Cross-tissue regulatory gene networks in coronary atherosclerosis

Coronary artery disease (CAD) is the underlying cause of myocardial infarction and stroke that together are responsible for nearly 30% of all global deaths. CAD is a common complex disease caused by the interactions of multiple genetic and environmental risk factors acting across several metabolic and vascular tissues. Owing to the complexity of these interactions, systems genetics is an increasingly recognized path to a better understanding of complex diseases. In this thesis, we applied systems genetics by integrating the analysis of genotype (DNA) and global gene expression (RNA) data from metabolic and vascular tissues with phenotype data from the clinically well-characterized subjects in the Stockholm Atherosclerosis Gene Expression (STAGE) study. We validated the initial findings using genome-wide association studies (GWAS) and several gene expression datasets from mice and cell models. As a result, we for the first time inferred regulatory gene networks (RGNs) with key drivers of CAD, several of its main risk factors and atherosclerosis regression. In paper I, we designed a computational pipeline to reconstruct RGNs with key drivers in CAD using the STAGE study. Then, by integrating expression quantitative traits (eQTLs) of these RGNs with genotype data from several GWAS, 30 CAD-causal RGNs interconnected in blood, vascular and metabolic tissues were identified. Twelve of these RGNs were further validated in gene expression and phenotype data from the Hybrid Mouse Diversity Panel. As proof of concept, by targeting the key drivers AIP, DRAP1, POLR2I, and PQBP1 in a crossspecies-validated, arterial-wall RGN involving RNA-processing genes, we re-identified this RGN in THP-1 foam cells and independent gene expression data from CAD macrophages and carotid lesions. In paper II, we developed a cross-tissue weighted gene co-expression network analysis (XWGCNA) method (used in Paper I) that reliably captures gene activities both within and across tissues. X-WGCNA is implemented as a package in R and is available online. In paper III, we inferred transcription factor (TF) RGNs from three plasma cholesterol lowering (PCL)-responsive gene sets causally related to regression of early, mature, and advanced mouse atherosclerosis. We then used THP-1 cells in an in vitro atherosclerosis regression model to successfully validate 3 key drivers in these RGNs driving regression in early (PPARG), mature (MLL5), and advanced (SRSF10/XRN2) atherosclerosis. In paper IV, we inferred the STAGE eQTLs (used in papers I and II) and identified subsets with gene regulatory effects across multiple tissues that according to GWAS were highly enriched in association with CAD. To better understand the pathophysiological role of these multi-tissue eQTLs, we identified and analyzed a number of associated gene sets. A key result of this thesis is a repository of RGNs with key drivers for CAD, CAD risk factors, and atherosclerosis regression. This repository together with the computational pipeline including X-WGCNA should be useful in future studies that aim to go beyond genetic loci identified by GWAS and provide opportunities for novel diagnostics and therapies. LIST OF SCIENTIFIC PAPERS I. Cross-Tissue Regulatory Gene Networks in Coronary Artery Disease. HUSAIN A. TALUKDAR, Hassan Foroughi Asl, Rajeev K. Jain, Raili Ermel, Arno Ruusalepp, Oscar Franzén, Brian A. Kidd, Ben Readhead, Chiara Giannarelli, Jason