Prevalence of the prothrombin G20210A mutation in the Irish populations: use of a novel polymerase chain reaction approach

The prothrombin G20210A polymorphism is associated with a threefold-increas ed risk of venous thrombosis. There is considerable variation in the report ed prevalence of this polymorphism within normal populations, ranging from 0 to 6.5%. The prevalence within the Irish population has not been determin ed. A restriction fragment length polymorphism (RFLP)-based assay is common ly used for the detection of the prothrombin 20210A allele, This assay does not include a control restriction digest fragment and, consequently, failu re of the enzyme activity or lack of addition of enzyme to the sample canno t be distinguished from wild-type prothrombin. We developed a RFLP-based as say, which incorporates an invariant digest site, resulting in the generati on of a control digest fragment. Furthermore, we developed a nested polymer ase chain reaction (PCR) method for the amplification and digestion of poor -quality or low-concentration DNA. In the Irish population studied, five of 385 (1.29%) were heterozygous and one patient was homozygous for the proth rombin 20210A polymorphism. This is the first reported data on an Irish or Celtic population and suggests that the allele frequency is similar to Angl o-Saxon populations. The nested PCR method successfully amplified and diges ted 100/100 (100%) of the archived samples; none of these samples could be analyzed by the standard single-round PCR method, In conclusion, nested PCR should be considered in the analysis of archived samples. Single-round PCR is appropriate for recently collected samples; however, an invariant contr ol digest site should be incorporated in RFLP-based assays to validate the integrity of the digestion enzyme and limit the risk of false-negative resu lts, Blood Coagul Fibrinolysis 11:669-672 (C) 2000 Lippincott Williams & Wi lkins.