6-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 the NT5C2 gene associated with 6-MP metabolism in patients with ALL. However, the full spectrum of genetic variation in NT5C2 is unclear and its impact on 6-MP drug activation has not been comprehensively examined. To this end, we performed targeted sequencing of NT5C2 in 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 activated NT5C2 transcription in-cis. Our results indicated that NT5C2 germline variation significantly contributes to inter-patient variability in thiopurine drug disposition.

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NT5C2种系变异对急性淋巴细胞白血病患儿6-巯基嘌呤代谢的影响

6-巯基嘌呤（6-MP）被广泛用于治疗急性淋巴细胞白血病（ALL），其细胞毒性主要由硫鸟嘌呤核苷酸代谢产物（TGN）介导。最近的全基因组关联研究已经确定了ALL患者NT5C2基因中与6-MP代谢相关的种系多态性（例如rs72846714）。然而，尚不清楚NT5C2的遗传变异的全谱，并且其对6-MP药物活化的影响尚未得到全面检查。为此，我们对588名ALL儿童进行了NT5C2的靶向测序，并确定了6-MP治疗期间名义上与红细胞TGN相关的121个单核苷酸多态性（SNP）（P <0.05）。其中，在372名ALL儿童的复制队列中验证了61种变异。在考虑连锁不平衡和多变量分析之后，我们确认了以rs72846714和rs58700372代表的两个变异簇，它们独立地影响6-MP代谢。功能研究表明，rs58700372直接改变了内含子增强子的活性，其变异等位基因与较高的转录活性和降低的6-MP代谢（较低的TGN）有关。相比之下，rs72846714不在调节元件中，而是通过与近端功能性变体rs12256506的连锁不平衡来解释其关联信号，该变体激活了顺式NT5C2转录。我们的结果表明，NT5C2种系变异显着影响了硫嘌呤药物处置的患者间变异性。独立影响6-MP代谢。功能研究表明，rs58700372直接改变了内含子增强子的活性，其变异等位基因与较高的转录活性和降低的6-MP代谢（较低的TGN）有关。相比之下，rs72846714不在调节元件中，而是通过与近端功能性变体rs12256506的连锁不平衡来解释其关联信号，该变体激活了顺式NT5C2转录。我们的结果表明，NT5C2种系变异显着影响了硫嘌呤药物处置的患者间变异性。独立影响6-MP代谢。功能研究表明，rs58700372直接改变了内含子增强子的活性，其变异等位基因与较高的转录活性和降低的6-MP代谢（较低的TGN）有关。相比之下，rs72846714不在调节元件中，而是通过与近端功能性变体rs12256506的连锁不平衡来解释其关联信号，该变体激活了顺式NT5C2转录。我们的结果表明，NT5C2种系变异显着影响了硫嘌呤药物处置的患者间变异性。相比之下，rs72846714不在调节元件中，而是通过与近端功能性变体rs12256506的连锁不平衡来解释其关联信号，该变体激活了顺式NT5C2转录。我们的结果表明，NT5C2种系变异显着影响了硫嘌呤药物处置的患者间变异性。相比之下，rs72846714不在调节元件中，而是通过与近端功能性变体rs12256506的连锁不平衡来解释其关联信号，该变体激活了顺式NT5C2转录。我们的结果表明，NT5C2种系变异显着影响了硫嘌呤药物处置的患者间变异性。