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 pharmaco-genetics-based dosing decisions may reduce the frequency of over- and undertreatment with vitamin K antagonists, especially during drug initiation, and thus improve patient safety.
Coumarin International Normalize Ratio Pharmacogenetic Testing Therapeutic International Normalize Ratio Range Pharmacogenetic Information
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Gabriele Spohn and Christof Geisen contributed equally to this work.
The work of the technicians of the molecular hemostaseology laboratory at the German Red Cross Blood Service Baden-Württemberg/Hesse and Institute for Transfusion Medicine and Immunohematology, who performed DNA isolation and sequencing, is gratefully acknowledged.
No funding has been received for the conduct of this study or the preparation of this manuscript. The authors have no conflicts of interest directly related to the content of this study.
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