Importance: Understanding antidepressant mechanisms could help design more effective and tolerated treatments. Objective: Identify DNA methylation (DNAm) changes associated with antidepressant exposure. Design: Case-control methylome-wide association studies (MWAS) of antidepressant exposure were performed from blood samples collected between 2006-2011 in Generation Scotland (GS). The summary statistics were tested for enrichment in specific tissues, gene ontologies and an independent MWAS in the Netherlands Study of Depression and Anxiety (NESDA). A methylation profile score (MPS) was derived and tested for its association with antidepressant exposure in eight independent cohorts, alongside prospective data from GS. Setting: Cohorts; GS, NESDA, FTC, SHIP-Trend, FOR2107, LBC1936, MARS-UniDep, ALSPAC, E-Risk, and NTR. Participants: Participants with DNAm data and self-report/prescription derived antidepressant exposure. Main Outcome(s) and Measure(s): Whole-blood DNAm levels were assayed by the EPIC/450K Illumina array (9 studies, Nexposed = 661, Nunexposed= 9,575) alongside MBD-Seq in NESDA (Nexposed= 398, Nunexposed= 414). Antidepressant exposure was measured by self-report and/or antidepressant prescriptions. Results: The self-report MWAS (N = 16,536, Nexposed = 1,508, mean age = 48, 59% female) and the prescription-derived MWAS (N = 7,951, Nexposed = 861, mean age = 47, 59% female), found hypermethylation at seven and four DNAm sites (p < 9.42x10-8), respectively. The top locus was cg26277237 (KANK1, pself-report= 9.3x10-13, pprescription = 6.1x10-3). The self-report MWAS found a differentially methylated region, mapping to DGUOK-AS1 (padj = 5.0x10-3) alongside significant enrichment for genes expressed in the amygdala, the synaptic vesicle membrane gene ontology and the top 1% of CpGs from the NESDA MWAS (OR = 1.39, p < 0.042). The MPS was associated with antidepressant exposure in meta-analysed data from external cohorts (Nstudies= 9, N = 10,236, Nexposed = 661,β = 0.196, p < 1x10-4). Conclusions and Relevance: Antidepressant exposure is associated with changes in DNAm across different cohorts. Further investigation into these changes could inform on new targets for antidepressant treatments.

REM is an advisor to the Epigenetic Clock Development Foundation and Optima Partners. HJG. has received travel grants and speaker honoraria from Fresenius Medical Care, Neuraxpharm, Servier, and Janssen Cilag, as well as research funding from Fresenius Medical Care. H.J.G. had personal contracts approved by the university administration for speaker honoraria and one IIT with Fresenius Medical Care. TK received unrestricted educational grants from Servier, Janssen, Recordati, Aristo, Otsuka. All other authors report no biomedical financial interests or potential conflicts of interest.

