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 a s a highly prevalent risk factor for the development of venous thrombo sis. In the majority of cases, APC resistance correlates with the pres ence of a single point mutation in the factor V gene (FV R506Q), The m utation 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 thrombosi s. In the current study, the authors have optimized and implemented fo r clinical diagnosis a method for detection of FV R506Q using the poly merase chain reaction coupled with restriction fragment length polymor phism analysis (PCR-RFLP), Forty-one healthy adults and 139 patients r eferred for hypercoagulability testing were genotyped and their APC re sistance ratios determined using commercially available reagents (COAT EST APC Resistance Kit), Comparative analysis indicated that if functi onal APC resistance was defined as per manufacturer's guidelines, a si gnificant number of individuals with a normal factor V genotype were c ategorized as APC resistant and conversely, a significant number of in dividuals heterozygous for FV R506Q were categorized as non-APC resist ant. These results indicate that comparative functional and genotypic analyses in the individual clinical laboratory setting are critical fo r establishing normal ranges and cut-off values for functional APC res istance due to FV R506Q. To facilitate molecular evaluation of APC res istance, Epstein-Barr virus (EBV) immortalized B-lymphocyte cell lines were established from individuals heterozygous and homozygous for FV R506Q.