Longitudinal changes in DNA methylation associated with clozapine use in treatment-resistant schizophrenia from two international cohorts

The second-generation antipsychotic clozapine is used as a medication for treatment-resistant schizophrenia. It has previously been associated with epigenetic changes in pre-clinical rodent models and cross-sectional studies of treatment-resistant schizophrenia. Cross-sectional studies are susceptible to confounding, however, and cannot disentangle the effects of diagnosis and medication. We therefore profiled DNA methylation in sequential blood samples (n=126) from two independent cohorts of patients (n=38) with treatment-resistant schizophrenia spectrum disorders who commenced clozapine after study enrolment and were followed up for up to six months. We identified significant non-linear changes in cell-type proportion estimates derived from DNA methylation data - specifically B-cells - associated with time on clozapine. Mixed effects regression models were used to identify changes in DNA methylation at specific sites associated with time on clozapine, identifying 37 differentially methylated positions (DMPs) (p < 5×10−5) in a linear model and 90 DMPs in a non-linear quadratic model. We compared these results to data from our previous epigenome-wide association study (EWAS) meta-analysis of psychosis, finding evidence that many previously identified DMPs associated with schizophrenia and treatment-resistant schizophrenia might reflect exposure to clozapine. In conclusion, our results indicate that clozapine exposure is associated with changes in DNA methylation and cellular composition. Our study shows that medication effects might confound many case-control studies of neuropsychiatric disorders performed in blood. ### Competing Interest Statement Unrelated to this work, A.L.G has received consultancy fees from Zogenix. A.H was member of advisory boards of Boehringer-Ingelheim, Lundbeck, Janssen, Otsuka, Rovi and Recordati and received paid speakership by these companies as well as by AbbVie and Advanz. He is editor of the German schizophrenia guideline. All other authors have nothing to disclose. ### Funding Statement This work was funded by the Medical Research Council, UK, Grant MR/L003988/1 to A.E. and by the European Community's Seventh Framework Programme (FP7/2007-2013), grant 279227 to J.H.M. This work was also supported by grants from the UK Medical Research Council (MRC; MR/K013807/1 and MR/R005176/1) to J.M. High-performance computing was supported by MRC Clinical Research Infrastructure Funding (MR/M008924/1) to J.M. This study presents independent research funded in part by the NIHR Maudsley Biomedical Research Centre at South London and Maudsley NHS Foundation Trust and King's College London. This work was also supported by the National Institute for Health and Care Research Exeter Biomedical Research Centre and Oxford Health 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. This study was endorsed by DZPG (German Center for Mental Health) (to A.H., FKZ: 01EE2303C). This work was supported by a personal Rudolf Magnus fellowship award to J.J.L (H-150) from the University Medical Centre Utrecht, Utrecht, The Netherlands. A.A. was supported by Fundacao para a Ciencia e a Tecnologia, scholarship ID SFRH/BD/115916/2016. ### 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: London South East NHS ethics committee gave ethical approval for this work (Ref: 13/LO/1857) 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 data produced are available online. GEO acession number GSE237561