Stability Over Time and Between Generations of Differential DNA Methylation Identified by Epigenome-Wide Association Study of Adolescent Adiposity

Background: Obesity predisposes to cardiovascular disease, itself influenced by perinatal factors. Epigenetics is hypothesized to link perinatal influences with later obesity. We aimed to identify, using an epigenome wide association study, differentially methylated CpGs measured at 17-years-old that associated with concurrent adiposity, then to ascertain whether the strongest of these associations were stable over time, between generations, and mediated the association between early life environment and later body composition. Methods: Genome-wide DNA methylation profiles were assayed using Infinium HumanMethylation 450K BeadChip arrays on blood samples from 1050 17-year-olds from The Raine Study (Generation 2). CpGs associated with adiposity measures (anthropometry, subcutaneous/visceral fat thickness by ultrasound) were pyrosequenced at 17 and 20-years-old (n=817), and in their mothers (n=406). Perinatal factors were examined as mediators of associations between CpG methylation and adiposity. Findings: Increased methylation of pyrosequenced CpGs within Musashi RNA Binding Protein 2 (MSI2), solute carrier family 25 member 10 (SLC25A10) and Ras Association (RalGDS/AF-6) Pleckstrin Homology Domains 1 (RAPH1) were associated with adiposity. Adjusting for perinatal factors, these CpGs independently predicted adolescent BMI accounting for 24% of the variance. MSI2 remained associated with BMI over time (β/SD, false discovery adjusted p-values: 17-years-old β=0.026, p=0.0025; 20-years-old β=0.027, p=0.0029), between generations (mother β=0.044, p=7.5e-04), with high sensitivity C-reactive protein (β=0.120, p=0.023) and leptin (β=0.149, p=0.0004). Interpretation: We report novel associations between DNA methylation within MSI2, SLC25A10 and RAPH1 with increased adolescent adiposity. For MSI2 these were stable over time, consistent across mother and child and, associated with inflammation. Funding Statement: The Raine Study Team, The NH&MRC, and The Telethon Kids Institute provided long term support of the Study. The University of Western Australia (UWA), Curtin University, The Telethon Kids Institute, Women and Infants Research Foundation, Edith Cowan University, Murdoch University, The University of Notre Dame Australia and Raine Medical Research Foundation provided funding for Core Management of the Raine Study. The DNA methylation work was supported by NHMRC grant 1059711. The 17 year follow-up was supported by National Health and Medical Research Council Program grant (ID353514) and Project grant (ID403981). RCH and TAM are supported by NHMRC Fellowships (grant number 1053384 and 1042255, respectively). S.R is supported by National Health and Medical Research Council EU grant (1142858) and the Department of Health, Western Australia FutureHealth fund in connection with the European Union's Horizon2020 grant 733206. This work was supported by resources provided by The Pawsey Supercomputing Centre with funding from the Australian Government and the Government of Western Australia. KMG is supported by the UK Medical Research Council (MC_UU_12011/4), the National Institute for Health Research (NIHR Senior Investigator (NF-SI-0515-10042) and the NIHR Southampton Biomedical Research Centre) and the European Union (Erasmus+ Programme Early Nutrition eAcademy Southeast Asia-573651-EPP-1-2016-1-DE-EPPKA2-CBHE-JP). Declaration of Interests: KMG has received reimbursement for speaking at conferences sponsored by companies selling nutritional products, and is part of an academic consortium that has received research funding from Abbott Nutrition, Nestec and Danone. All other authors declare no conflicts of interest. Ethics Approval Statement: Institutional ethics approval has been obtained through the University of Western Australia Ethics Committee (RA/4/1/6613, 1214-EP, RA-4-1-2646). Informed consent was obtained from legal guardians or parents of those under the age of 17 years with assent of the child. Informed consent was obtained for those 17 years and older.