Validation of a Rapid and Inexpensive Allele-Specific Amplification (ASA)-PCR Genotyping Assay for Vitamin K Antagonist
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Mol Diagn Ther 2011; 15 (1): 13-22 1177-1062/11/0001-0013/$49.95/0
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Validation of a Rapid and Inexpensive Allele-Specific Amplification (ASA)-PCR Genotyping Assay for Vitamin K Antagonist Pharmacogenomics Gabriele Spohn, Christof Geisen, Beate Luxembourg, Katja Sittinger, Erhard Seifried and Halvard Bo¨nig German Red Cross Blood Service Baden-Wu¨rttemberg/Hesse and Institute for Transfusion Medicine and Immunohematology, Goethe University, Frankfurt, Germany
Abstract
Background: Variant alleles of vitamin K epoxide reductase complex subunit 1 gene (VKORC1), the target molecule of vitamin K antagonists, and of cytochrome P450 (CYP) 2C9, an enzyme involved in coumarin metabolism, affect the anticoagulant response of coumarins, which have a narrow therapeutic window. Genotyping for these variants allows for prediction of therapeutic drug doses. The discussion of the clinical role of genotype-guided coumarin dosing is ongoing. For pharmacogenetic information to be useful, results must be available quickly. Methods: Here we report on the establishment of an allele-specific amplification (ASA)-PCR assay for the three most relevant polymorphisms for coumarin pharmacogenetics. The assay was validated against sequencing data on 100 random samples from Caucasian blood donors, incorporating all genotypes. Divergent results were confirmed by repeating the analysis with both methods. One hundred percent congruence with DNA sequencing was determined as the ‘pass’ criterion for the assay. Results: The ASA-PCR assay reproducibly identified the three informative single nucleotide polymorphisms. Discrepancies between ASA-PCR and sequencing were clarified by retrospective analysis as being due to erroneous analysis or documentation. In summary, the congruence of sequencing and duplex ASA-PCR was 100%. Conclusion: ASA-PCR is significantly faster and less expensive than sequencing. We expect that pharmacogenetics-based dosing decisions may reduce the frequency of over- and undertreatment with vitamin K antagonists, especially during drug initiation, and thus improve patient safety.
Introduction Oral anticoagulant treatment with coumarins, such as warfarin, acenocoumarol or phenprocoumon, is universally used for treatment and prevention of thromboembolic events. Besides the narrow therapeutic concentration range, clinical use is complicated by a high variability in dose requirements among individuals. Many patient-specific factors – such as age, body mass, diet, concurrent medication, or diseases – contribute to the variability in dose, but genetic factors have an even greater and more predictable impact on the inter-patient variation in responsiveness to warfarin.[1] Therefore, the possibility and potential benefit of pharmacogenomic testing were recently added as a black box warning to warfarin preparations in the US. For pharmacogenetic testing to achieve its full benefit,
ubiquitous availability of such tests, rapid turn-over time, and limited cost are critical prerequisites
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