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


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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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




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).

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