A methylome-wide association study of major depression with out-of-sample case-control classification and trans-ancestry comparison

Major Depression (MD) is a leading cause of global disease burden, and both experimental and population-based studies suggest that differences in DNA methylation (DNAm) may be associated with the condition. However, previous DNAm studies have not so far been widely replicated, suggesting a need for larger meta-analysis studies. In the present study, the Psychiatric Genomics Consortium Major Depressive Disorder working group conducted a meta-analysis of methylome-wide association analysis (MWAS) for life-time MD across 18 studies of 24,754 European-ancestry participants (5,443 MD cases) and an East Asian sample (243 cases, 1846 controls). We identified fifteen CpG sites associated with lifetime MD with methylome-wide significance (p < 6.42×10-8). Top CpG effect sizes in European ancestries were positively correlated with those from an independent East Asian MWAS (r = 0.482 and p = 0.068 for significant CpG sites, r = 0.261 and p = 0.009 for the top 100 CpG sites). Methylation score (MS) created using the MWAS summary statistics was significantly associated with MD status in an out-of-sample classification analysis (β = 0.122, p = 0.005, AUC = 0.53). MS was also associated with five inflammatory markers, with the strongest association found with Tumor Necrosis Factor Beta (β=-0.154, p=1.5×10-5). Mendelian randomisation (MR) analysis demonstrated that 23 CpG sites were potentially causally associated with MD and six of those were replicated in an independent mQTL dataset (Wald’s ratio test, absolute β ranged from 0.056 to 0.932, p ranged from 7×10-3 to 4.58×10-6). CpG sites located in the Major Histocompatibility complex (MHC) region showed the strongest evidence from MR analysis of being associated with MD. Our study provides evidence that variations in DNA methylation are associated with MD, and further evidence supporting involvement of the immune system. Larger sample sizes in diverse ancestries are likely to reveal replicable associations to improve mechanistic inferences with the potential to inform molecular target identification. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement Generation Scotland (GS) This work is supported by three Wellcome Trust grants (220857/Z/20/Z, 104036/Z/14/Z, 216767/Z/19/Z). DNAm profiling was supported by funding from NARSAD (Ref 27404, DMH) and the Royal College of Physicians of Edinburgh (SIM Fellowship, HCW). Genotyping of the GS samples was funded by the MRC and Wellcome Trust [104036/Z/14/Z]. GS also receives support from the Chief Scientist Office of the Scottish Government Health Directorates [CZD/16/6] and the Scottish Funding Council [HR03006]. Avon Longitudinal Study of Parents and Children (ALSPAC) / Accessible Resource for Integrated Epigenomic Studies (ARIES) The UK Medical Research Council and Wellcome (Grant Ref: 217065/Z/19/Z) and the University of Bristol provide core support for ALSPAC. A comprehensive list of grants funding is available on the ALSPAC website (). Part of this data was collected using REDCap, see the REDCap website for details ). We thank all the families who took part in this study, the midwives for their help in recruiting them, and the whole ALSPAC team, which includes interviewers, computer and laboratory technicians, clerical workers, research scientists, volunteers, managers, receptionists and nurses. BeCOME/OPTIMA Funding: OPTIMA and BeCOME are funded by the Max Planck Society. Acknowledgements: We would like to thank all contributors to the research project including physicians, psychologists, study nurses, researchers and research assistants, and of course patients of the hospital of the Max Planck Institute of Psychiatry in Munich. BiDirect The BiDirect Study is supported by grants of the German Ministry of Research and Education (BMBF) to the University of Muenster (01ER0816 and 01ER1506). Biopsychosocial factors of major depression in youth (BioMD-Y/LMU) BioMD-Y was supported by the Randebrock Stiftung associated with the kbo-Heckscher-Klinikum, Munich, Germany during the years 2009-2011 upon its inception, and has since been financed through intramural funds. DNA extraction, genome-wide genotyping and DNA methylation analyses were supported by the Max Planck Society. DNA Methylation of the BioMD-Y/LMU cohort was funded in part by Sabine Schaefer, private Donor of the Max-Planck-Foundation. Dublin cohort This research was supported by a grant from AbbVie (no. 10118) to HM, TMM and EMcD. EMcD was the recipient of the Boston Scientific Newman Fellowship awarded by the UCD Foundation. The authors wish to thank all the participants in the Dublin Cohort and all researchers involved in the original IBD Study. Environmental Risk (E-Risk) Longitudinal Twin Study 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. This project represents independent research part funded by the NIHR Maudsley Biomedical Research Centre at South London and Maudsley NHS Foundation Trust and King's College London. The views expressed are those of the authors and not necessarily those of the NIHR or the Department of Health and Social Care [NIHR203318]. Helen L. Fisher was supported by the UK ESRC Centre for Society and Mental Health at King's College London [ES/S012567/1]. The views expressed are those of the authors and not necessarily those of the ESRC or King's College London. We are grateful to the E-Risk study mothers and fathers, the twins, and the twins' teachers for their participation. Our thanks to the E-Risk team for their dedication, hard work, and insights. EXTEND This study was funded by the National Institute for Health and Care Research Exeter Clinical Research Facility. This