Adam W Turner1, Amy Martinuk1, Anada Silva1, Paulina Lau1, Majid Nikpay1, Per Eriksson1, Lasse Folkersen1, Ljubica Perisic1, Ulf Hedin1, Sebastien Soubeyrand1, Ruth McPherson2. 1. 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.). 2. 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.
Abstract
OBJECTIVE: 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. APPROACH AND RESULTS: 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. CONCLUSIONS: 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.
OBJECTIVE: 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. APPROACH AND RESULTS: 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. CONCLUSIONS: 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: Ilakya Selvarajan; Anu Toropainen; Kristina M Garske; Maykel López Rodríguez; Arthur Ko; Zong Miao; Dorota Kaminska; Kadri Õunap; Tiit Örd; Aarthi Ravindran; Oscar H Liu; Pierre R Moreau; Ashik Jawahar Deen; Ville Männistö; Calvin Pan; Anna-Liisa Levonen; Aldons J Lusis; Sami Heikkinen; Casey E Romanoski; Jussi Pihlajamäki; Päivi Pajukanta; Minna U Kaikkonen Journal: Am J Hum Genet Date: 2021-02-23 Impact factor: 11.025
Authors: Boxiang Liu; Milos Pjanic; Ting Wang; Trieu Nguyen; Michael Gloudemans; Abhiram Rao; Victor G Castano; Sylvia Nurnberg; Daniel J Rader; Susannah Elwyn; Erik Ingelsson; Stephen B Montgomery; Clint L Miller; Thomas Quertermous Journal: Am J Hum Genet Date: 2018-08-23 Impact factor: 11.025
Authors: Thomas R Webb; Jeanette Erdmann; Kathleen E Stirrups; Nathan O Stitziel; Nicholas G D Masca; Henning Jansen; Stavroula Kanoni; Christopher P Nelson; Paola G Ferrario; Inke R König; John D Eicher; Andrew D Johnson; Stephen E Hamby; Christer Betsholtz; Arno Ruusalepp; Oscar Franzén; Eric E Schadt; Johan L M Björkegren; Peter E Weeke; Paul L Auer; Ursula M Schick; Yingchang Lu; He Zhang; Marie-Pierre Dube; Anuj Goel; Martin Farrall; Gina M Peloso; Hong-Hee Won; Ron Do; Erik van Iperen; Jochen Kruppa; Anubha Mahajan; Robert A Scott; Christina Willenborg; Peter S Braund; Julian C van Capelleveen; Alex S F Doney; Louise A Donnelly; Rosanna Asselta; Pier A Merlini; Stefano Duga; Nicola Marziliano; Josh C Denny; Christian Shaffer; Nour Eddine El-Mokhtari; Andre Franke; Stefanie Heilmann; Christian Hengstenberg; Per Hoffmann; Oddgeir L Holmen; Kristian Hveem; Jan-Håkan Jansson; Karl-Heinz Jöckel; Thorsten Kessler; Jennifer Kriebel; Karl L Laugwitz; Eirini Marouli; Nicola Martinelli; Mark I McCarthy; Natalie R Van Zuydam; Christa Meisinger; Tõnu Esko; Evelin Mihailov; Stefan A Escher; Maris Alver; Susanne Moebus; Andrew D Morris; Jarma Virtamo; Majid Nikpay; Oliviero Olivieri; Sylvie Provost; Alaa AlQarawi; Neil R Robertson; Karen O Akinsansya; Dermot F Reilly; Thomas F Vogt; Wu Yin; Folkert W Asselbergs; Charles Kooperberg; Rebecca D Jackson; Eli Stahl; Martina Müller-Nurasyid; Konstantin Strauch; Tibor V Varga; Melanie Waldenberger; Lingyao Zeng; Rajiv Chowdhury; Veikko Salomaa; Ian Ford; J Wouter Jukema; Philippe Amouyel; Jukka Kontto; Børge G Nordestgaard; Jean Ferrières; Danish Saleheen; Naveed Sattar; Praveen Surendran; Aline Wagner; Robin Young; Joanna M M Howson; Adam S Butterworth; John Danesh; Diego Ardissino; Erwin P Bottinger; Raimund Erbel; Paul W Franks; Domenico Girelli; Alistair S Hall; G Kees Hovingh; Adnan Kastrati; Wolfgang Lieb; Thomas Meitinger; William E Kraus; Svati H Shah; Ruth McPherson; Marju Orho-Melander; Olle Melander; Andres Metspalu; Colin N A Palmer; Annette Peters; Daniel J Rader; Muredach P Reilly; Ruth J F Loos; Alex P Reiner; Dan M Roden; Jean-Claude Tardif; John R Thompson; Nicholas J Wareham; Hugh Watkins; Cristen J Willer; Nilesh J Samani; Heribert Schunkert; Panos Deloukas; Sekar Kathiresan Journal: J Am Coll Cardiol Date: 2017-02-21 Impact factor: 24.094