PDF herunterladen
Thromb Haemost 1995; 74(03): 874-878
DOI: 10.1055/s-0038-1649840
Original Article
Coagulation
Schattauer GmbH Stuttgart

The Polymerase Chain Reaction with Sequence Specific Primers for the Detection of the Factor V Mutation Associated with Activated Protein C Resistance

Nancy E Kirschbaum
1   The Hemostasis Reference Laboratory of the Blood Center of Southeastern Wisconsin, USA
,
Paul A Foster
1   The Hemostasis Reference Laboratory of the Blood Center of Southeastern Wisconsin, USA
2   The Blood Research Institute of the Blood Center of Southeastern Wisconsin, USA
3   The Departments of Medicine, Pathology and Pediatrics of the Medical College of Wisconsin, Milwaukee, Wisconsin,USA
› Institutsangaben
Weitere Informationen

Publikationsverlauf

Received 21. März 1995

Accepted after revision 23. Mai 1995

Publikationsdatum:
09. Juli 2018 (online)

Auch verfügbar auf
  als PDF herunterladen  Lizenzen und Reprints

Summary

The prevalence of the Factor V (FV) mutation associated with activated protein C resistance (FV Leiden) and its significance as a genetic risk factor for venous thrombosis have necessitated the development of a simple, rapid, and accurate assay for its detection. The polymerase chain reaction with sequence specific primers (PCR-SSP) provides a powerful technique for the discrimination of alleles resulting from single base substitutions. PCR amplification was performed using a sense primer complementary to both FV alleles coupled with either of two antisense allele specific primers, one complementary to the normal FV allele and one complementary to the FV Leiden allele. PCR conditions were developed that favored amplification only in the case of perfect complementation between template DNA and allele specific primer. The FV genotype was assigned based on whether or not each allele specific primer set produced an amplified product. Assignment of genotypes correlated 100% with those determined by the method of PCR amplification followed by MnII digestion. PCR-SSP allows the rapid and accurate identification of carriers of the Factor V Leiden mutation by a simple PCR reaction without the need for the usual post-amplification specificity step.

 

