Reliable Genotyping of the G-20210-A Mutation of Coagulation Factor II (Prothrombin)

Abstract

1 Prothrombin (coagulation factor II) is the precursor of thrombin, which participates as a serine protease (factor II) in the coagulation cascade. Thrombin is essential in the processes of hemostasis and thrombosis (1)(2)(3). The gene that codes for prothrombin is 21 kb in size and contains 14 exons (4). The gene has been mapped on chromosome 11 at position 11p11-q12 (5). Recently Poort et al. (6) reported a PCR-mediated site-directed mutagenesis method for the detection of the factor II G-20210-A mutation. This single-point mutation (G→A) at position 20210, the last nucleotide of the 3′-UT region [ 4 , 6] , has been shown to be associated with an increased risk of deep vein thrombosis. The prevalences for the heterozygous genotype 20210 AG found in the Leiden Thrombophilia Study were 2.3% in healthy control subjects and 6.2% in patients. A homozygous AA genotype was not found (expected prevalence 0.014%) (6). The relative risk for thrombosis being associated with the heterozygous state AG was 2.8 (6). The primers and enzyme were chosen in such a way that only the amplification product of the mutant allele was digested; the PCR product of the wild-type allele was not digested. The reverse primer PR 95–315 (5′-ATA gCA CTg ggA gCA TTg AAg C-3′) (6) contains a single-base mismatch G→A (asterisk), thereby creating a novel Hin dIII restriction site (A↓AgCTT) in the amplified product of the mutant allele. Because only the amplified product of the mutant allele is cut, no internal control for the digestion of Hin dIII is available in case of an amplified product of the wild-type allele. This could result in wrong conclusions in cases when digestion activity is decreased or absent, for example by influence of inhibitory factors in the reaction mix, and a false-negative result …