Factor V Leiden and Factor V R2 Allele: High-throughput Analysis and Association with Venous Thromboembolism

Jane M. Benson; Dorothy Ellingsen; Muhydine El-Jamil; Meredith Jenkins; Connie H. Miller; Anne Dilley; Bruce L. Evatt; Craig W. Hooper

doi:10.1055/s-0037-1616049

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2001; 86(05): 1188-1192
DOI: 10.1055/s-0037-1616049

Review Article

Schattauer GmbH

Factor V Leiden and Factor V R2 Allele: High-throughput Analysis and Association with Venous Thromboembolism

Authors

Jane M. Benson

¹Hematologic Diseases Branch, Division of AIDS, STD, and TB Laboratory Research, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, USA
Dorothy Ellingsen

¹Hematologic Diseases Branch, Division of AIDS, STD, and TB Laboratory Research, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, USA
Muhydine El-Jamil

¹Hematologic Diseases Branch, Division of AIDS, STD, and TB Laboratory Research, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, USA
Meredith Jenkins

¹Hematologic Diseases Branch, Division of AIDS, STD, and TB Laboratory Research, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, USA
Connie H. Miller

¹Hematologic Diseases Branch, Division of AIDS, STD, and TB Laboratory Research, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, USA
Anne Dilley

¹Hematologic Diseases Branch, Division of AIDS, STD, and TB Laboratory Research, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, USA
Bruce L. Evatt

¹Hematologic Diseases Branch, Division of AIDS, STD, and TB Laboratory Research, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, USA
Craig W. Hooper

¹Hematologic Diseases Branch, Division of AIDS, STD, and TB Laboratory Research, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, USA

Abstract

Summary

Thrombophilia is a multigenic disease in which the combination of genetic polymorphisms increases the risk of deep vein thrombosis (DVT). The rapid identification of these genetic combinations requires high-throughput analysis of single nucleotide polymorphisms (SNPs). The TaqMan^® fluorogenic 5’→3’ nuclease assay (PE/Applied Bio-systems, Foster City, CA) with custom-designed primers, probes and controls has provided a highly efficient platform for high throughput. This assay was used to rapidly detect two SNPs, FV Leiden (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 samples 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 while the initial sets of reactions were in the PCR machines, the output could correspondingly be increased. The TaqMan^® assay was extremely accurate, avoided contamination by using uracil-N-glycolase (UNG) in a single, closed tube, and offered the possibility for additional automation with robotic equipment to implement the PCR. This TaqMan^® assay facilitates high throughput 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. Caucasians, 5.6% in African-Americans, 13.4% in Asian or Pacific Islanders and 11.3% in Hispanics. No association between venous thromboembolism and the R2 allele was noted, and furthermore no interaction with FV Leiden was observed.

Keywords

Factor V Leiden - R2 allele - single nucleotide polymorphisms (SNPs) - TaqMan^® - venous thrombosis

Full Text

References

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