study was supported* by the National Institute for Health and Care Research Exeter Biomedical Research Centre. The views expressed are those of the author(s) and not necessarily those of the NIHR or the Department of Health and Social Care. Finnish Twin Cohort Phenotype and genotype data collection in FinnTwin12 and FinnTwin16 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 Jessica Salvatore) and the Academy of Finland (grants 100499, 205585, 118555, 141054, 264146, 308248 to JK, 328685, 307339, 297908 and 251316 to MO, 309119 to TK) and Sigrid Juselius Foundation to MO and JK. and the Centre of Excellence in Complex Disease Genetics (grants 312073, 336823, and 352792 to JKaprio). JKaprio acknowledges support by the Academy of Finland (grants 265240, 263278). Tellervo Korhonen acknowledges support from the Juho Vainio Foundation. We would like to thank the active participation of the twins, which has made the studies possible. 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 (KI588/14-1, KI588/14-2,, KI588/20-1, KI588/22-1), Udo Dannlowski (DA1151/5-1, DA1151/5-2), Axel Krug (KR3822/5-1, KR3822/7-2), Igor Nenadic (NE2254/1-2,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. We are deeply indebted to all study participants and staff. A list of acknowledgments can be found here: [www.for2107.de/acknowledgements][1]. Janssen Funding: The Janssen cohort was funded by Janssen Research & Development, LLC. Acknowledgements: We thank the clinical investigators and research coordinators who ran the clinical study and collected the blood samples used in this study, as well as study participants and their families, whose help and participation made this work possible. We thank the staff from Cancer Genetic Institute and HD Bioscience for performing the DNA extraction from whole blood samples, plating, QC, and the staff from Illumina to perform the epigenetic and genotyping assays to enable the data generation. Conflict of interest: QSL is an employee of Janssen Research & Development, LLC. QSL owns stock and/or stock options in Johnson & Johnson. Munich Antidepressant Response Signature (MARS/GSK) Funding: 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. Acknowledgements: We would like to thank all contributors to the research project including physicians, psychologists, study nurses, researchers and research assistants, and of course patients of the hospital of the Max Planck Institute of Psychiatry in Munich and psychiatric hospitals in Augsburg and Ingolstadt. 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. Netherlands Twin Register (NTR) We warmly thank all twin families of the Netherlands Twin Register who make this research possible. 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). Study of Health in Pomerania (SHIP) 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. The SHIP authors are grateful to Paul S. DeVries for his support with the EWAS pipeline. Conflict of interest: HJG has received travel grants and speakers' honoraria from Fresenius Medical Care, Neuraxpharm, Servier and Janssen Cilag. Taiwan Biobank This work is supported by Population Health Research Center from Featured Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education in Taiwan (grant number NTU-112L9004). We thank all the participants and investigators of the Taiwan Biobank. TwinsUK The study received support from the ESRC (ES/N000404/1 to J.T.B) and European HDHL Joint Programming Initiative funding scheme DIMENSION project (BBSRC BB/S020845/1 and BB/T019980/1 to J.T.B.). The TwinsUK study is funded by the Wellcome Trust, Medical Research Council, Versus Arthritis, European Union Horizon 2020, Chronic Disease Research Foundation (CDRF), ZOE LIMITED, and the National Institute for Health Research (NIHR) Clinical Research Network (CRN) and Biomedical Research Centre based at Guy's and St Thomas' NHS Foundation Trust in partnership with King's College London. Understanding Society/ UK Household Longitudinal Study (UKHLS) We acknowledge the Wellcome Trust Sanger Institute and Ele Zeggini for generating the genotype data and the School of Life Sciences, University of Essex for QC of the methylation data. Both genotyping and DNA methylation in UKHLS were funded through enhancements to the Economic and Social Research Council (ESRC) grants ES/K005146/1 and ES/N00812X/1. MK is partially supported by the ESRC (ES/S012486/1). AD and ERW are Soc-B students (ESRC project reference numbers 2765580 and 2604212 respectively). We would also like to acknowledge the summary statistics provided by the Psychiatric Genomics Consortium. In addition, we would like to thank the research participants and employees of 23andMe, Inc. for making this work possible. ### 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: Written informed consent was obtained for all participants. Individual ethical approval for summary statistics were obtained and detailed in the cited protocol studies. Individual-level data from Generation Scotland used in the study was approved by the NHS Tayside Research Ethics committee (05/s1401/89). Ethics permission for LBC1921 was obtained from the Lothian Research Ethics Committee (LREC/1998/4/183). Ethics permission for LBC1936 was obtained from the Multi-Centre Research Ethics Committee for Scotland (MREC/01/0/56) and the Lothian Research Ethics Committee (LREC/2003/2/29) [25, 26]. 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, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All summary statistics produced in the present study are available upon reasonable request to the authors upon publication. Individual-level data requires approval of proposed analyses and data transfer agreement (if applicable). [1]: http://www.for2107.de/acknowledgements