Allelic Discrimination of Factor V Leiden Using a 5’ Nuclease Assay

D. Happich; R. Schwaab; P. Hanfland; D. Hoernschemeyer

doi:10.1055/s-0037-1614378

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 1999; 82(04): 1294-1296
DOI: 10.1055/s-0037-1614378

Review Article

Schattauer GmbH

Allelic Discrimination of Factor V Leiden Using a 5’ Nuclease Assay

D. Happich

¹From the Institute of Experimental Haematology and Transfusion Medicine, University of Bonn, Germany

,

R. Schwaab

¹From the Institute of Experimental Haematology and Transfusion Medicine, University of Bonn, Germany

,

P. Hanfland

¹From the Institute of Experimental Haematology and Transfusion Medicine, University of Bonn, Germany

,

D. Hoernschemeyer

¹From the Institute of Experimental Haematology and Transfusion Medicine, University of Bonn, Germany

› Author Affiliations

Abstract

Summary

The G1691A (Leiden) mutation of the factor V gene is the most prevalent identified cause of venous thrombosis. Therefore, we developed a new genetic test using the TaqMan system. With this assay which combines PCR amplification and detection reaction in one closed tube, a cohort of 234 patients with a history of thrombosis was screened. In parallel, amplification products of the same patients were screened with a previously described test using endonuclease digestion of PCR products followed by gel electrophoresis. Identical results were obtained by both methods. Among cases, 122 (52%) individuals were homozygous normal, 99 (42%) were heterozygous affected and 13 (5.5%) showed homozygous pattern for the Factor V Leiden mutation. Thus, it could be demonstrated that the new TaqMan assay is a robust, rapid and automated method for high throughput application which avoids time consuming and difficult post-PCR steps.

Full Text

References

References
1 Dahlbäck B. Resistance to activated protein C, the Arg⁵⁰⁶ to Gln mutation in the factor V gene, and venous thrombosis. Thromb Haemost 1995; 73: 739-42.
2 Dahlbäck B, Carlsson M, Svensson PJ. Familial thrombophilia due to a previously unrecognized mechanism characterized by poor anticoagulant response to activated protein C: prediction of a cofactor to activated protein. Proc Natl Acad Sci USA 1993; 90: 1004-8.
3 Griffin JH, Evatt B, Widemann C, Fernandez JA. Anticoagulant protein C pathway defective in majority of thrombophilic patients. Blood 1993; 82: 1989-93.
4 Svensson PJ, Dahlbäck B. Resistance to activated protein C as a basis for venous thrombosis. N Engl J Med 1994; 330: 517-22.
5 Koster T, Rosendaal FR, de Ronde H, Brief E, Vandenbroucke JP, Bertina RM. Venous thrombosis due to poor anticoagulant response to activated protein C: Leiden Thrombophilia Study. Lancet 1993; 342: 1503-6.
6 Beauchamp NJ, Daly ME, Hampton KK, Cooper PC, Preston E, Peake IR. High prevalence of a mutation in the factor V gene within the UK population: Relationship to activated protein C resistance and familial thrombosis. Brit J Haematol 1994; 88: 219-22.
7 Rosendaal FR, Siscovick DS, Schwartz SM, Beverly RK, Psaty BM, Longstreth WT. Factor V Leiden (resistance to activated protein C) increases the risk of myocardial infarction in young woman. Blood 1997; 89: 2817-21.
8 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.
9 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.
10 Rabes JP, Trossaert 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.
11 Bowen DM, Standen GR, Granville S, Bowly S, Wood NAP, Bidwell J. Genetic diagnosis of Factor V Leiden using heteroduplex technology. Thromb Haemost 1997; 77: 119-22.
12 Ulvik A, Ren J, Refsum H, Ueland PM. Simultaneous determination of methylenetetrahydrofolate reductase C677T and factor V G1691A genotypes by mutagenically separated PCR and multiple-injection capillary electrophoresis. Clin Chem 1998; 44: 264-69.
13 Holland PM, Abramson RD, Watson R, Gelfand DH. Detection of specific polymerase chain reaction products by utilizing the 5’ to 3’ exonuclease activity of Thermus aquaticus polymerase. Proc Natl Acad Sci USA 1991; 88: 7276-80.
14 Livak KJ, Flood SAJ, Marmaro J, Giusti 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 Lee LG, Connell CR, Bloch W. Allelic discrimination by nick translation PCR with fluorogenic probes. Nucleic Acids Res 1993; 21: 3761-6.
16 Bowen DJ, Bowley S, John M, Collins PW. Factor V Leiden (G1691), the prothrombin 3’-untranslated region variant (G20210A) and thermolabile methylenetetrahydrofolate reductase (C677T): a single genetic test genotypes all three loci – determination of frequencies in the S. Wales population of the UK. Thromb Haemost 1998; 79: 949-54.
17 Hezard N, Cornillet-Lefebvre P, Gillot L, Potron G, Nguyen P. Multiplex ASA PCR for a simultaneous determination of Factor V Leiden Gene, G→A 20210 Prothrombine Gene and C→T 677 MTHFR Gene mutations. Thromb Haemost 1998; 79: 1054-5.
18 Chakravarty A, Hansen TS, Horder M, Kristensen SR. A fast and robust dual label nonradioactive oligonucleotide ligation assay for detection of Factor V Leiden. Thromb Haemost 1997; 78: 1234-6.
19 Behn M, Schuermann M. Simple and reliable factor V genotyping by PNA-mediated PCR clamping. Thromb Haemost 1998; 79: 773-7.
20 Whitcombe D, Brownie J, Gillard HL, McKechnie D, Theaker J, Newton CR, Little S. A homogenous fluorescence assay for PCR amplicons: its application to real-time, single-tube genotyping. Clin Chem 1998; 44: 918-23.
21 Lay MJ, Wittwer CT. Real-time fluorescence genotyping of Factor V Leiden during rapid-cycle PCR. Clin Chem 1997; 43: 2262-7.
22 Frosst P, Blom HJ, Milos R, Goyette P, Sheppard CA, Mattews RG. Identification of a candidate gene risk factor for vascular disease: a common mutation at the methylenetetrahydrofolate reductase locus. Nat Genet 1995; 10: 111-3.
23 Poort SR, Rosendaal FR, 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.