Antithrombin, protein C and protein S: Genome and transcriptome wide association studies identify 7 novel loci regulating plasma levels

Objective Antithrombin, protein C (PC) and protein S (PS) are circulating natural-anticoagulant proteins that regulate hemostasis and of which partial deficiencies are causes of venous thromboembolism. Previous genetic association studies involving antithrombin, PC, and PS were limited by modest sample sizes or by being restricted to candidate genes. In the setting of the Cohorts for Heart and Aging Research in Genomic Epidemiology consortium, we meta-analyzed across ancestries the results from 10 genome-wide association studies (GWAS) of plasma levels of antithrombin, PC, PS free and PS total. Approach and Results Study participants were of European and African ancestries and genotype data were imputed to TOPMed, a dense multi-ancestry reference panel. Each of 10 studies conducted a GWAS for each phenotype and summary results were meta-analyzed, stratified by ancestry. We also conducted transcriptome-wide association analyses and multi-phenotype analysis to discover additional associations. Novel GWAS findings were validated by in vitro functional experiments. Mendelian randomization was performed to assess the causal relationship between these proteins and cardiovascular outcomes. GWAS meta-analyses identified 4 newly associated loci: 3 with antithrombin levels ( GCKR, BAZ1B , and HP-TXNL4B ) and 1 with PS levels ( ORM1 - ORM2 ). TWAS identified 3 newly associated genes: 1 with antithrombin level ( FCGRT ), 1 with PC ( GOLM2 ), and 1 with PS ( MYL7 ). In addition, we replicated 7 independent loci reported in previous studies. Functional experiments provided evidence for the involvement of GCKR, SNX17 , and HP genes in antithrombin regulation. Conclusion The use of larger sample sizes, diverse populations, and a denser imputation reference panel allowed the detection of 7 novel genomic loci associated with plasma antithrombin, PC, and PS levels. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This study is supported in part by the National Heart, Lung, and Blood Institute grant HL134894 and HL139553; infrastructure for the CHARGE Consortium is supported in part by HL105756. The views expressed in this manuscript are those of the authors and do not necessarily represent the views of the National Heart, Lung, and Blood Institute; the National Institutes of Health; or the US Department of Health and Human Services. G. Temprano-Sagrera is supported by the Pla Estrategic de Recerca i Innovacio en Salut (PERIS) grant from the Catalan Department of Health for junior research personnel (SLT017/20/000100). P de Vries is supported by American Heart Association grant 17POST33350042. M. Sabater-Lleal is supported by a Miguel Servet contract from the ISCIII Spanish Health Institute (CP17/00142) and co-financed by the European Social Fund, and acknowledges funding from the CERCA Programme/Generalitat de Catalunya. Sources of funding for the specific cohorts can be found in the online-only Data Supplement. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The ARIC study was approved by the UNC Office of Human Research Ethics/Institutional Review Board (OHRE/IRB); University of Mississippi Medical Center IRB, Wake Forest University Health Sciences IRB, University of Minnesota IRB, and John Hopkins University IRB. CHRIS study was approved by the Ethical Committee of the Healthcare System of the Autonomous Province of Bolzano CHS was approved by the Wake Forest University Health Sciences IRB, University of California, Davis IRB, John Hopkins University IRB, and University of Pittsburgh IRB, and University of Washington IRB GAIT was approved by the Institutional Review Board of the Hospital de la Santa Creu i Sant Pau, Barcelona, Spain The HVH study is part of data repository which is under the oversight of the University of Washington Human Subjects Division. LURIC was approved by the Ethics Committee at the Arztekammer Rheinland-Pfalz MARTHA was approved by the Health Department of the General Directorate for the French Ministry of Research and Innovation (Projects DC: 2008-880 and 09.576) RETROVE was approved by Institutional Review Board of the Hospital de la Santa Creu i Sant Pau, Barcelona, Spain The Genes and Blood Clotting study GABC was approved by the University of Michigan IRB. #HUM00043243. For the Trinity Student Study (TSS), ethical approval was obtained from the Dublin Federated Hospitals Research Ethics Committee, which is affiliated with the Trinity College, and reviewed by the Office of Human Subjects Research at the National Institutes of Health. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors and will be available through dbGAPs after publication. * TOPMed : Trans-Omic for Precision Medicine PC : protein C PS : protein S VTE : venous thromboembolism CAD : coronary artery disease PAD : peripheral artery disease IS : ischemic stroke GWAS : genome-wide association study TWAS : transcription-wide association study EA : European ancestry AA : African ancestry eQTL : expression quantitative trait locus