Pyrosequencing Analysis of Thrombosis-Associated Risk Markers

Abstract

The factor V Leiden and prothrombin G20210A polymorphisms are established risk factors for thrombosis (1)(2). General screening for these polymorphisms in persons with additional risk factors has been discussed (3), but a significant proportion of familial cases with deep vein thrombosis/venous thromboembolism is not explained by carriage of either of these mutations (4). There is accumulating evidence that multiple coexisting defects are present in persons with the most marked tendency to thrombosis (5). The current lack of a clear consensus regarding the clinical roles for several of the additional polymorphisms studied (1)(2) could reflect that most studies have addressed these independently. We developed a pyrosequencing-based genotyping protocol for parallel analysis of the β-fibrinogen (−455G/A and −854 G/A), prothrombin (G20210A), coagulation factor V Leiden (G1691; Arg506Gln), coagulation factor VII (−401G/T and −402 G/A), coagulation factor XIII (G163T; Val34Leu), plasminogen activator inhibitor-1 ( PAI-1 ; −675 4G/5G), methylenetetrahydrofolate reductase ( MTHFR ; C677T; Ala222Val), glycoprotein IIIa ( GPIIIa ; C1565T; Leu33Pro; also known as PlA1/PlA2), and endothelial nitric oxide synthase ( eNOS ; G894T; Glu298Asp) polymorphisms, together with the cytochrome P450 2C9 [ CYP2C9*1 (wild type)], CYP2C9*2 (C430T; Cys144Arg), CYP2C9*3 (A1075C; Ile359Leu), and CYP2C9*4 (T1076C; Ile359Thr) isoforms, which modulate the effect of warfarin in antithrombotic therapy. To start with subnanogram amounts of genomic DNA, we developed an outer nested PCR for simultaneous amplification of 11 gene fragments covering these single-nucleotide polymorphisms (SNPs). Genomic DNA samples were arrayed in 96-well plates together with negative controls. PCR primers were designed based on available GenBank entries and searched against publicly available nucleotide databases to ensure specificity for the selected primer annealing regions. Individual …