NUDT15c.415C>T和TPMT*3C基因多态性检测方法的比较与临床应用

Comparison and Clinical Application of Genotyping Approaches for Azoioprine Induced Leukocyte Reduction Alleles NUTT15C.415C>T and TPMT*3C

关键词:  硫唑嘌呤  白细胞减少  NUDT15c.415C>T  TPMT*3C  基因多态性检测

Key words:  azathioprine  leukocyte  NUDT15 c.415C>T  TPMT*3C  gene polymorphism detection

摘要:

目的 建立准确、快速、经济的方法，检测NUDT15 c.415C>T和TPMT*3C基因多态性，探讨临床应用价值。方法 收集2017年5月-2018年5月期间福建汉族患者服用硫唑嘌呤2周以上的血清样本，提取DNA或白细胞后分别采用PCR-RFLP法、PCR-Sanger测序法和荧光定量PCR法对NUDT15 c.415C>T和TPMT*3C进行基因多态性分型，比较这3种方法的准确性、简便性及经济性。根据白细胞值分组，结合临床资料，探讨基因多态性等因素与硫唑嘌呤致白细胞减少的相关性。结果 共纳入129例患者，其中硫唑嘌呤致白细胞减少15例（11.6%）。3种方法的基因多态性检测结果一致，TPMT*3C未发现突变纯合子。携带NUDT15c.415C>T突变等位基因者服用硫唑嘌呤致白细胞减少的风险高于携带野生等位基因者（OR=6.2，95%CI：2.5~15.4，P=0.000 054），而携带TPMT*3C突变等位基因者与野生等位基因者出现白细胞减少比例并无显著性差异（P=0.393）。NUDT15c.415C>T基因多态性预测白细胞减少敏感度为53.3%，特异度为85.1%，ROC曲线AUC为0.69。结论 3种方法都可用于临床检测NUDT15 c.415C>T和TPMT*3C基因多态性。PCR-RFLP法不需要专用试剂盒，也不需要昂贵的仪器设备，成本较低，过程简单，易于操作，特别适合条件有限的单位开展工作。福建汉族患者在服用硫唑嘌呤前进行NUDT15c.415C>T基因多态性检测比TPMT*3C更具临床价值。

Abstract:

OBJECTIVE To establish an accurate, rapid and economical method for detecting NUDT15 c.415C>T and TPMT*3C gene polymorphisms and evaluate the value of their clinical application. METHODS Serum samples of patients taking azathioprine for more than 2 weeks from May 2017 to May 2018 in Fujian were collected. NUDT15 c.415C>T and TPMT*3C were genotyping by PCR-restriction fragment length polymorphism (PCR-RFLP), PCR-Sanger, and real-time PCR. The accuracy, simplicity and economy of the three methods were compared. The association between genetic polymorphisms and azathioprine-induced leukocyte reduction was analyzed in combination with clinical data. RESULTS A total of 129 patients were included, 15 (11.6%) of whom developed leukopenia. The results of the methods were essentially equivalent. No mutant homozygote genotype was found in TPMT*3C gene. There was strong associations of NUDT15 c.415C>T with azathioprine-induced leukopenia (OR=6.2, 95%CI:2.5~15.4, P=0.000 054), whereas there was no relevance for TPMT*3C (P=0.393). The predictive sensitivity of NUDT15 c.415C>T was 53.3%,specificity was 85.1%, and AUC of ROC curve was 0.69. CONCLUSION The three methods can be used for clinical detection of NUDT15 c.415C>T and TPMT*3C gene polymorphisms. The PCR-RFLP method does not require special kit, no expensive equipment, low cost, simple process, easy operability, and especially applicable to the basic medical institutions or scientific research units with limited conditions. The detection of NUDT15c.415C>T gene polymorphisms for Fujian Han patients with azathioprine has better clinical value than the detection of TMPT*3C.