Factor V R506Q gene mutation analysis by PCR-RFLP: optimization, comparison with functional testing for resistance to activated protein C, and establishment of cell line controls.

Resistance to activated protein C (APC) has been recently identified as a highly prevalent risk factor for the development of venous thrombosis. In the majority of cases, APC resistance correlates with the presence of a single point mutation in the factor V gene (FV R506Q). The mutation is present in 3% to 5% of the general population and in up to 50% of patients with a personal and family history of venous thrombosis. In the current study, the authors have optimized and implemented for clinical diagnosis a method for detection of FV R506Q using the polymerase chain reaction coupled with restriction fragment length polymorphism analysis (PCR-RFLP). Forty-one healthy adults and 139 patients referred for hypercoagulability testing were genotyped and their APC resistance ratios determined using commercially available reagents (COATEST APC Resistance Kit). Comparative analysis indicated that if functional APC resistance was defined as per manufacturer's guidelines, a significant number of individuals with a normal factor V genotype were categorized as APC resistant and conversely, a significant number of individuals heterozygous for FV R506Q were categorized as non-APC resistant. These results indicate that comparative functional and genotypic analyses in the individual clinical laboratory setting are critical for establishing normal ranges and cut-off values for functional APC resistance due to FV R506Q. To facilitate molecular evaluation of APC resistance, Epstein-Barr virus (EBV) immortalized B-lymphocyte cell lines were established from individuals heterozygous and homozygous for FV R506Q.