A GENOME WIDE ASSOCIATION ANALYSIS ON NITRENIUM-DERIVED CLOZAPINE METABOLITES – A STUDY WITH 306 SUBJECTS WITH KNOWN SMOKING STATUS

Background and aim Clozapine is the most effective antipsychotic for treatment of schizophrenia, yet its clinical use is limited by potentially life-threatening side effects including agranulocytosis and myocarditis. It has been suggested that bioactivation of clozapine to its reactive toxic metabolites, i.e., nitrenium ions, may be underlying these side effects. Nitrenium ions are detoxified by glutathione S-transferase mediated conjugation pathways to clozapine cysteinyl metabolites. Currently, the extent to which genetic variation modulates the formation or inactivation of cysteinyl metabolites remains unknown. Therefore, we performed a genome-wide association study on nitrenium-derived cysteinyl metabolites of clozapine. Methods The study included genotyped subjects treated with clozapine from the therapeutic drug monitoring and pharmacogenetics service at Center for Psychopharmacology, Diakonhjemmet Hospital, Oslo, Norway during 2018-2025. A novel ultra performance liquid chromatography and high-resolution mass spectrometry (UPLC-HRMS) assay was used for identification and semi-quantitative measurements of nitrenium-derived clozapine metabolites (i.e., clozapine-Cys1+, clozapine-NOX-Cys1+, norclozapine-Cys1+, and clozapine-8Cys1+). The metabolite-to-clozapine metabolic ratios were included as phenotypes in the GWAS adjusting for participants’ smoking habits, which is the most important non-genetic determinant of clozapine metabolism. Results The study included 306 subjects treated with clozapine. A genome-wide significant locus, represented by the lead intronic rs59698651 C > G polymorphism (ROBO1 gene), was significantly associated with the clozapine-8Cys1+-to-clozapine metabolic ratio (p = 4.28e-08). Previous studies suggest that ROBO1 plays a role in regulating cell migration in hematological malignancies and inflammatory diseases. Conclusion Our findings may indicate that pharmacogenetic variability may underlie individual variability in formation or inactivation of clozapine nitrenium metabolites, which potentially modulate susceptibility to clozapine toxicity.