Atypical Melting Curve Resulting from Genetic Variation in the 3′ Untranslated Region at Position 20218 in the Prothrombin Gene Analyzed with the LightCycler Factor II (Prothrombin) G20210A Assay

Carriers of the G20210A allele have high plasma prothrombin concentrations, which in turn are associated with an almost 3-fold increased risk of venous thrombosis (1). As it is the second most common genetic change associated with inherited thrombophilia, the variant site in the 3′ untranslated region is routinely examined in at-risk patients. We use the factor II (prothrombin) G20210A assay (Roche Diagnostics) for genotyping. In this assay, a 165-bp fragment of the prothrombin gene is amplified, and the different allelic variants are distinguished by melting-curve analysis using the fluorescence resonance energy transfer principle. The wild-type and variant alleles have melting peaks at ∼59 ± 2.5 °C and ∼49 ± 2.5 °C, respectively. Heterozygous samples exhibit a distinct combination of both melting peaks. We have genotyped genomic DNA from more than 2000 patients. Of these, 97% were homozygous wild type, ∼3% were heterozygous, and <0.1% were homozygous mutant. We have experienced no difficulties …