Simple and Reliable Factor V Genotyping by PNA-Mediated PCR Clamping

Summary

Resistance to activated protein C (APC resistance) is the most common cause of thrombophilia and linked to a single point mutation in the factor V gene (G>A transition at nucleotide 1691). In the past, several PCR based methods have been proposed to determine the allelostatus of individual patients from small amounts of blood DNA including PCR followed by restriction fragment length polymorphism detection (PCR-RFLP), PCR using sequence-specific primers (PCR-SSP) and oligonucleotide ligation assay (OLA). Here, we present a novel approach based on the method of peptide nucleic acid(PNA)-mediated PCR clamping which is extremely sensitive to base pair mismatches. If PNAs specific for the two allelic variants are applied separately in each case a clear discrimination between a heterozygous or homozygous normal or homozygous Factor V Leiden status is possible and no further confirmation step is required. In a prospective study, 60 patients with suspected venous thrombosis events were tested and compared to the conventional PCR-RFLP technique. The concordance between both methods was 100%. PNA-based factor V genotyping, therefore, should be considered for large scale screening of those patients considered to be at risk for deep venous thrombosis.

• References

• 1 Dahlbäck B, Carlsson M, Svensson PJ. Familial thrombophilia due to a previuosly 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: 1004-8.
• 2 Griffin JH, Evatt B, Wideman C, Fernandez JA. Anticoagulant protein C pathway defective in majority of thrombophilic patients. Blood 1993; 82: 1989-93.
• 3 Koster T, Rosendaal FR, de Ronde H, Briët E, Vandenbroucke JP, Bertina RM. Venous thrombosis due to poor anticoagulant response to activated protein C: The Leiden Study. Lancet 1993; 342: 1503-6.
• 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 Dahlbäck B. New molecular insights into the genetics of thrombophilia. Resistance to activated Protein C caused by Arg⁵⁰⁶ to Gln mutation in factor V as a prognostic risk factor for venous thrombosis. Thromb Haemost 1995; 74: 139-48.
• 6 Bertina RM, Koeleman BPC, Koster T, Rosendaal FR, Dirven RJ, de Ronde H, van der Velden PA. Reitsma. Mutation in blood coagulation factor V associated with resistance to activated protein C. Nature 1994; 369: 64-7.
• 7 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: 1361-2.
• 8 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 Arg⁵⁰⁶ of factor V. Lancet 1994; 343: 1535-6.
• 9 Zöller B, Dahlbäck B. Linkage between inherited resistance to activated protein C and factor V gene mutation in venous thrombosis. Lancet 1994; 343: 1536-8.
• 10 Kalafatis M, Bertina RM, Rand MD, Mann KG. Characterisation of the molecular defect in factor VR⁵⁰⁶Q. J Biol Chem 1995; 270: 4053-7.
• 11 Kirschbaum NE, Foster PA. The polymerase chain reaction with sequence specific primers for the detection of the factor V mutation associated with activated protein C resistance. Thromb Haemost 1995; 74: 874-8.
• 12 Van-de-Locht LT, Kuypers AW, Verbruggen BW, Linssen PC, Novakova IR, Mensink EJ. Semi-automated detection of the factor V mutation by allele specific amplification and capillary electrophoresis. Thromb Haemost 1995; 74: 1276-9.
• 13 Engel H, Zwang L, van Vliet HHDM, Michiels JJ, Stibbe J, Lindemans J. Phenotyping and genotyping of coagulation factor V Leiden. Thromb Haemost 1996; 75: 267-9.
• 14 Zotz RB, Maruhn-Debowski B, Scharf RE. Mutation in the gene coding for coagulation factor V and resistance to activated protein C: detection of the genetic mutation by oligonucleotide ligation assay using a semi-automated system. Thromb Haemost 1996; 76: 53-5.
• 15 Orum H, Nielsen PE, Egholm M, Berg RH, Buchardt O, Stanley C. Single base pair mutation analysis by PNA directed PCR clamping. Nucleic Acids Res 1993; 21: 5332-6.
• 16 Nielsen PE. DNA analogues with nonphosphodiester backbones. Annu Rev Biophys Biomol Struct 1995; 24: 167-83.
• 17 Agarwal S, Iyer RP. Modified oligonucleotides as therapeutic and diagnostic agents. Curr Opin Biotechnol 1995; 6: 12-9.
• 18 Buchardt O, Egholm M, Berg RH, Nielsen PE. Peptide nucleic acids and their potential applications in biotechnology. Trends Biotechnol 1993; 11: 384-6.
• 19 Thiede C, Bayerdorffer E, Blasczyk R, Wittig B, Neubauer A. Simple and sensitive detection of mutations in the ras proto-oncogenes using PNA-mediated PCR clamping. Nucleic Acids Res 1996; 24: 983-4.