1931–1939 (2007).

    Article  CAS  Google Scholar 

  22. Vieira, A.R. et al. Medical sequencing of candidate genes for nonsyndromic cleft lip and palate. PLoS Genet. 1, e64 (2005).

    Article  Google Scholar 

  23. Riley, B.M. et al. Impaired FGF signaling contributes to cleft lip and palate. Proc. Natl. Acad. Sci. USA 104, 4512–4517 (2007).

    Article  CAS  Google Scholar 

  24. Thomas, J.W. et al. Comparative analyses of multi-species sequences from targeted genomic regions. Nature 424, 788–793 (2003).

    Article  CAS  Google Scholar 

  25. Schwartz, S. et al. MultiPipMaker and supporting tools: alignments and analysis of multiple genomic DNA sequences. Nucleic Acids Res. 31, 3518–3524 (2003).

    Article  CAS  Google Scholar 

  26. Margulies, E.H., Blanchette, M., Haussler, D. & Green, E.D. Identification and characterization of multi-species conserved sequences. Genome Res. 13, 2507–2518 (2003).

    Article  CAS  Google Scholar 

  27. Nickerson, D.A., Tobe, V.O. & Taylor, S.L. PolyPhred: automating the detection and genotyping of single nucleotide substitutions using fluorescence-based resequencing. Nucleic Acids Res. 25, 2745–2751 (1997).

    Article  CAS  Google Scholar 

  28. Weinberg, C.R. Methods for detection of parent-of-origin effects in genetic studies of case-parents triads. Am. J. Hum. Genet. 65, 229–235 (1999).

    Article  CAS  Google Scholar 

  29. Dudbridge, F. Likelihood-based association analysis for nuclear families and unrelated subjects with missing genotype data. Hum. Hered. 66, 87–98 (2008).

    Article  Google Scholar 

  30. Provenzano, M.J. et al. AP-2 participates in the transcriptional control of the amyloid precursor protein (APP) gene in oral squamous cell carcinoma. Exp. Mol. Pathol. 83, 277–282 (2007).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We would like to thank A. Kinoshita, K. Frees, A. Mansilla, J. L'Heureux, M. Johnson, H. Morrison, G. Wehby, N. Rorick, K. Bedell and L. Powers for technical assistance and S. McConnell, D. Benton and M. DeVore for their administrative assistance. We would also like to thank A. Klingelhutz (University of Iowa) for kindly providing us with HFK cell line. This work was supported by grants from the National Institutes of Health (NIH): P50 DE16215 (J.C.M., M.L.M., B.C.S.), P30 ES05605 (J.C.M.), R37 DE08559 (J.C.M., M.L.M.), R01-DE13513 (B.C.S.), 1 UL1 RR024979-01 (J.C.M., B.C.S.), R01-CA73612 (F.E.D.), R01-HG003988 administered under Department of Energy Contract DE-AC02-05CH11231 (L.A.P.) as well as by the Intramural Research Program of the National Human Genome Research Institute (E.D.G.), in part by the Intramural Research Program of the NIH, National Institute of Environmental Health Sciences (A.J.W.), and European Commission FP5: EUROCRAN Project (contract no. QLG1-CT-2000-01019) (M.R., P.A.M., J.L., R.P.S.-T.). A.V. was supported by the American Heart Association. M.J.H. received salary support from T32 CA078586.