  • References

  • 1 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 C. Proc Natl Acad Sci USA 1993; 90: 1004-1008
    CrossrefPubMedSuche in Google Scholar
  • 2 Griffin JH, Evatt B, Wideman C, Fernandez JA. Anticoagulant protein C pathway defective in majority of thrombophilic patients. Blood 1993; 82: 1989-1993
    PubMedSuche in Google Scholar
  • 3 Koster T, Rosendaal FR, de Ronde H, Briet E, Vandenbroucke JP, Bertina RM. Venous thrombosis due to poor anticoagulant response to activated protein C: Leiden Thrombophilia Study. Lancet 1993; 342: 1503-1506
    CrossrefPubMedSuche in Google Scholar
  • 4 Svensson PJ, Dahlbäck B. Resistance to activated protein C as a basis for venous thrombosis. N Engl J Med 1994; 330: 517-521
    CrossrefPubMedSuche in Google Scholar
  • 5 Cadroy Y, Sié P, Boneu B. Frequency of a defective response to activated protein C in patients with a history of venous thrombosis. Blood 1994; 83: 2008-2009
    PubMedSuche in Google Scholar
  • 6 Halbmeyer W-M, Haushofer A, Schon R, Fischer M. The prevalence of poor anticoagulant response to activated protein C resistance (APC resistance) among patients suffering from stroke or venous thrombosis and among healthy subjects. Blood Coagul Fibrinolysis 1994; 5: 51-57
    CrossrefPubMedSuche in Google Scholar
  • 7 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: 64-67
    CrossrefPubMedSuche in Google Scholar
  • 8 Beauchamp NJ, Daly ME, Hampton KK, Cooper PC, Preston FE, Peake IR. High prevalence of a mutation in the factor V gene within the U.K. population: relationship to activated protein C resistance and familial thrombosis. Br J Haemotol 1994; 88: 219-222
    CrossrefPubMedSuche in Google Scholar
  • 9 Greengard JS, Sun X, Xu X, Fernandez JA, Griffin JH, Evatt B. Activated protein C resistance caused by Arg506GIn mutation in factor Va. Lancet 1994; 343: 1361-1362
    PubMedSuche in Google Scholar
  • 10 Voorberg J, Roelse J, Koopman R, Buller H, Berends F, ten Cate JW, Mertens K, van Mourik JA. Association of idiopathic venous thromboembolism with single point-mutation at Arg506of factor V. Lancet 1994; 343: 1535-1536
    CrossrefPubMedSuche in Google Scholar
  • 11 Zoller B, Dahlbäck B. Linkage between inherited resistance to activated protein C and factor V gene mutation in venous thrombosis. Lancet 1994; 343: 1536-1538
    CrossrefPubMedSuche in Google Scholar
  • 12 Kalafatis M, Bertina RM, Rand MD, Mann KG. Characterization of the molecular defect in FV R506Q. J Biol Chem 1995; 270: 4053-4057
    CrossrefPubMedSuche in Google Scholar
  • 13 Rosen S, Johansson K, Lindberg K, Dahlbäck B. Mutlicenter evaluation of a kit for activated protein C resistance on various coagulation instruments using plasma from healthy individuals. Thromb Haemost 1994; 72: 255-260
    Thieme ConnectPubMedSuche in Google Scholar
  • 14 Alhenc-Gelas M, Gandrille S, Aubry M-L, Emmerich J, Fiessinger J-N, Aiach M. Unexplained thrombosis and factor V Leiden mutation. Lancet 1994; 344: 555-556
    PubMedSuche in Google Scholar
  • 15 Zoller B, Svensson PJ, He X, Dahlbäck B. Identification of the same factor V gene mutation in 47 out of 50 thrombosis-prone families with inherited resistance to activated protein C. J Clin Invest 1994; 94: 2521-2524
    CrossrefPubMedSuche in Google Scholar
  • 16 Jorquera JI, Montoro JM, Fernandez MA, Aznar JA, Aznar J. Modified test for activated Protein C resistance. Lancet 1994; 344: 1162
    PubMedSuche in Google Scholar
  • 17 Trossaert M, Conard J, Horellou MH, Samama MM, Ireland H, Bayston TA, Lane DA. Modified APC resistance assay for patients on oral anticoagulants. Lancet 1994; 344: 1709
    CrossrefPubMedSuche in Google Scholar
  • 18 Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 1977; 74: 5463-5467
    CrossrefPubMedSuche in Google Scholar
  • 19 Chen EY, Liao YC, Smith DH, Barrera-Saldana HA, Gelinas RE, Seeburg PH. The human growth hormone locus: nucleotide sequence, biology, and evolution. Genomics 1989; 4: 479-497
    CrossrefPubMedSuche in Google Scholar
  • 20 Cripe LD, Moore KD, Kane WH. Structure of the gene for human coagulation factor V. Biochemistry 1992; 31: 3777-3785
    CrossrefPubMedSuche in Google Scholar
  • 21 Wu DY, Ugozzoli L, Pal BK, Wallace RB. Allele-specific enzymatic amplification of β-globin genomic DNA for diagnosis of sickle cell anemia. Proc Natl Acad Sci USA 1989; 86: 2757-2760
    CrossrefPubMedSuche in Google Scholar
  • 22 Newton CR, Graham A, Heptinstall LE, Powell SJ, Summers C, Kalsheker N, Smith JC, Markham AF. Analysis of any point mutation in DNA. The amplification refractory mutation system (ARMS) Nucleic Acids Res 1989; 17: 2503-2517
    CrossrefPubMedSuche in Google Scholar
  • 23 Sarkar G, Cassady J, Bottema CD K, Sommer SS. Characterization of polymerase chain reaction amplification of specific alleles. Anal Biochem 1990; 186: 64-68
    CrossrefPubMedSuche in Google Scholar
  • 24 Olerup O, Zetterquist H. HLA-DR typing by PCR amplification with sequence-specific primers (PCR-SSP) in 2 hours: an alternative to serological DR typing in clinical practice including donor-recipient matching in cadaveric transplantation. Tissue Antigens 1992; 39: 225-235
    CrossrefPubMedSuche in Google Scholar
  • 25 Skogen B, Bellissimo DB, Hessner MJ, Santoso S, Aster RH, Newman PJ, McFarland JG. Rapid determination of platelet alloantigen genotypes by polymerase chain reaction using allele-specific primers. Transfusion 1994; 34: 955-960
    CrossrefPubMedSuche in Google Scholar
  • 26 Beauchamp NJ, Daly ME, Cooper PC, Preston FE, Peake IR. Rapid two-stage PCR for detecting factor V G1691A mutation. Lancet 1994; 344: 694-695
    PubMedSuche in Google Scholar
  • 27 Jenny RJ, Pittman DD, Toole JJ, Kriz RW, Aldape RA, Hewick R, Kaufman RJ, Mann KG. Complete cDNA and derived amino acid sequence of human factor V. Proc Natl Acad Sci USA 1987; 84: 4846-4850
    CrossrefPubMedSuche in Google Scholar