Abstract
Background: Inherited thrombophilia is caused by mutations in genes central to the clotting cascade. Analysis of the factor V Leiden (FVL) and prothrombin G20210A mutations are the most prevalent in thrombophilia.
Methods and Results: We have optimized an allele-specific PCR assay for the simultaneous detection of both wild-type and mutant alleles. This method is adapted for clinical use with the FVL and prothrombin G20210A assays and is significant in its intentional use of nucleotide mismatches at the 3′ end of allele-specific primers. Two internal allele-specific primers are designed to amplify in opposite directions on opposite strands that reduce differential amplification. Our results show concordance with methods involving PCR with restriction endonuclease digestion, yet are simpler to perform.
Conclusion: The simultaneous allele-specific amplification method allows simultaneous detection of wild-type and mutant alleles by PCR using four distinct primers. Nucleotide mismatches in the primers reduce competitive amplification.
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References
Bertina RM, Koeleman BP, Koster T, et al.: Mutation in blood coagulation factor V associated with resistance to activated protein C. Nature 1994;369: 64–67
Bowen DJ, Bowley S, John M, Collins PW: Factor V Leiden (G1691A), 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;79:949–954
Faioni EM, Franchi F, Bucciarelli P, et al.: Coinheritance of the HR2 haplotype in the factor V gene confers an increased risk for venous thromboembolism to carriers of factor V R506Q (factor V Leiden). Blood 1999;94:3062–3066
Williamson D, Brown K, Luddington R, Baglin C, Baglin T: Factor V Cambridge: A new mutation (Arg306→Thr) associated with resistance to activated protein C. Blood 1998;91:1140–1144
Dahlback 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 U S A 1993;90:1004–1008
Zoller B, Garcia de Frutos P, Hillarp A, Dahlback B: Thrombophilia as a multigenic disease. Haematologica 1999;84:59–70
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;88:3698–3703
Rosendaal FR, Doggen CJ, Zivelin A, et al.: Geographic distribution of the 20210 G to A prothrombin variant. Thromb Haemost 1998;79:706–708
De Stefano V, Chiusolo P, Paciaroni K, et al.: Prothrombin G20210A mutant genotype is a risk factor for cerebrovascular ischemic disease in young patients. Blood 1998;91:3562–3565
Bloemenkamp KW, Rosendaal FR, Heimerhorst FM, Koster T, Bertina RM, Vandenbroucke JP: Hemostatic effects of oral contraceptives in women who developed deep-vein thrombosis while using oral contraceptives. Thromb Haemost 1998;80:382–387
Arruda VR, Siquiera LH, Chiaparini LC, et al.: Prevalence of the prothrombin gene variant 20210 G→A among patients with myocardial infarction. Cardiovasc Res 1998;37:42–45
Lindqvist P, Dahlback B, Marsal K: Thrombotic risk during pregnancy: A population study. Obstet Gynecol 1999;94:595–599
De Groot CJ, Bloemenkamp KW, Duvekot EJ, et al.: Preeclampsia and genetic risk factors for thrombosis: A case-control study. Am J Obstet Gynecol 1999; 181:975–980
Behn M, Schuermann M: Simple and reliable factor V genotyping by PNA-mediated PCR clamping. Thromb Haemost 1998;79:773–777
Muriel G, Christine B, Philippe J, Christophe M, Francois SJ, Patricia AM: An even easier method for one-step detection of both FV Leiden and FII G20210A transition [letter; comment]. Blood 1998; 92:3478–3479
Patri S, Salmeron S, Caillon M, Kitzis A, Chomel JC: Multiplex PCR for one-step determination of the G20210A variation and the factor V Leiden mutation by denaturing gradient gel electrophoresis (DGGE) [letter]. Thromb Haemost 1999;81:313–314
Ripoll L, Paulin D, Thomas S, Drouet LO: Multiplex PCR-mediated site-directed mutagenesis for one-step determination of factor V Leiden and G20210A transition of the prothrombin gene [letter]. Thromb Haemost 1997;78:960–961
Mitterer M, Lanthaler AJ, Mair W, Giacomuzzi K, Coser P: Simultaneous detection of FV Q506 and prothrombin 20210 A variation by allele-specific PCR. Haematologica 1999;84:204–207
Poort SR, Bertina RM, Vos HL: Rapid detection of the prothrombin 20210 A variation by allele specific PCR [letter]. Thromb Haemost 1997;78:1157–1158
Liu Q, Thorland EC, Heit JA, Sommer SS: Overlapping PCR for bidirectional PCR amplification of specific alleles: A rapid one-tube method for simultaneously differentiating homozygotes and heterozygotes. Genome Res 1997;7:389–398
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 prothrombin gene and C→T 677 MTHFR gene mutations [letter]. Thromb Haemost 1998;79:1054–1055
Gomez E, van der Poel SC, Jansen JH, van der Reijden BA, Lowenberg B: Rapid simultaneous screening of factor V Leiden and G20210A prothrombin variant by multiplex polymerase chain reaction on whole blood [letter]. Blood 1998;91: 2208–2209
Dubreuil Lastrucci RM, Dawson DA, Bowden JH, Marion Munster M: Development of a simple multiplex polymerase chain reaction for the simultaneous detection of factor V Leiden and prothrombin 20210A mutations. Mol Diagn 1999;4:247–250
Blasczyk R, Wehling J, Ritter M, Neubauer A, Riess H: Allele-specific PCR amplification of factor V Leiden to identify patients at risk for thromboembolism. Beitr Infusionsther Transfusionsmed 1997; 34:236–241
Ye S, Humphries S, Green F: Allele specific amplification by tetra-primer PCR. Nucleic Acids Res 1992;20:1152
Hersberger M, Marti-Jaun J, Rentsch K, Hanseler E: Rapid detection of the CYP2D6*3, CYP2D6*4, and CYP2D6*6 alleles by tetra-primer PCR and of the CYP2D6*5 allele by multiplex long PCR. Clin Chem 2000;46:1072–1077
Hyndman D, Cooper A, Pruzinsky S, Coad D, Mitsuhashi M: Software to determine optimal oligo-nucleotide sequences based on hybridization simulation data. Biotechniques 1996;20:1090–1094, 1096-1097
Nicodeme P, Steyaert JM: Selecting optimal oligo-nucleotide primers for multiplex PCR. ISMB 1997; 5:210–213
Kwog S, Kellogg DE, McKinney N, et al.: Effects of primer-template mismatches on the polymerase chain reaction: Human immunodeficiency virus type 1 model studies. Nucleic Acids Res 1990; 18:999–1005
Jenny RJ, Pittman DD, Toole JJ, et al.: Complete cDNA and derived amino acid sequence of human factor V. Proc Natl Acad Sci U S A 1987;84:4846–4850
Degen SJ, Davie EW: Nucleotide sequence of the gene for human prothrombin. Biochemistry 1987; 26:6165–6177
Dieffenbach CW, Lowe TM, Dveksler GS: General concepts for PCR primer design. PCR Methods Appl 1993;3:S30–S37
Lewandowski K, Rozek M, Zawilska K, Markiewicz WT: An alternative method for identifying the factor V gene Leiden mutation. Thromb Res 1997;85:105–113
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Delrio-Lafreniere, S.A., McGlennen, R.C. Simultaneous Allele-specific Amplification: A Strategy Using Modified Primer-template Mismatches for SNP Detection—Application to Prothrombin 20210A (Factor II) and Factor V Leiden (1691A) Gene Mutations. Molecular Diagnosis 6, 201–209 (2001). https://doi.org/10.1007/BF03262054
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DOI: https://doi.org/10.1007/BF03262054