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Letters to the Editor |
Institute of Clinical Chemistry, and Pathobiochemistry, RWTH-University Hospital, Aachen, Germany
aAddress correspondence to this author at: Institute of Clinical Chemistry and Pathobiochemistry, RWTH-University Hospital, D-52074 Aachen, Germany. Fax 49-241-80-82512; e-mail rweiskirchen{at}ukaachen.de.
To the Editor:
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 in assigning the genotype with this method, but we have identified a 60-year-old woman of Slavic ancestry 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 reference interval (70%–130%).
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To sequence the 3' part of the prothrombin gene, we performed a standard PCR using primers Fac2for (5'-CCG CTG GTA TCA AAT GGG-3') and Fac2rev (5'-CCA GTA GTA TTA CTG GCT CTT CCT G-3'), which led to amplification of the reported 291-bp fragment (2). The amplicons were purified, subjected to the Big Dye Terminator Cycle Sequencing protocol, and analyzed on the ABI PRISM 310 genetic analyzer (PE Applied Biosystems). Sequences revealed 1 wild-type allele and 1 with an A-to-G transition at position 20218 (Fig. 1B
). This mutation was identified previously in a screen evaluating an electronically addressable microarray carrying genetic risk factors of venous thrombosis (3). However, the obtained melting curve is indistinguishable from those reported for 2 other mutations within the 3'-untranslated region of the prothrombin gene (Fig. 1C
), revealing that the patient carries an alteration in close proximity to position 20210.
Among the previously reported variants was a C
T change at position 20221 that was found in a 9-year-old boy of Lebanese/Syrian origin who developed chronic renal failure and acute vascular rejection of a renal transplant (4). The other variant, C20209T, was first noticed in 4 unrelated African-American individuals; 3 of these patients had a history of venous thrombosis or stroke, whereas the fourth had severe liver disease, which may have masked a thrombotic predisposition (5). Subsequently, 3 other non-Caucasian patients who had obstetric complications, including recurrent spontaneous abortions, intrauterine growth retardation, and neonatal demise, tested positive for the C20221T or C20209T mutation (6). In summary, all 3 mutations (C20209T, A20218G, and C20221T) give LightCycler melting curves that are clearly distinguishable from those obtained in wild-type or G20210A samples. Therefore, the conscientious hints included in the factor II assay package insert noting that these rare mutations will possibly lead to a false-positive result might be exaggerated. Furthermore, it is tempting to speculate that all 3 variants are rare and possibly have different frequencies in different ethnic groups.
Because the patient identified in our laboratory as carrying the A20218G transition also tested heterozygous for factor V Leiden (G1691A) and the methylenetetrahydrofolate reductase (C677T) mutation, the clinical significance of this mutation is currently unknown. However, based on the knowledge that the G20210A substitution represents in vitro a gain-of-function mutation causing mRNA accumulation and increased protein synthesis (7), creates in vitro but not in vivo a more effective polyadenylation site and cleavage site (7)(8)(9), and is proposed to be associated with different mRNA structures leading to abnormal mRNA function (9), it will be worthwhile to initiate additional studies to estimate the impact of this genetic variant on risk assessment for thrombotic events and adverse pregnancy outcomes.
References
The following articles in journals at HighWire Press have cited this article:
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  E. Lyon Discovering Rare Variants by Use of Melting Temperature Shifts Seen in Melting Curve Analysis Clin. Chem., August 1, 2005; 51(8): 1331 - 1332. [Full Text] [PDF]
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