Factor V leiden and factor V R2 allele: High-throughput analysis and association with venous thromboembolism

Thrombophilia is a multigenic disease in which the combination of genetic p olymorphisms increases the risk of deep vein thrombosis (DVT). The rapid id entification of these genetic combinations requires high-throughput analysi s of single nucleotide polymorphisms (SNPs). The TaqMan (R) fluorogenic 5'- ->3' nuclease assay (PE/Applied Biosystems, Foster City, CA) with custom-de signed primers, probes and controls has provided a highly efficient platfor m for high throughput. This assay was used to rapidly detect two SNPs, FV L eiden (G1691A) and FV A4070G (R2 allele), in a study of 6295 subjects. With one thermal cycler, we completed sample set-up, PCR and analysis on 84 sam ples in 3 h with an additional 12 wells containing 4 "no template controls" (NTC), 4 "allele-1 controls", and 4 "allele-2 controls" in a 96-well plate . When additional thermal cyclers were used and more assays were set up whi le the initial sets of reactions were in the PCR machines, the output could correspondingly be increased. The TaqMan (R) assay was extremely accurate, avoided contamination by using uracil-N-glycolase (UNG) in a single, close d tube, and offered the possibility for additional automation with robotic equipment to implement the PCR. This TaqMan (R) assay facilitates high thro ughput to screen large populations quickly and economically while utilizing a simple protocol that requires minimal expenditure of personnel time. Our results demonstrated a prevalence of the R2 allele of 11.9% in U. S. Cauca sians, 5.6% in African-Americans, 13.4% in Asian or Pacific Islanders and 1 1.3% in Hispanics. No association between venous thromboembolism and the R2 allele was noted, and furthermore no interaction with FV Leiden was observ ed.