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, Inhaltsverzeichnis

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

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

› Institutsangaben

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

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Referenzen

References
1 De Stefano V, Chiusolo P, Paciaroni K. et al. Prevalence of the factor II G20210A mutation in symptomatic patients with inherited thrombophilia. Thromb Haemost 1998; 80: 342-2.
2 De Stefano V, Martinelli I, Mannuccio P. et al. The risk of recurrent deep vein thrombosis among heterozygous carriers of both Factor V Leiden and the G20210A prothrombin mutation. N Engl J Med 1999; 341: 801-6.
3 Greengard JS, Sun X, Xu X, Fernandez JA, Griffin JH, Evatt B. Activated protein C resistance caused by Arg506Gln mutation in factor Va. Lancet 1994; 343: 1535-6.
4 Bertina RM, Koeleman BPC, Koster T, Rosendaal FR, Dirven RJ, De Ronde H, van der Velden PA, Reitsma PH. Mutation in blood coagulation factor V associated with resistance to activated protein C. Nature 1994; 369: 64-7.
5 Poort SR, Rosendaal F, Reitsma PH, Bertina RM. A common genetic variation in the 3′-untranslated region of the prothrombin gene is associated with elevated plasma prothrombin levels and an increase in venous thrombosis. Blood 1996; 88: 3698-703.
6 Lunghi B, Iacoviello L, Gemmati D, Dilasio MG, Castoldi E, Pinotti M, Castaman G, Redaelli R, Mariani G, Marchetti G, Bernardi F. Detection of new polymorphic markers in the factor V gene: association with factor V levels in plasma. Thromb Haemost 1996; 75: 45-8.
7 Bernardi F, Faioni EM, Castoldi E, Lunghi B, Castaman G, Sacchi E, Mannucci PM. A factor V genetic component differing from Factor V R506Q contributes to the activated protein C resistance phenotype. Blood 1997; 90 (Suppl. 04) 1552-7.
8 Alhenc-Gelas M, Nicaud V, Gandrille S, van Dreden P, Amiral J, Aubry M, Fiessinger J, Emmerich J, Aiach M. The factor V gene A4040G mutation and the risk of venous thrombosis. Thromb Haemost 1999; 81: 193-7.
9 Faioni EM, Franchi F, Bucciarelli P, Margaglione M, De Stefano V, Castaman G, Finazzi G, Mannucci PM. Coinheritance of HR2 haplotype in the factor V gene confers an increased risk of venous thromboembolism to carriers of Factor V R506Q (Factor V Leiden). Blood 1999; 94 (Suppl. 09) 3062-6.
10 Blasczyk R, Ritter M, Thiede C, Wehling J, Hintz G, Neubauer A, Riess H. Simple and rapid detection of Factor V Leiden by allele-specific PCR amplification. Thromb Haemost 1996; 75: 757-9.
11 Rabes JP, Trossaer M, Conrad J, Samama M, Giraudet P, Boileau C. Single point mutation at Arg⁵⁰⁶ of factor V associated with APC resistance and venous thromboembolism: improved detection by PCR-mediated site-directed mutagenesis. Thromb Haemost 1995; 74: 1379-80.
12 Benson JM, Phillips DJ, Holloway BP, Evatt BL, Hooper WC. Oligonucleotide ligation assay for detection of the Factor V Leiden mutation (Arg5⁰⁶→Gln) causing protein C resistance. Thromb Res 1996; 83: 87-96.
13 Livak KJ. Allelic discrimination using fluorogenic probes and the 5′ nuclease assay. Genet Anal 1999; 14: 143-9.
14 Livak KJ, Flood SJA, Marmo J, Giusto W, Deetz K. Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization. PCR Methods and Applications 1995; 4: 357-62.
15 Benson JM, Ellingsen D, Renshaw MA, Resler AG, Evatt BL, Hooper WC. Multiplex analysis of mutations in four genes using fluorescence scanning technology. Thromb Res 1999; 96: 57-64.
16 Hooper WC, Dilley A, Ribeiro MJA, Benson J, Austin H, Silva V, Rawlins P, Wenger NK, Evatt BL. A racial difference in the prevalence of the Arg⁵⁰⁶→Gln mutation. Thromb Res 1996; 81: 577.
17 Happich D, Schwaab Hanfland P, Hoernschemeyer D. Allelic discrimination of Factor V Leiden using a 5′nuclease assay. Thromb Haemost 1999; 82: 1294-6.