title: Coronary heart disease-associated variation in TCF21 disrupts a miR-224 binding site and miRNA-mediated regulation.
abstract: Genome-wide association studies (GWAS) have identified chromosomal loci that affect risk of coronary heart disease (CHD) independent of classical risk factors. One such association signal has been identified at 6q23.2 in both Caucasians and East Asians. The lead CHD-associated polymorphism in this region, rs12190287, resides in the 3' untranslated region (3'-UTR) of TCF21, a basic-helix-loop-helix transcription factor, and is predicted to alter the seed binding sequence for miR-224. Allelic imbalance studies in circulating leukocytes and human coronary artery smooth muscle cells (HCASMC) showed significant imbalance of the TCF21 transcript that correlated with genotype at rs12190287, consistent with this variant contributing to allele-specific expression differences. 3' UTR reporter gene transfection studies in HCASMC showed that the disease-associated C allele has reduced expression compared to the protective G allele. Kinetic analyses in vitro revealed faster RNA-RNA complex formation and greater binding of miR-224 with the TCF21 C allelic transcript. In addition, in vitro probing with Pb2+ and RNase T1 revealed structural differences between the TCF21 variants in proximity of the rs12190287 variant, which are predicted to provide greater access to the C allele for miR-224 binding. miR-224 and TCF21 expression levels were anti-correlated in HCASMC, and miR-224 modulates the transcriptional response of TCF21 to transforming growth factor-β (TGF-β) and platelet derived growth factor (PDGF) signaling in an allele-specific manner. Lastly, miR-224 and TCF21 were localized in human coronary artery lesions and anti-correlated during atherosclerosis. Together, these data suggest that miR-224 interaction with the TCF21 transcript contributes to allelic imbalance of this gene, thus partly explaining the genetic risk for coronary heart disease associated at 6q23.2. These studies implicating rs12190287 in the miRNA-dependent regulation of TCF21, in conjunction with previous studies showing that this variant modulates transcriptional regulation through activator protein 1 (AP-1), suggests a unique bimodal level of complexity previously unreported for disease-associated variants.
authors.affiliation: Department of Medicine, Division of Cardiovascular Medicine, and Cardiovascular Institute, Stanford University School of Medicine, Stanford, California, United States of America.
authors.forename: Clint L
authors.initials: CL
authors.lastname: Miller
authors.affiliation: Institut für Molekulare Medizin, Universität zu Lübeck, Lübeck, Germany.
authors.forename: Ulrike
authors.initials: U
authors.lastname: Haas
authors.affiliation: Department of Medicine, Division of Cardiovascular Medicine, and Cardiovascular Institute, Stanford University School of Medicine, Stanford, California, United States of America.
authors.forename: Roxanne
authors.initials: R
authors.lastname: Diaz
authors.affiliation: Department of Medicine, Division of Cardiovascular Medicine, and Cardiovascular Institute, Stanford University School of Medicine, Stanford, California, United States of America.
authors.forename: Nicholas J
authors.initials: NJ
authors.lastname: Leeper
authors.affiliation: Department of Medicine, Division of Cardiovascular Medicine, and Cardiovascular Institute, Stanford University School of Medicine, Stanford, California, United States of America.
authors.forename: Ramendra K
authors.initials: RK
authors.lastname: Kundu
authors.affiliation: Department of Medicine, Division of Cardiothoracic Surgery, and Cardiovascular Institute, Stanford University School of Medicine, Stanford, California, United States of America.
authors.forename: Bhagat
authors.initials: B
authors.lastname: Patlolla
authors.affiliation: Department of Medicine, Division of Cardiovascular Medicine, and Cardiovascular Institute, Stanford University School of Medicine, Stanford, California, United States of America.
authors.forename: Themistocles L
authors.initials: TL
authors.lastname: Assimes
authors.affiliation: Institut für Humangenetik, Universität zu Lübeck, Lübeck, Germany; DZHK (German Research Centre for Cardiovascular Research), partner site Hamburg/Lubeck/Kiel, Lubeck, Germany.
authors.forename: Frank J
authors.initials: FJ
authors.lastname: Kaiser
authors.affiliation: Center for Molecular Medicine, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Ljubica
authors.initials: L
authors.lastname: Perisic
authors.affiliation: Center for Molecular Medicine, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Ulf
authors.initials: U
authors.lastname: Hedin
authors.affiliation: Center for Molecular Medicine, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Lars
authors.initials: L
authors.lastname: Maegdefessel
authors.affiliation: Deutsches Herzzentrum München, Technische Universität München, Munich, DZHK, partner site Munich Heart Alliance, Munich, Germany.
authors.forename: Heribert
authors.initials: H
authors.lastname: Schunkert
authors.affiliation: DZHK (German Research Centre for Cardiovascular Research), partner site Hamburg/Lubeck/Kiel, Lubeck, Germany; Institut für Integrative und Experimentelle Genomik, Universität zu Lübeck, Lübeck, Germany.
authors.forename: Jeanette
authors.initials: J
authors.lastname: Erdmann
authors.affiliation: Department of Medicine, Division of Cardiovascular Medicine, and Cardiovascular Institute, Stanford University School of Medicine, Stanford, California, United States of America.
authors.forename: Thomas
authors.initials: T
authors.lastname: Quertermous
authors.affiliation: Institut für Molekulare Medizin, Universität zu Lübeck, Lübeck, Germany; DZHK (German Research Centre for Cardiovascular Research), partner site Hamburg/Lubeck/Kiel, Lubeck, Germany.
authors.forename: Georg
authors.initials: G
authors.lastname: Sczakiel
mesh_terms: D020413:3' Untranslated Regions; D000483:Alleles; D044466:Asian People / Q000235:genetics; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics*; D001665:Binding Sites; D002454:Cell Differentiation; D002896:Chromosomes, Human, Pair 6 / Q000235:genetics; D003327:Coronary Disease / Q000209:etiology / Q000235:genetics; D005786:Gene Expression Regulation; D055106:Genome-Wide Association Study; D006801:Humans; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism; D020641:Polymorphism, Single Nucleotide; D012307:Risk Factors; D015398:Signal Transduction / Q000235:genetics*
chemical_list: D020413:3' Untranslated Regions; D051792:Basic Helix-Loop-Helix Proteins; D035683:MicroRNAs; C546140:MIRN224 microRNA, human; C491825:TCF21 protein, human