title: Identification of common non-coding variants at 1p22 that are functional for non-syndromic orofacial clefting.
abstract: Genome-wide association studies (GWAS) do not distinguish between single nucleotide polymorphisms (SNPs) that are causal and those that are merely in linkage-disequilibrium with causal mutations. Here we describe a versatile, functional pipeline and apply it to SNPs at 1p22, a locus identified in several GWAS for non-syndromic cleft lip with or without cleft palate (NS CL/P). First we amplified DNA elements containing the ten most-highly risk-associated SNPs and tested their enhancer activity in vitro, identifying three SNPs with allele-dependent effects on such activity. We then used in vivo reporter assays to test the tissue-specificity of these enhancers, chromatin configuration capture to test enhancer-promoter interactions, and genome editing in vitro to show allele-specific effects on ARHGAP29 expression and cell migration. Our results further indicate that two SNPs affect binding of CL/P-associated transcription factors, and one affects chromatin configuration. These results translate risk into potential mechanisms of pathogenesis.
authors.affiliation: Department of Anatomy and Cell Biology, College of Medicine, University of Iowa, Iowa City, Iowa 52242, USA.|State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key Laboratory for Oral Biomedicine of Ministry of Education, School and Hospital of Stomatology, Wuhan University, Wuhan, Hubei 430079, China.
authors.forename: Huan
authors.initials: H
authors.lastname: Liu
authors.affiliation: Center for Craniofacial and Dental Genetics, Department of Oral Biology, School of Dental Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15219, USA.
authors.forename: Elizabeth J
authors.initials: EJ
authors.lastname: Leslie
authors.affiliation: Department of Biostatistics, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA.
authors.forename: Jenna C
authors.initials: JC
authors.lastname: Carlson
authors.affiliation: Department of Epidemiology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland 21205, USA.
authors.forename: Terri H
authors.initials: TH
authors.lastname: Beaty
authors.affiliation: Center for Craniofacial and Dental Genetics, Department of Oral Biology, School of Dental Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15219, USA.|Department of Human Genetics, Graduate School of Public Health and Clinical and Translational Science Institute, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15219, USA.
authors.forename: Mary L
authors.initials: ML
authors.lastname: Marazita
authors.affiliation: Department of Orthodontics, College of Dentistry, University of Iowa, Iowa City, Iowa 52246, USA.
authors.forename: Andrew C
authors.initials: AC
authors.lastname: Lidral
authors.affiliation: Department of Anatomy and Cell Biology, College of Medicine, University of Iowa, Iowa City, Iowa 52242, USA.
authors.forename: Robert A
authors.initials: RA
authors.lastname: Cornell
mesh_terms: D000483:Alleles; D000818:Animals; D030801:Animals, Genetically Modified; D001681:Biological Assay; D002843:Chromatin / Q000737:chemistry; D002878:Chromosomes, Human, Pair 1 / Q000737:chemistry*; D002971:Cleft Lip / Q000175:diagnosis / Q000235:genetics* / Q000473:pathology; D002972:Cleft Palate / Q000175:diagnosis / Q000235:genetics* / Q000473:pathology; D004742:Enhancer Elements, Genetic; D020690:GTPase-Activating Proteins / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D017930:Genes, Reporter; D056426:Genetic Loci; D020022:Genetic Predisposition to Disease; D055106:Genome-Wide Association Study; D006801:Humans; D015810:Linkage Disequilibrium; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D020641:Polymorphism, Single Nucleotide; D011401:Promoter Regions, Genetic; D012307:Risk Factors; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D015027:Zebrafish
chemical_list: C500429:ARHGAP29 protein, human; D002843:Chromatin; D020690:GTPase-Activating Proteins; D014157:Transcription Factors; D008156:Luciferases