Integrative multi-omics analysis of genomic, epigenomic, and metabolomics data leads to new insights for Attention-Deficit/Hyperactivity Disorder

The evolving field of multi-omics combines data across omics levels and provides methods for simultaneous analysis. We integrated genomics (transmitted and non-transmitted polygenic scores), epigenomics and metabolomics data in a multi-omics framework to identify biomarkers for ADHD and investigated the connections among omics levels. We trained single- and multi-omics models to differentiate between cases and controls in 596 twins (cases=14.8%) from the Netherlands Twin Register (NTR) demonstrating reasonable in-sample prediction through cross-validation. The multi-omics model selected 30 PGSs, 143 CpGs, and 90 metabolites. We confirmed previous associations of ADHD with glucocorticoid exposure and the transmembrane protein family TMEM , show that the DNA methylation of the MAD1L1 gene associated with ADHD has a relation with parental smoking behavior and present novel findings including associations between indirect genetic effects and CpGs of the STAP2 gene. Out-of-sample prediction in NTR participants (N=258, cases=14.3%) and in a clinical sample (N=145, cases=51%) did not perform well (range misclassification: 0.40-0.57). The results highlighted connections between omics levels, with the strongest connections between indirect genetic effects, CpGs, and amino acid levels and show that multi-omics designs considering interrelated omics levels can help unravel the complex biology underlying ADHD. ### Competing Interest Statement Erik A. Ehli was employed by the Avera Institute for Human Genetics (Sioux Falls, SD, United States). The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. ### Funding Statement The current work is supported by the Aggression in Children: Unraveling gene-environment interplay to inform Treatment and InterventiON strategies project (ACTION) and the Consortium on Individual Development (CID). ACTION received funding from the European Union Seventh Framework Program (FP7/2007-2013) under grant agreement no 602768. NH is supported by the Royal Netherlands Academy of Science Professor Award (PAH/6635) to DIB. We acknowledge the CID Gravitation Program of the Dutch Ministry of Education, Culture, and Science and the Netherlands Organization for Scientific Research (NWO grant number 024-001-003). The Netherlands Twin Register is supported by multiple grants from the Netherlands Organizations for Scientific Research (NWO) and Medical Research (ZonMW): Netherlands Twin Registry Repository (NWO 480-15-001/674); Genetic influences on stability and change in psychopathology from childhood to young adulthood (ZonMw 912-10-020); Twin family database for behavior genomics studies (NWO 480-04-004); Twin research focusing on behavior (NWO 400-05-717); Longitudinal data collection from teachers of Dutch twins and their siblings (NWO 481-08-011), Twin-family-study of individual differences in school achievement (NWO-FES, 056-32-010), Genotype/phenotype database for behavior genetic and genetic epidemiological studies (ZonMw Middelgroot 911-09-032); the Biobank-based integrative omics study (BIOS) funded by BBMRI-NL (NWO projects 184.021.007 and 184.033.111); the European Science Council (ERC) Genetics of Mental Illness (ERC Advanced, 230374, PI Boomsma); Developmental trajectories of psychopathology (NIMH 1RC2 MH089995); the Avera Institute for Human Genetics, Sioux Falls, USA. A research visit to the University of Toulouse (France) by FAH was supported by the Faculty of Behavioural and Movement Sciences (FGB) Talent Fund (2019). JvD is supported by NWO Large Scale infrastructures, X-omics (184.034.019). MB is supported by an ERC consolidator grant (WELL-BEING 771057, PI Bartels). ### 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: Parents provided written informed consent for their children and twin parents provided written informed consent for their own participation. Study approval was obtained from the Central Ethics Committee on Research Involving Human Subjects of the VU University Medical Center, Amsterdam (NTR 25th of May 2007 and ACTION 2013/41 and 2014.252), 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), and the Medical Ethical Committee of Leiden University Medical Center (B15.017, B17.031, B17.032 and B17.040). 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 The standardized protocol for large scale collection of urine and buccal-cell samples in the home situation as developed for the ACTION Biomarker Study in children available at . The data of the Netherlands Twin Register (NTR) ACTION Biomarker Study may be accessed, upon approval of the data access committee, through the NTR ([https://tweelingenregister.vu.nl/information\_for\_researchers/working-with-ntr-data][1]) [https://tweelingenregister.vu.nl/information\_for\_researchers/working-with-ntr-data][1] [1]: https://tweelingenregister.vu.nl/information_for_researchers/working-with-ntr-data