School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai, 200127, P.R. China.
Department of Laboratory Medicine, Shanghai Children's Medical Center, Shanghai Jiao Tong University School of Medicine, Shanghai, 200127, P.R. China.
Sci Rep. 2017 Nov 22;7(1):16021. doi: 10.1038/s41598-017-16310-8.
Clopidogrel is an antiplatelet medicine used to prevent blood clots in patients who have had a heart attack, stroke, or other symptoms. Variability in the clinical response to clopidogrel treatment has been attributed to genetic factors. In particular, five SNPs of rs4244285, rs4986893, rs12248560, rs662 and rs1045642 have been associated with resistance to clopidogrel therapy in Chinese population. This work involves the development of a multiplex high-resolution melting (HRM) assay to genotype all five of these loci in 2 tubes. Amplicons corresponding to distinct SNPs in a common tube were designed with the aid of uMelt prediction software to have different melting temperatures Tm by addition of a GC-rich tail to the 5' end of the certain primers. Two kinds of commercial methods, Digital Fluorescence Molecular Hybridization (DFMH) and Sanger sequencing, were used as a control. Three hundred sixteen DFMH pretested samples from consecutive acute coronary syndrome patients were used for a blinded study of multiplex HRM. The sensitivity of HRM was 100% and the specificity was 99.93% reflecting detection of variants other than the known resistance SNPs. Multiplex HRM is an effective closed-tube, highly accurate, fast, and inexpensive method for genotyping the 5 clopidogrel resistance associated SNPs.
氯吡格雷是一种抗血小板药物，用于预防心脏病发作、中风或其他症状的患者发生血栓。氯吡格雷治疗的临床反应存在变异性，这归因于遗传因素。特别是，rs4244285、rs4986893、rs12248560、rs662 和 rs1045642 这五个 SNP 与中国人群对氯吡格雷治疗的耐药性有关。这项工作涉及开发一种多重高分辨率熔解（HRM）分析方法，在 2 个管中对所有 5 个基因座进行基因分型。在 uMelt 预测软件的辅助下，设计了对应于同一管中不同 SNP 的扩增子，通过在特定引物的 5' 端添加富含 GC 的尾巴，使它们具有不同的熔解温度 Tm。使用两种商业方法，数字荧光分子杂交（DFMH）和 Sanger 测序，作为对照。对 316 例来自连续急性冠脉综合征患者的 DFMH 预测试样本进行了多重 HRM 盲法研究。HRM 的灵敏度为 100%，特异性为 99.93%，反映了除已知耐药 SNP 以外的变异体的检测。多重 HRM 是一种有效的闭管、高度准确、快速且廉价的方法，可用于对 5 个与氯吡格雷耐药相关的 SNP 进行基因分型。
