title: High resolution melting method to detect single nucleotide polymorphism of VKORC1 and CYP2C9.
abstract: 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.
authors: Department of Laboratory Medicine, West China Hospital, Sichuan University Chengdu 610041, China.
authors: Chunxia
authors: C
authors: Chen
authors: Department of Laboratory Medicine, West China Hospital, Sichuan University Chengdu 610041, China.
authors: Siyue
authors: S
authors: Li
authors: Department of Laboratory Medicine, West China Hospital, Sichuan University Chengdu 610041, China.
authors: Xiaojun
authors: X
authors: Lu
authors: Department of Laboratory Medicine, West China Hospital, Sichuan University Chengdu 610041, China.
authors: Bin
authors: B
authors: Tan
authors: Department of Laboratory Medicine, West China Hospital, Sichuan University Chengdu 610041, China.
authors: Chunyan
authors: C
authors: Huang
authors: Department of Laboratory Medicine, West China Hospital, Sichuan University Chengdu 610041, China.
authors: Li
authors: L
authors: Qin
mesh_terms: D000328:Adult; D000368:Aged; D000925:Anticoagulants / Q000627:therapeutic use; D044466:Asian People / Q000235:genetics; D002681:China / Q000453:epidemiology; D065729:Cytochrome P-450 CYP2C9 / Q000235:genetics*; D005260:Female; D005787:Gene Frequency; D005838:Genotype; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D008297:Male; D008875:Middle Aged; D010597:Pharmacogenetics; D010641:Phenotype; D020641:Polymorphism, Single Nucleotide; D060888:Real-Time Polymerase Chain Reaction; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA / Q000379:methods*; D064417:Vitamin K Epoxide Reductases / Q000235:genetics*; D014859:Warfarin / Q000627:therapeutic use; D055815:Young Adult
chemical_list: D000925:Anticoagulants; D014859:Warfarin; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; C578523:VKORC1 protein, human; D064417:Vitamin K Epoxide Reductases
keywords: CYP2C9; HRM; VKORC; genotyping; warfarin