Rapid Detection of Factor XIII Val34Leu by Allele Specific PCR

Coagulation factor XIII contributes to clot stability and to its resistance to fibrinolysis by crosslinking fibrin monomers and other substrates such as a2 antiplasmin, collagen and fibronectin. A variation GÕT in codon 34 of exon 2 which codes for a ValineÕLeucine (Factor XIIIVal34Leu) change in the catalytic A-subunit close to the thrombin activation site has been described (1). By interfering with factor XIII activation, Factor XIIIVal34Leu could contribute to the pathogenesis of thrombotic disorders (2). Indeed, it has been shown to be protective against myocardial infarction (MI) in a British population, the prevalence of the mutation being lower in patients with MI than without (32% vs 50%) (2). In that study, to detect this gene variant, a 183 bp fragment of exon 2 / intron B of Factor XIII was amplified by PCR and analyzed by single strand conformational polymorphism (SSCP) technique. Here, we present an alternative method which uses allele specific PCR. This method is rapid, simple and especially useful for the screening of large number of samples. Two external primers (primer 1 and 2) and two internal forward primers (primer 3 and 4) have been employed: Primer 1 forward: 5’GACCTTGTAAAGTCAAAAATGTC 3’; primer 2 a reverse primer: 5’ACCCAGAGTGGTGGGGAAG 3’; primer 3 a forward primer G: GGAGCTTCAGGGCGT and primer 4 a forward primer T: GGAGCTTCAGGGCTT. Primers 1 and 2 correspond to those used in the SSCP technique and amplify a 183 bp fragment. The allele specific primers 3 and 4 produced a PCR fragment of 76 bp which determine the factor XIII genotype for the Val 34 Leu polymorphism. Amplification was carried out in 25 μl in a Thermocycler 9600 Perkin Elmer (Applied Biosystems CA, USA). Each sample contained about 62 ng genomic DNA in 1X Taq polymerase buffer (2.5 mM MgCl2),0.77 mM dNTPs, 5 pmoles of each primer (1, 2 and 3 for genotype G or 1, 2 and 4 for genotype T determination), and 0.38 units Taq polymerase (Biotaq, Quantum Bioprobe, Quebec Canada). A first denaturation at 95° C for 2 min was followed by 40 cycles of 59° C 1min for allele G amplification or 61° C 1min for allele T amplification (annealing), 72° C 1min 30 (extension), 95° C 45 sec (denaturation); then 72° C 5 min. Five ml of each amplification sample was loaded on a 2% agarose gel ethidium bromide stained. Results obtained with the different genotypes are presented on Fig.1, they were all confirmed by DNA sequencing. It is to note a strong decrease in the 183 bp band intensity in homozygous individuals. This is due to the choice of the annealing temperature that favours primer 3 or 4 hybridization compared to primer 1. This makes the differential diagnosis easier between homozygotes and heterozygotes. Among healthy blood donors from the south of France (mean age 47.9 + 9.1), all caucasians, the T allele was found in 115 out of 244 individuals (47%); 9 subjects (4%) being homozygotes, which represents an allele frequency of 26%. These results are very similar to those observed in the British as well as in the Finnish population (1-2-3). The present method is especially useful for the identification of the FXIII Val34Leu in large series of individuals and to assess more easily the role of this polymorphism in thrombotic disorders.