To the Editor:
Activated protein C resistance (APCR) has emerged as the most common inherited risk factor for venous thrombosis. Heterozygosity for the underlying DNA mutation Factor V Leiden (Arg 506 Gln) is associated with a 5- to 10-fold increased risk of developing a venous thrombotic episode, whereas homozygosity is associated with a 50- to 100-fold increased risk.1 Interaction of APCR with other co-inherited risk factors such as the recently described prothrombin gene variant (G → A transition at position 20210) is expected to synergistically increase the thrombotic risk.2
Therefore, a one-step detection of both genetic variants is very attractive for the diagnosis and management of deep vein thrombosis. We read with interest the recent report by Gomez3 about the rapid screening of Factor V Leiden and the G20210A prothrombin variant. The investigators described a multiplex polymerase chain reaction followed by a combined restriction digest of both the factor V gene and the prothrombin gene PCR products. They simultaneously use two different endonucleases, leading to a complex restriction pattern with several cleavage sites for wild-type and mutated amplified fragments.
In our laboratory, we have developed a very simple method based on multiplex polymerase chain reaction (PCR)-mediated site-directed mutagenesis using primers with mismatched 3′-ends. This duplex PCR is followed by restriction using a single and inexpensive endonuclease,HindIII. The electrophoretic patterns can be easily identified whatever the genotypic combination. This method is used as a routine diagnosis strategy for both mutations.
We use either genomic DNA prepared by a standard salting out procedure4 or a rapid extraction procedure using Chelex resin (20 μL of extraction product from whole blood).5
Primers for the factor V gene G1691A determination are those described by Gandrille et al6 and primers for the prothrombin gene 20210A determination have been described by Poort et al.7For each pair of primers, the 3′ antisense has been modified to create a single HindIII cleavage site in the presence of both mutated PCR products.
After HindIII restriction, the digested products can be easily separated on a 2% agarose gel. Figure 1 shows the different migration patterns observed. For both factor V and factor II alleles, the normal genotypes produce undigested PCR products (241 and 345 bp, respectively), whereas mutated homozygous lead to restricted fragments (209 + 32 and 322 + 23 bp, respectively). The heterozygous patterns are characterized by the presence of undigested and digested amplified fragments.
Electrophoretic patterns for multiplex FV and FII PCR. The PCR mixture in a final volume of 50 μL consisted of 1 μg genomic DNA, PCR buffer (16.6 mmol/L amonium sulphate, 67 mmol/L Tris-HCl, pH 8.8, 6.7 mmol/L magnesium chloride, 67 μmol/L Na2EDTA, 170 μg bovine serum albumin per mL, 10 mmol/L β-mercaptoethanol), 400 μmol/L of each desoxynucleotide triphosphate, 30 pmol of each FV primer, 10 pmol of each FII primer, and 2 U Taq polymerase. Thermocycling conditions are 94°C (1 minute), 58°C (1 minute), 72°C (2 minutes) for 40 cycles. Fifteen microliters of PCR product is digested with 15 U HindIII restriction enzyme. The restricted products are separated by electrophresis through a 2% agarose gel stained with ethidium bromide and directly visualized under UV light. The smallest restricted fragments (32 and 23 bp) are not visible on the gel. Lane 1, size marker (1-kb ladder); lane 2, undigested PCR products; lanes 3 through 8, digested PCR products. UD, undigested PCR products; N, normal allele; m, mutated allele.
Electrophoretic patterns for multiplex FV and FII PCR. The PCR mixture in a final volume of 50 μL consisted of 1 μg genomic DNA, PCR buffer (16.6 mmol/L amonium sulphate, 67 mmol/L Tris-HCl, pH 8.8, 6.7 mmol/L magnesium chloride, 67 μmol/L Na2EDTA, 170 μg bovine serum albumin per mL, 10 mmol/L β-mercaptoethanol), 400 μmol/L of each desoxynucleotide triphosphate, 30 pmol of each FV primer, 10 pmol of each FII primer, and 2 U Taq polymerase. Thermocycling conditions are 94°C (1 minute), 58°C (1 minute), 72°C (2 minutes) for 40 cycles. Fifteen microliters of PCR product is digested with 15 U HindIII restriction enzyme. The restricted products are separated by electrophresis through a 2% agarose gel stained with ethidium bromide and directly visualized under UV light. The smallest restricted fragments (32 and 23 bp) are not visible on the gel. Lane 1, size marker (1-kb ladder); lane 2, undigested PCR products; lanes 3 through 8, digested PCR products. UD, undigested PCR products; N, normal allele; m, mutated allele.
To check the accuracy of the results, we systematically include in each assay two positive controls corresponding to homozygous patients for FV or FII mutations, respectively. Furthermore, a single reaction mix for the digestion, including the restriction endonuclease, is prepared for each sample set. This allows both samples and controls to be assayed with the same conditions.
Our method has at least two main advantages. First, the same inexpensive restriction endonuclease is used for the detection of the mutated alleles in both FV and FII, the size of the PCR digests allowing a simple detection on agarose gels. Second, the electrophoretic patterns can be easily and unambiguously recognized as they are very simple and identical for both mutations. This method thus offers a reliable tool for routine diagnosis.
