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 To the Editor:

high plasma prothrombin concentra-tions, 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 thrombo-philia, the variant site in the 3 un-translated region is routinely exam-ined in at-risk patients. We use the factor II (prothrombin) G20210A as-say (Roche Diagnostics) for genotyp-ing. In this assay, a 165-bp fragment of the prothrombin gene is amplified, and the different allelic variants are distinguished by melting-curve anal-ysis 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 combina-tion 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 in assigning the genotype with this method, but we have identified a 60-year-old woman of Slavic ances-try in whom the test showed a wild-type allele and an allele with an unexpected melting peak at 55 °C (Fig. 1A), which corresponds to none of the common prothrombin alleles. Although this patient had a strong history of thrombosis, her factor II activity (101%) was within the refer-ence interval (70%–130%). To sequence the 3 part of the prothrombin gene, we performed a standard PCR using primers Fac2fo