title: Functional Analysis of a Novel Genome-Wide Association Study Signal in SMAD3 That Confers Protection From Coronary Artery Disease.
abstract: A recent genome-wide association study meta-analysis identified an intronic single nucleotide polymorphism in SMAD3, rs56062135C>T, the minor allele (T) which associates with protection from coronary artery disease. Relevant to atherosclerosis, SMAD3 is a key contributor to transforming growth factor-β pathway signaling. Here, we seek to identify ≥1 causal coronary artery disease-associated single nucleotide polymorphisms at the SMAD3 locus and characterize mechanisms whereby the risk allele(s) contribute to coronary artery disease risk.
  
  
  
  By genetic and epigenetic fine mapping, we identified a candidate causal single nucleotide polymorphism rs17293632C>T (D', 0.97; r(2), 0.94 with rs56062135) in intron 1 of SMAD3 with predicted functional effects. We show that the sequence encompassing rs17293632 acts as a strong enhancer in human arterial smooth muscle cells. The common allele (C) preserves an activator protein (AP)-1 site and enhancer function, whereas the protective (T) allele disrupts the AP-1 site and significantly reduces enhancer activity (P<0.001). Pharmacological inhibition of AP-1 activity upstream demonstrates that this allele-specific enhancer effect is AP-1 dependent (P<0.001). Chromatin immunoprecipitation experiments reveal binding of several AP-1 component proteins with preferential binding to the (C) allele. We show that rs17293632 is an expression quantitative trait locus for SMAD3 in blood and atherosclerotic plaque with reduced expression of SMAD3 in carriers of the protective allele. Finally, siRNA knockdown of SMAD3 in human arterial smooth muscle cells increases cell viability, consistent with an antiproliferative role.
  
  
  
