Multicenter Evaluation of PCR Methods for the Detection of Factor V Leiden (R506Q) Genotypes

Abstract

Activated protein C (APC) resistance is reported to be present in 20–60% of individuals with a history of venous thrombosis (1). Thus, the prevalence of APC resistance in Caucasian patients with a history of venous thromboembolism is much greater than the prevalence of protein C deficiency, protein S deficiency, or antithrombin deficiency (1). The APC resistance phenotype is associated with a guanine-to-adenine substitution at nucleotide 1691 of the factor V gene (1). This mutation produces a glutamine-for-arginine (R506Q) substitution at factor V residue 506 (factor V Leiden). The risk of thrombosis is increased five- to eightfold in individuals heterozygous for factor V Leiden and 30- to 140-fold in homozygous individuals (1). Coagulation-based assays for the detection of APC resistance have been described and are commercially available. The APC resistance ratios from some of these assay systems, however, have demonstrated overlap between unaffected individuals and individuals heterozygous for factor V Leiden (1). Newer coagulation-based assays have been reported, but it is not clear that they can be used in all clinical settings. Unlike coagulation-based assays for APC resistance, DNA-based assays are not affected by pregnancy, the therapeutic use of anticoagulants, the use of oral contraceptives, or the presence of inhibitors such as lupus anticoagulants. Therefore, a rapid and accurate nucleic-acid-based assay to detect the factor V Leiden mutation is highly desirable. Bertina et al. (2) reported the first assay for the detection of factor V Leiden, a PCR-based assay that detects the guanine-to-adenine substitution at nucleotide 1691 by taking advantage of the loss of an Mnl I restriction site in the PCR product if the mutation is present. This assay has been used in several published reports on APC resistance. Other DNA-based assays have also been reported, including …