等位基因特异性PCR检测CYP3A5和MDR-1基因多态性方法的建立及临床应用

摘要

目的 用等位基因特异性PCR(ARMS-PCR)检测细胞色素P450酶CYP3A5 (A6986G)和多药耐药基因MDR-1 (C3435T)基因多态性,探讨其与肾移植受者他克莫司(Tac)血药浓度和剂量比的相关性.方法 根据CYP3A5 (A6986G)和MDR-1(C3435T)基因多态性位点分别设计ARMS-PCR引物,分析72例肾移植受者外周血基因组DNA中该2个基因位点的多态性,同时以DNA测序法为金标准进行验证.化学发光微粒子免疫分析法测定肾移植受者血Tac浓度,并比较术后1个月时不同基因型受者之间血Tac浓度、Tac剂量/Tac用量的差异.结果 建立的ARMS-PCR法与DNA测序法检测符合率为100％.72例肾移植受者中,CYP3A5*1/*1、*1/*3和*3/*3基因型的发生频率分别为18.1％、31.9％和50.0％,MDR-1 C/C、C/T和T/T基因型的发生频率分别为27.8％、58.3％和13.9％.此外,不同CYP3A5基因型肾移植受者的血Tac浓度(P=0.014)和Tac浓度/Tac用量(P=0.019)均存在明显差异,进一步两两比较发现,CYP3A5*3/*3基因型受者血Tac浓度/Tac用量明显高于*1/*1和*1/*3基因型(P均＜0.05).结论 成功建立检测CYP3A5和MDR-1基因多态性的ARMS-PCR法,肾移植受者CYP3A5*3基因多态性与Tac的药代动力学明显相关.%Objective To establish an allele-specific PCR method for the detection of cytochrome P-450 CYP3A5 (A6986G) and multiding resistance gene MDR-1 (C3435T) polymorphisms,and investigate the correlations of their polymorphisms with blood tacrolimus (Tac) concentration/dose (C/D) ratio in renal transplant recipients.Methods The allele-specific PCR primers were designed according to the polymorphism sites of CYP3A5 (A6986G) and MDR-1 (C3435T) genes.Then,their polymorphisms in the genomic DNA of peripheral blood samples from 72 renal transplant recipients were analyzed,and the results were validated by DNA sequencing.The blood Tac concentration was determined by the chemiluminescence microparticle immunoassay and the differences of concentration,dose and C/D ratio of blood Tac in renal transplant recipients with different genotypes were compared at 1 month after transplantation.Results The coincidence rate between the established allele-specific PCR and DNA sequencing was 100％.The frequencies of CYP3A5 * 1/* 1,* 1/* 3 and * 3/* 3 genotypes in 72 renal transplant recipients were 18.1％,31.9％ and 50.0％,respectively,and those of MDR-1 C/C,C/T and T/T genotypes were 27.8％,58.3％ and 13.9％,respectively.There were significant differences in blood Tac concentration (P =0.014) and Tac C/D ratio (P =0.019) between different CYP3A5 genotypes of renal transplant recipients.Further analysis found that the Tac C/D ratio of CYP3A5 * 3/* 3 genotype was significantly higher than that of CYP3A5 * 1/* 1 and * 1/* 3 genotypes (P ＜ 0.05).Conclusion The allele-specific PCR method for the detection of CYP3A5 and MDR-1 polymorphisms is successfully established and the polymorphism of CYP3A5 * 3 gene in renal transplant recipients is obviously correlated with the pharmacokinetics of Tac.