  The coronary artery disease-associated rs17293632C>T single nucleotide polymorphism represents a novel functional cis-acting element at the SMAD3 locus. The protective (T) allele of rs17293632 disrupts a consensus AP-1 binding site in a SMAD3 intron 1 enhancer, reduces enhancer activity and SMAD3 expression, altering human arterial smooth muscle cell proliferation.
authors.affiliation: From the Atherogenomics Laboratory (A.W.T., A.M., A.S., P.L., S.S., R.M.) and Department of Medicine, Ruddy Canadian Cardiovascular Genetics Centre (M.N., R.M.), University of Ottawa Heart Institute, Ottawa, Canada; Atherosclerosis Research Unit (P.E., L.F.) and Department of Molecular Medicine and Surgery (L.P., U.H.), Karolinska University Hospital, Stockholm, Sweden; and Department of Systems Biology, Technical University of Denmark, Copenhagen, Denmark (L.F.).
authors.forename: Adam W
authors.initials: AW
authors.lastname: Turner
authors.affiliation: From the Atherogenomics Laboratory (A.W.T., A.M., A.S., P.L., S.S., R.M.) and Department of Medicine, Ruddy Canadian Cardiovascular Genetics Centre (M.N., R.M.), University of Ottawa Heart Institute, Ottawa, Canada; Atherosclerosis Research Unit (P.E., L.F.) and Department of Molecular Medicine and Surgery (L.P., U.H.), Karolinska University Hospital, Stockholm, Sweden; and Department of Systems Biology, Technical University of Denmark, Copenhagen, Denmark (L.F.).
authors.forename: Amy
authors.initials: A
authors.lastname: Martinuk
authors.affiliation: From the Atherogenomics Laboratory (A.W.T., A.M., A.S., P.L., S.S., R.M.) and Department of Medicine, Ruddy Canadian Cardiovascular Genetics Centre (M.N., R.M.), University of Ottawa Heart Institute, Ottawa, Canada; Atherosclerosis Research Unit (P.E., L.F.) and Department of Molecular Medicine and Surgery (L.P., U.H.), Karolinska University Hospital, Stockholm, Sweden; and Department of Systems Biology, Technical University of Denmark, Copenhagen, Denmark (L.F.).
authors.forename: Anada
authors.initials: A
authors.lastname: Silva
authors.affiliation: From the Atherogenomics Laboratory (A.W.T., A.M., A.S., P.L., S.S., R.M.) and Department of Medicine, Ruddy Canadian Cardiovascular Genetics Centre (M.N., R.M.), University of Ottawa Heart Institute, Ottawa, Canada; Atherosclerosis Research Unit (P.E., L.F.) and Department of Molecular Medicine and Surgery (L.P., U.H.), Karolinska University Hospital, Stockholm, Sweden; and Department of Systems Biology, Technical University of Denmark, Copenhagen, Denmark (L.F.).
authors.forename: Paulina
authors.initials: P
authors.lastname: Lau
authors.affiliation: From the Atherogenomics Laboratory (A.W.T., A.M., A.S., P.L., S.S., R.M.) and Department of Medicine, Ruddy Canadian Cardiovascular Genetics Centre (M.N., R.M.), University of Ottawa Heart Institute, Ottawa, Canada; Atherosclerosis Research Unit (P.E., L.F.) and Department of Molecular Medicine and Surgery (L.P., U.H.), Karolinska University Hospital, Stockholm, Sweden; and Department of Systems Biology, Technical University of Denmark, Copenhagen, Denmark (L.F.).
authors.forename: Majid
authors.initials: M
authors.lastname: Nikpay
authors.affiliation: From the Atherogenomics Laboratory (A.W.T., A.M., A.S., P.L., S.S., R.M.) and Department of Medicine, Ruddy Canadian Cardiovascular Genetics Centre (M.N., R.M.), University of Ottawa Heart Institute, Ottawa, Canada; Atherosclerosis Research Unit (P.E., L.F.) and Department of Molecular Medicine and Surgery (L.P., U.H.), Karolinska University Hospital, Stockholm, Sweden; and Department of Systems Biology, Technical University of Denmark, Copenhagen, Denmark (L.F.).
authors.forename: Per
authors.initials: P
authors.lastname: Eriksson
authors.affiliation: From the Atherogenomics Laboratory (A.W.T., A.M., A.S., P.L., S.S., R.M.) and Department of Medicine, Ruddy Canadian Cardiovascular Genetics Centre (M.N., R.M.), University of Ottawa Heart Institute, Ottawa, Canada; Atherosclerosis Research Unit (P.E., L.F.) and Department of Molecular Medicine and Surgery (L.P., U.H.), Karolinska University Hospital, Stockholm, Sweden; and Department of Systems Biology, Technical University of Denmark, Copenhagen, Denmark (L.F.).
authors.forename: Lasse
authors.initials: L
authors.lastname: Folkersen
authors.affiliation: From the Atherogenomics Laboratory (A.W.T., A.M., A.S., P.L., S.S., R.M.) and Department of Medicine, Ruddy Canadian Cardiovascular Genetics Centre (M.N., R.M.), University of Ottawa Heart Institute, Ottawa, Canada; Atherosclerosis Research Unit (P.E., L.F.) and Department of Molecular Medicine and Surgery (L.P., U.H.), Karolinska University Hospital, Stockholm, Sweden; and Department of Systems Biology, Technical University of Denmark, Copenhagen, Denmark (L.F.).
authors.forename: Ljubica
authors.initials: L
authors.lastname: Perisic
authors.affiliation: From the Atherogenomics Laboratory (A.W.T., A.M., A.S., P.L., S.S., R.M.) and Department of Medicine, Ruddy Canadian Cardiovascular Genetics Centre (M.N., R.M.), University of Ottawa Heart Institute, Ottawa, Canada; Atherosclerosis Research Unit (P.E., L.F.) and Department of Molecular Medicine and Surgery (L.P., U.H.), Karolinska University Hospital, Stockholm, Sweden; and Department of Systems Biology, Technical University of Denmark, Copenhagen, Denmark (L.F.).
authors.forename: Ulf
authors.initials: U
authors.lastname: Hedin
authors.affiliation: From the Atherogenomics Laboratory (A.W.T., A.M., A.S., P.L., S.S., R.M.) and Department of Medicine, Ruddy Canadian Cardiovascular Genetics Centre (M.N., R.M.), University of Ottawa Heart Institute, Ottawa, Canada; Atherosclerosis Research Unit (P.E., L.F.) and Department of Molecular Medicine and Surgery (L.P., U.H.), Karolinska University Hospital, Stockholm, Sweden; and Department of Systems Biology, Technical University of Denmark, Copenhagen, Denmark (L.F.).
authors.forename: Sebastien
authors.initials: S
authors.lastname: Soubeyrand
authors.affiliation: From the Atherogenomics Laboratory (A.W.T., A.M., A.S., P.L., S.S., R.M.) and Department of Medicine, Ruddy Canadian Cardiovascular Genetics Centre (M.N., R.M.), University of Ottawa Heart Institute, Ottawa, Canada; Atherosclerosis Research Unit (P.E., L.F.) and Department of Molecular Medicine and Surgery (L.P., U.H.), Karolinska University Hospital, Stockholm, Sweden; and Department of Systems Biology, Technical University of Denmark, Copenhagen, Denmark (L.F.). rmcpherson@ottawaheart.ca.
authors.forename: Ruth
authors.initials: R
authors.lastname: McPherson
mesh_terms: D001665:Binding Sites; D049109:Cell Proliferation; D002470:Cell Survival; D002478:Cells, Cultured; D003324:Coronary Artery Disease / Q000175:diagnosis / Q000235:genetics* / Q000378:metabolism / Q000517:prevention & control*; D003331:Coronary Vessels / Q000378:metabolism / Q000473:pathology; D004742:Enhancer Elements, Genetic; D020022:Genetic Predisposition to Disease; D055106:Genome-Wide Association Study; D006239:Haplotypes; D006801:Humans; D007438:Introns; D009131:Muscle, Smooth, Vascular / Q000378:metabolism / Q000473:pathology; D032389:Myocytes, Smooth Muscle / Q000378:metabolism; D010641:Phenotype; D058226:Plaque, Atherosclerotic; D020641:Polymorphism, Single Nucleotide; D011401:Promoter Regions, Genetic; D065840:Protective Factors; D040641:Quantitative Trait Loci; D034622:RNA Interference; D012307:Risk Factors; D051900:Smad3 Protein / Q000235:genetics* / Q000378:metabolism; D018808:Transcription Factor AP-1 / Q000235:genetics / Q000378:metabolism; D014162:Transfection
chemical_list: C497790:SMAD3 protein, human; D051900:Smad3 Protein; D018808:Transcription Factor AP-1
keywords: SMAD3 protein; binding sites; coronary artery disease; genome-wide association study; genomics