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Authors and Affiliations

  1. Department of Pediatrics, University of Iowa, 2182 ML, S Grand Ave, Iowa City, 52242, Iowa, USA

    Fedik Rahimov, Brian C Schutte & Jeffrey C Murray

  2. Department of Oral Biology, Center for Craniofacial and Dental Genetics, School of Dental Medicine, University of Pittsburgh, Suite 500, Bridgeside Point Building, Pittsburgh, 15219, Pennsylvania, USA

    Mary L Marazita, Margaret E Cooper & Manika Govil

  3. Genomics Division, Lawrence Berkeley National Laboratory, Berkeley, 94720, California, USA

    Axel Visel & Len A Pennacchio

  4. Department of Radiation Oncology, University of Iowa, 4202 MERF, Iowa City, 52242, Iowa, USA

    Michael J Hitchler & Frederick E Domann

  5. Department of Experimental Diagnostic Medicine, Medical Genetics Unit, University of Ferrara, Ferrara, 44100, Italy

    Michele Rubini

  6. Center for the Prevention of Congenital Malformations, Institute of Public Health, University of Southern Denmark, J.B. Winsløws Vej 9B, Odense C, DK-5000, Denmark

    Kaare Christensen & Camille Bille

  7. Department of Epidemiology Research, Danish Epidemiology Science Center, Statens Serum Institute, Copenhagen, DK-2300, Denmark

    Mads Melbye

  8. Department of Public Health and Primary Health Care, Section for Epidemiology and Medical Statistics, University of Bergen, Kalfarveien 31, Bergen, N-5018, Norway

    Astanand Jugessur & Rolv T Lie

  9. Epidemiology Branch, National Institute of Environmental Health Sciences, National Institutes of Health, 111 T.W. Alexander Drive, Durham, 27709, North Carolina, USA

    Allen J Wilcox

  10. Medical Research Council Human Genetics Unit, Western General Hospital, Edinburgh, EH4 2XU, UK

    David R Fitzpatrick

  11. Genome Technology Branch and US National Institutes of Health Intramural Sequencing Center, National Human Genome Research Institute, National Institutes of Health, Bethesda, 20892, Maryland, USA

    Eric D Green

  12. Dental Hospital & School, University of Dundee, Dundee, DD1 4HR, Scotland, UK

    Peter A Mossey

  13. Department of Epidemiology and Community Medicine, University of Ottawa, 451 Smyth Road, Room 3227, Ottawa, K1H 8M5, Ontario, Canada

    Julian Little

  14. University Medical Center, Dr. Molewaterplein 40, Rotterdam, 3015, GD, The Netherlands

    Regine P Steegers-Theunissen

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  1. Fedik Rahimov
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Consortia

NISC Comparative Sequencing Program

Contributions

F.R. performed sequencing, SNP genotyping and statistical analysis, comparative sequence analysis, functional assays, drafted the manuscript and prepared figures and tables including supplementary material. M.L.M. conducted the statistical analyses, wrote several sections and edited the manuscript. M.E.C. and M.G. assisted with the statistical analyses. A.V., L.A.P. and D.R.F. performed transgenic mouse enhancer assay and contributed critical revisions. M.J.H. and F.E.D. provided samples for ChIP assay. E.D.G. and NISC generated sequences for comparative sequence analysis. M.R., K.C., C.B., M.M., A.J., R.T.L. A.J.W., P.A.M., J.L. and R.P.S.-T. coordinated the recruitment, phenotyping and sample collection. M.R. genotyped EUROCRAN samples. B.C.S. was involved in study design, data interpretation and contributed critical revisions. J.C.M. is the principal investigator and obtained funding, carried out patient recruitment, established key collaborations, provided study design and supervision and oversaw manuscript preparation and revision.

Corresponding author

Correspondence to Jeffrey C Murray.

Supplementary information

Supplementary Text and Figures

Supplementary Methods, Supplementary Tables 1–4, Supplementary Figures 1 and 2 (PDF 4608 kb)

Supplementary Video 1

Embryo 3a (MPG 27826 kb)

Supplementary Video 2

Embryo 3b (MPG 27838 kb)

Supplementary Video 3

Embryo 3c (MPG 20900 kb)

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Rahimov, F., Marazita, M., Visel, A. et al. Disruption of an AP-2α binding site in an IRF6 enhancer is associated with cleft lip. Nat Genet 40, 1341–1347 (2008). https://doi.org/10.1038/ng.242

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