C. Kovacic, Torbjörn Ivert, Joel T. Dudley, Mete Civelek, Aldons J. Lusis, Eric E. Schadt, Josefin Skogsberg, Tom Michoel, and Johan L.M. Björkegren Cell Systems, 2016, 2, 3:196-208 † Editorial Preview: Deciphering the Dark Matter of Complex Genetic Inheritance; Crawford, Nigel P.S. ; Cell Systems, 2016, 2, 3:144-146 ‡ ECCB 2016: True scientific highlights of the year in bioinformatics and computational biology (Systems category) II. X-WGCNA: an R package for Cross-Tissue Weighted Gene Coexpression Network Analysis. HUSAIN A. TALUKDAR, Yifan Mo, Hassan Foroughi Asl, Josefin Skogsberg, Johan Bjorkegren and Tom Michoel. Manuscript III. Plasma Cholesterol–Induced Lesion Networks Activated before Regression of Early, Mature, and Advanced Atherosclerosis. Johan L. M. Björkegren, Sara Hägg, HUSAIN A. TALUKDAR*, Hassan Foroughi Asl*, Rajeev K. Jain, Cecilia Cedergren, Ming-Mei Shang, Aránzazu Rossignoli, Rabbe Takolander, Olle Melander, Anders Hamsten, Tom Michoel, and Josefin Skogsberg PLOS GENETICS, 2014, 10(2), e1004201 *Equal contribution IV. Expression quantitative trait Loci acting across multiple tissues are enriched in inherited risk for coronary artery disease. Hassan Foroughi Asl, HUSAIN A. TALUKDAR, Alida S.D. Kindt, Rajeev K. Jain, Raili Ermel, Arno Ruusalepp, Khanh-Dung H. Nguyen, Radu Dobrin, Dermot F. Reilly, Heribert Schunkert, Nilesh J. Samani, Ingrid Braenne, Jeanette Erdmann, Olle Melander, Jianlong Qi, Torbjörn Ivert, Josefin Skogsberg, Eric E. Schadt, Tom Michoel, Johan L.M. Björkegren, CARDIoGRAM Consortium. Circulation: Cardiovascular Genetics, 2015, 8, 305-315 Related scientific papers but not included in this thesis I. Lim domain binding 2: a key driver of transendothelial migration of leukocytes and atherosclerosis. Shang MM, HUSAIN A. TALUKDAR, Hofmann JJ, Niaudet C, Asl HF, Jain RK, Rossignoli A, Cedergren C, Silveira A, Gigante B, Leander K, de Faire U, Hamsten A, Ruusalepp A, Melander O, Ivert T, Michoel T, Schadt EE, Betsholtz C, Skogsberg J, Björkegren JL. Arterioscler Thromb Vasc Biol. 2014 Sep;34(9):2068-77 II. Cardiometabolic Risk Loci Share Downstream Cisand Trans-Genes Across Tissues and Diseases. The Stockholm-Tartu Atherosclerosis Network Engineering Task (STARNET) Study Oscar Franzén, Raili Ermel, Ariella Cohain, Nicholas K. Akers, Antonio Di Narzo, HUSAIN A. TALUKDAR, Hassan Foroughi-Asl, Claudia Giambartolomei, John F. Fullard, Katyayani Sukhavasi, Sulev Köks, Li-Ming Gan, Chiara Gianarelli, Jason C. Kovacic, Christer Betsholtz, Bojan Losic, Tom Michoel, Ke Hao, Panos Roussos, Josefin Skogsberg, Arno Ruusalepp, Eric E. Schadt and Johan L.M. Björkegren Accepted in Science *Equal contribution III. Poliovirus Receptor-Related 2– A Cholesterol Responsive Gene Affecting Atherosclerosis Development by Modulating Leukocyte Migration Aránzazu Rossignoli, Ming-Mei Shang, Christine Mössinger, Hassan Foroughi Asl, HUSAIN A. TALUKDAR, Oscar Franzén, Erika Folestad, Johan L.M. Björkegren, Josefin Skogsberg Manuscript -in revision IV. Alternatively Spliced Tissue Factor Isoform is a Critical Driver of Foam Cell Formation and Plaque Burden Daniel Alicea Delgado, Aleksey Chudnovskiy, Claudia Calcagno, HUSAIN A. TALUKDAR, Dong Kwong Yang, Dongtak Jeong, Yifan Mo, Saboor Hekmaty, Gustav J. Strijkers, Gerald A. Soff , Jason C. Kovacic, Thomas Weber, Zahi A. Fayad, Juan Badimon, Valentin Fuster, Josefin Skogsberg, Miriam Merad, Johan L.M. Björkegren, Roger J. Hajjar, Chiara Giannarelli Manuscript -in revision TABLE OF CONTENTS