Chen Chunxia, Li Siyue, Lu Xiaojun, Tan Bin, Huang Chunyan, Qin Li
Department of Laboratory Medicine, West China Hospital, Sichuan University Chengdu 610041, China.
Int J Clin Exp Pathol. 2014 Apr 15;7(5):2558-64. eCollection 2014.
Single nucleotide polymorphisms (SNPs) of VKORC (1173T/C, rs9934438) and CYP2C9 (1075A/C, rs1057910) are major contributory factors on the sensitivity of warfarin in Chinese. Analysis of the two genomic loci could help warfarin treatment individual from bleeding or thrombosis events. An assay with the advantages of simplicity, speed, high sensitivity and low cost for genotyping is calling for clinical laboratories. High resolution method (HRM) meets these callings, but no study with large sample tested its performance in genotyping of rs9934438 and rs1057910. In this study, we identified polymorphisms of rs9934438 and rs1057910 in 255 unrelated Chinese heart valve replacement patients of Han ethnic group from West China Hospital. The two genomic loci were genotyped by HRM using LightCycler® 480 High Resolution Melting Master on LightCycler® 480 Real-Time PCR instruments (Roche Diagnostics), and all amplified PCR products were sent for direct DNA sequencing. The genotyping of rs1057910 between HRM and sequencing showed perfect 100% concordance. While the concordance of rs9934438 between HRM and sequencing was 99.2%. Unexpected mutation interfered genotyping results of HRM when genotyping rs9934438. HRM is a valuable technique for genotype detection of rs9934438 and rs1057910 to assess individual sensitivity to warfarin, where DNA sequencing is added inevitably sometimes.
维生素K环氧化物还原酶复合体亚单位1（VKORC）（1173T/C，rs9934438）和细胞色素P450 2C9（CYP2C9）（1075A/C，rs1057910）的单核苷酸多态性是影响华法林在中国人群中敏感性的主要因素。对这两个基因位点进行分析有助于避免华法林治疗个体出现出血或血栓形成事件。临床实验室需要一种具有操作简单、速度快、灵敏度高和成本低等优点的基因分型检测方法。高分辨率熔解曲线分析（HRM）方法符合这些要求，但尚无大样本研究测试其对rs9934438和rs1057910基因分型的性能。在本研究中，我们对来自华西医院的255名汉族非亲缘关系心脏瓣膜置换患者的rs9934438和rs1057910基因多态性进行了鉴定。使用罗氏诊断公司的LightCycler® 480实时荧光定量PCR仪上的LightCycler® 480高分辨率熔解曲线分析试剂盒，通过HRM对这两个基因位点进行基因分型，所有扩增的PCR产物均进行直接DNA测序。HRM与测序结果显示，rs1057910的基因分型一致性达100%。而rs9934438的HRM与测序结果的一致性为99.2%。在对rs9934438进行基因分型时，意外突变干扰了HRM的基因分型结果。HRM是一种用于检测rs9934438和rs1057910基因分型以评估个体对华法林敏感性的有价值技术，不过有时不可避免地需要进行DNA测序辅助。