ED was supported by the UK Research and Innovation (Grant No. EP/S02431X/1), UK Research and Innovation Centre for Doctoral Training in Biomedical AI at the University of Edinburgh, School of Informatics. This work was supported by the United Kingdom Research and Innovation (grant EP/S02431X/1), UKRI Centre for Doctoral Training in Biomedical AI at the University of Edinburgh, School of Informatics. For the purpose of open access, the author has applied a creative commons attribution (CC BY) licence to any author accepted manuscript version arising. AMM is also supported by a UK Research and Innovation award (Grant No. MR/W014386/1) and by European Union Horizon 2020 funding (Grant No. 847776). GS & STRADL This work was supported by 3 Wellcome Trust grants (220857/Z/20/Z, 226770/Z/22/Z and 104036/Z/14/Z [AMM]). Funding from the Biotechnology and Biological Sciences Research Council and Medical Research Council (Grant Nos. MR/X003434/1 and MR/W014386/1) and the European Union (Grant agreement 847776) is also gratefully acknowledged. The Psychiatric Genomics Consortium (including AMM) has received major funding from the U.S. National Institute of Mental Health (Grant No. 5 U01MH109528-03). Finnish Twin Cohort (FTC) Phenotype and genotype data collection in FT12 and FT16 studies of the Finnish twin cohort has been supported by the Wellcome Trust Sanger Institute, the Broad Institute, ENGAGE, European Network for Genetic and Genomic Epidemiology, FP7-HEALTH-F4-2007, grant agreement number 201413, National Institute of Alcohol Abuse and Alcoholism (grants AA-12502, AA-00145, and AA-09203 to R J Rose; AA15416 and K02AA018755 to D M Dick; R01AA015416 to J Salvatore) and the Academy of Finland (grants 100499, 205585, 118555, 141054, 264146, 308248, 265240, 263278 to J Kaprio, 328685, 307339, 297908 and 251316 to M Ollikainen, and Centre of Excellence in Complex Disease Genetics grants 312073, 336823, and 352792 to J Kaprio), NIH/NHLBI (grant HL104125 to X Wang), and Sigrid Juselius Foundation to M Ollikainen and J Kaprio. Study of Health in Pomerania (SHIP-Trend) SHIP is part of the Community Medicine Research net of the University of Greifswald which is funded by the Federal Ministry of Education and Research (01ZZ9603, 01ZZ0103, and 01ZZ0403), the Ministry of Cultural Affairs and the Social Ministry of the Federal State of Mecklenburg-West Pomerania. DNA methylation data have been supported by the DZHK (grant 81X3400104). The University of Greifswald is a member of the Cache Campus program of the InterSystems GmbH. FOR2107 The German multicenter consortium Neurobiology of Affective Disorders. A translational perspective on brain structure and function is funded by the German Research Foundation (Research Unit FOR2107). Principal investigators are Tilo Kircher (speaker FOR2107, DFG grant numbers KI588/14-1, KI588/14-2, KI588/20-1, KI588/22-1, KI 588/15-1, KI 588/17-1), Udo Dannlowski (co-speaker FOR2107; DA1151/5-1, DA1151/5-2, DA1151/6-1), Axel Krug (KR3822/5-1, KR3822/7-2), Igor Nenadic (NE2254/1-2, NE2254/2-1, NE2254/3-1, NE2254/4-1), Carsten Konrad (KO4291/3-1), Marcella Rietschel (RI 908/11-1, RI 908/11-2), Markus Nothen (NO 246/10-1, NO 246/10-2), Stephanie Witt (WI 3439/3-1, WI 3439/3-2). Tilo Kircher received unrestricted educational grants from Servier, Janssen, Recordati, Aristo, Otsuka, neuraxpharm. NTR This work was supported by the Royal Dutch Academy for Arts and Science (KNAW) Academy Professor Award (PAH/6635) to DIB; the Netherlands Organization for Scientific Research (NWO 480-15-001/674) and Biobanking and Biomolecular Resources Research Infrastructure (BBMRI-NL: 184.021.007; 184.033.111). MARS-UniDep The MARS cohort was sponsored by the Max Planck Society. The UniDep cohort was funded by the Bavarian Ministry of Commerce and by the Federal Ministry of Education and Research in the framework of the National Genome Research Network, Foerderkennzeichen 01GS0481 and the Bavarian Ministry of Commerce. DNA methylation analysis of a subset of both cohorts was financed by ERA-NET NEURON. Lothian Birth Cohort 1936 (LBC1936) The LBC1936 is supported by the Biotechnology and Biological Sciences Research Council, and the Economic and Social Research Council [BB/W008793/1] (which supports SEH), Age UK (Disconnected Mind project), the Medical Research Council [G0701120, G1001245, MR/M013111/1, MR/R024065/1], the Milton Damerel Trust, and the University of Edinburgh. SRC is supported by a Sir Henry Dale Fellowship jointly funded by the Wellcome Trust and the Royal Society (221890/Z/20/Z). Methylation typing of was supported by Centre for Cognitive Ageing and Cognitive Epidemiology (Pilot Fund award), Age UK, The Wellcome Trust Institutional Strategic Support Fund, The University of Edinburgh, and The University of Queensland. ALSPAC The UK Medical Research Council and Wellcome (Grant Ref: 217065/Z/19/Z) and the University of Bristol provide core support for ALSPAC. ASFK is funded by a Wellcome Early Career Award (Grant ref: 227063/Z/23/Z). A comprehensive list of grants funding is available on the ALSPAC website (http://www.bristol.ac.uk/alspac/external/documents/grant-acknowledgements.pdf). This publication is the work of the authors and ED, PY & ASFK will serve as guarantors for the ALSPAC contents of this paper. PY's work is supported by the National Institute for Health and Care Research Bristol Biomedical Research Centre and the Medical Research Council Integrative Epidemiology Unit at the University of Bristol (MC_UU_00032/4, MC_UU_00032/6). The views expressed are those of the authors and not necessarily those of the NIHR or the Department of Health and Social Care. E-Risk The E-Risk Study is funded by grants from the UK Medical Research Council [G1002190; MR/X010791/1]. Additional support was provided by the US National Institute of Child Health and Human Development [HD077482] and the Jacobs Foundation. Chloe C. Y. Wong was supported by the National Institute for Health and Care Research (NIHR) Maudsley Biomedical Research Centre at South London and Maudsley NHS Foundation Trust and Kings College London [NIHR203318]. Helen L. Fisher was supported by the Economic and Social Research Council (ESRC) Centre for Society and Mental Health at Kings College London [ES/S012567/1]. The views expressed are those of the authors and not necessarily those of the NIHR, the Department of Health and Social Care, the ESRC, or Kings College London.

