Effects ofNT5C2germline variants on 6-mecaptopurine metabolism in children with acute lymphoblastic leukemia

Abstract

Abstract6-mercaptopurine (6-MP) is widely used in the treatment of acute lymphoblastic leukemia (ALL), and its cytotoxicity is primarily mediated by thioguanine nucleotide metabolites (TGN). Recent genome-wide association study has identified germline polymorphisms (e.g., rs72846714) in theNT5C2gene associated with 6-MP metabolism in patients with ALL. However, the full spectrum of genetic variation inNT5C2is unclear and its impact on 6-MP drug activation has not been comprehensively examined. To this end, we performed targeted sequencing ofNT5C2in 588 children with ALL and identified 121 single nucleotide polymorphisms (SNPs) nominally associated with erythrocyte TGN during 6-MP treatment (P< 0.05). Of these, 61 variants were validated in a replication cohort of 372 children with ALL. After considering linkage disequilibrium and multivariate analysis, we confirmed two clusters of variants, represented by rs72846714 and rs58700372, that independently affected 6-MP metabolism. Functional studies showed that rs58700372 directly altered the activity of an intronic enhancer, with the variant allele linked to higher transcription activity and reduced 6-MP metabolism (lower TGN). By contrast, rs72846714 was not located in a regulatory element and instead its association signal was explained by linkage disequilibrium with a proximal functional variant rs12256506 that activatedNT5C2transcription in-cis. Our results indicated thatNT5C2germline variation significantly contributes to inter-patient variability in thiopurine drug disposition.