Taqman-MGB法检测CYP3A5基因rs776746位点单核苷酸多态性及该位点对他克莫司代谢的影响

Taqman-MGB method for determining CYP3A5 rs776746 site SNP and the influence of this site on the metabolism of tacrolimus

目的建立检测CYP3A5基因rs776746位点单核苷酸多态性(SNP)的Taqman-MGB实时荧光聚合酶链反应(PCR),并分析该位点对他克莫司药物代谢的影响。方法针对CYP3A5基因rs776746位点设计相应引物及双标记探针,建立并评价检测该位点SNP的Taqman-MGB实时荧光PCR;收集170例服用他克莫司患者的全血DNA样本,用建立的Taqman-MGB实时荧光PCR检测CYP3A5基因型,并分析不同CYP3A5基因型与他克莫司血药浓度剂量比的相关性。结果建立的Taqman-MGB基因分析法能够对CYP3A5 rs776746位点基因型进行高效区分,可检测最低含量为0.01 ng水平的DNA样本,并且具有较好的重复性和特异性,其检测结果与测序结果完全一致。170例患者中CYP3A5*1/*1型18例(10.59%)、CYP3A5*1/*3型65例(38.24%)、CYP3A5*3/*3型87例(51.18%);其中104例异体肾移植患者3种基因型的血药浓度剂量比分别为(52.47±35.96)、(77.79±33.80)、(134.80±79.35)(ng/m L)/(mg·kg-1·d-1),各组之间差异有统计学意义(P<0.05)。结论建立了一种敏感性高、特异性好、重复性佳、适用于临床快速检测CYP3A5 rs776746 SNP位点的Taqman-MGB方法。初步分析显示CYP3A5*3基因型的存在会减弱患者对他克莫司的代谢能力。

Objective To establish Taqman-MGB real-time fluorescence polymerase chain reaction （PCR） for determining CYP3A5 rs776746 site single nucleotide polymorphism （SNP）, and to analyze the influence on the metabolism of tacrolimus. Methods The primers and double-labeled probes were designed according to the genotype of CYP3A5 rs776746 site, and the Taqman-MGB real-time fluorescence PCR for SNP was established and evaluated. A total of 170 whole blood DNA samples from patients who took tacrolimus were collected, and CYP3A5 genotype was determined by Taqman-MGB real-time fluorescence PCR. The relationship between CYP3A5 genotype and tacrolimus concentration to dose ratio was analyzed. Results The established Taqman-MGB method can discriminate CYP3A5 rs776746 site efficiently with good reproducibility and specificity and had a detection limit of 0.01 ng for DNA. The results of Taqman-MGB method were consistent with those of sequencing. Among the 170 samples, 18 samples belonged to CYP3A5 ＊ 1/＊ 1 （10.59%）, 65 samples were CYP3A5 ＊ 1/＊ 3 （38.24%） and 87 samples were CYP3A5 ＊ 3/＊ 3 （51.18%）. Among the samples collected, 104 samples belonged to renal allograft recipients, and the mean tacrolimus concentrations to dose ratios for 3 genotypes were （52.47 ±35.96 ）, （77.79 ± 33.80） and （ 134.80 ± 79.35 ） （ ng/ mL）/（ mg · kg^-1· d^-1）, and there was statistical significance （P 〈 0.05 ）. Conclusions The Taqman-MGB method for determining CYP3A5 rs776746 site SNP is established, which has high sensitivity, specificity and reproducibility and is a rapid method for clinical practice. Through primary analysis, the existence of CYP3A5 ＊ 3 genotype do weaken the ability of metabolizing tacrolimus.