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:

Generation Scotland: Ethical approval was provided by the Tayside Research Ethics Committee (REC reference 05/S1401/89). STRADL: All components of STRADL received formal, national ethical approval from the NHS Tayside committee on research ethics (reference 14/SS/0039). Finnish Twin Cohort (s): Ethics approvals have been granted for multiple studies concerning the FTC twins by the ethics committees of Helsinki University Central Hospital (113/E3/2001, 249/E5/2001, 346/E0/05, 270/13/03/01/2008, and 154/13/03/00/2011) with the last one on the transfer of biological samples to the THL Biobank in 2018 (HU51179912017). Study of Health in Pomerania (SHIP-Trend):The Ethics Committee of the University Medicine Greifswald, Germany provided ethical approval for the study (BB 39/08). FOR2107: The ethics committees of the Medical Faculties, University of Marburg (AZ: 07/14) and University of Munster (2014-422-b-S) provided ethical approval for the study. Netherlands Twin Study: The study was approved by the Central Ethics Committee on Research Involving Human Subjects of the VU university Medical Centre, Amsterdam, an Institutional Review Board certified by the U.S Office of Human Research Protections (IRB number IRB00002991 under Federal-wide Assurance-FWA00017598, IRB/institute codes, NTR 03-180). Munich Antidepressant Response Study (MARS): The study was approved by the local Ethics Committee of the Ludwig Maximilians University, Munich, Germany, and carried out in accordance with the latest version of the Declaration of Helsinki. All participants provided written consent after the study protocol and potential risks were explained. UniPolar Depression Study: The study was approved by the Ethics committee of the Ludwig Maximillans University in Munich, Germany and written informed consent was obtained from all subjects. Lothian Birth Cohort 1936: Ethical approval was obtained from the Multicentre Research Ethics Committee for Scotland (baseline, MREC/01/0/56), the Lothian Research Ethics Committee (age 70, LREC/2003/2/29), and the Scotland A Research Ethics Committee (ages 73, 76, 79, 07/MRE00/58). Avon Longitudinal Study of Parents and Children: Ethical approval for the study was obtained from the ALSPAC Ethics and Law Committee and the Local Research Ethics Committees. Consent for biological samples has been collected in accordance with the Human Tissue Act (2004). Environmental Risk (E-Risk) Longitudinal Twin Study: The Joint South London and Maudsley and Institute of Psychiatry Research Ethics Committee approved each study phase. Parents gave informed consent, and twins gave assent between 5 and 12 years and then informed consent at age 18.

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