OBJECTIVE: The aims of this pharmacoepigenetic study were to evaluate differences in peripheral blood DNA methylation of the TPMT gene and in azathioprine pharmacokinetics in patients with VEO-IBD compared to aIBD.
METHODS: The association of age with whole genome DNA methylation profile was evaluated in a pilot group of patients and confirmed by a meta-analysis on 3 cohorts of patients available on the public functional genomics data repository. Effects of candidate CpG sites in the TPMT gene were validated in a larger cohort using pyrosequencing. TPMT activity and azathioprine metabolites (TGN) were measured in patients\' erythrocytes by HPLC and associated with patients\' age group and TPMT DNA methylation.
RESULTS: Whole genome DNA methylation pilot analysis, combined with the meta-analysis revealed cg22736354, located on TPMT downstream neighboring region, as the only statistically significant CpG whose methylation increases with age, resulting lower in VEO-IBD patients compared to aIBD (median 9.6% vs 12%, p = 0.029). Pyrosequencing confirmed lower cg22736354 methylation in VEO-IBD patients (median 4.0% vs 6.0%, p = 4.6 ×10-5). No differences in TPMT promoter methylation were found. Reduced cg22736354 methylation was associated with lower TGN concentrations (rho = 0.31, p = 0.01) in patients with VEO-IBD and aIBD.
CONCLUSIONS: Methylation of cg22736354 in TPMT gene neighborhood is lower in patients with VEO-IBD and is associated with reduced azathioprine inactivation and increased TGN concentrations.
目的:这项药物表观遗传学研究的目的是评估与aIBD相比,VEO-IBD患者外周血TPMT基因DNA甲基化和硫唑嘌呤药代动力学的差异。
方法:在一组试验患者中评估了年龄与全基因组DNA甲基化谱的关联,并通过对公共功能基因组学数据储存库上3组患者的荟萃分析证实了这一关联。使用焦磷酸测序在更大的队列中验证了TPMT基因中候选CpG位点的作用。通过HPLC测量患者红细胞中的TPMT活性和硫唑嘌呤代谢物(TGN),并与患者年龄组和TPMTDNA甲基化相关。
结果:全基因组DNA甲基化先导分析,结合荟萃分析显示,cg22736354位于TPMT下游相邻区域,作为甲基化随年龄增长而增加的唯一具有统计学意义的CpG,与aIBD相比,VEO-IBD患者的发病率较低(中位数为9.6%vs12%,p=0.029)。焦磷酸测序证实VEO-IBD患者的cg22736354甲基化较低(中位数为4.0%vs6.0%,p=4.6×10-5)。没有发现TPMT启动子甲基化的差异。在VEO-IBD和aIBD患者中,降低的cg22736354甲基化与较低的TGN浓度(rho=0.31,p=0.01)相关。
结论:在VEO-IBD患者中,TPMT基因邻域中cg22736354的甲基化程度较低,并且与硫唑嘌呤失活减少和TGN浓度增加有关。