title: Validation of a rapid and inexpensive allele-specific amplification (ASA)-PCR genotyping assay for vitamin K antagonist pharmacogenomics.
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%.
  
  
  CONCLUSIONS
  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.
authors.affiliation: German Red Cross Blood Service Baden-Württemberg/Hesse and Institute for Transfusion Medicine and Immunohematology, Goethe University, Frankfurt, Germany.
authors.forename: Gabriele
authors.initials: G
authors.lastname: Spohn
authors.forename: Christof
authors.initials: C
authors.lastname: Geisen
authors.forename: Beate
authors.initials: B
authors.lastname: Luxembourg
authors.forename: Katja
authors.initials: K
authors.lastname: Sittinger
authors.forename: Erhard
authors.initials: E
authors.lastname: Seifried
authors.forename: Halvard
authors.initials: H
authors.lastname: Bönig
mesh_terms: D000483:Alleles; D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics*; D001483:Base Sequence; D003374:Coumarins / Q000627:therapeutic use; D065729:Cytochrome P-450 CYP2C9; D005260:Female; D005784:Gene Amplification; D005838:Genotype; D006801:Humans; D008297:Male; D006899:Mixed Function Oxygenases / Q000235:genetics*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D020641:Polymorphism, Single Nucleotide; D017422:Sequence Analysis, DNA; D014812:Vitamin K / Q000037:antagonists & inhibitors; D064417:Vitamin K Epoxide Reductases
chemical_list: D003374:Coumarins; D014812:Vitamin K; D006899:Mixed Function Oxygenases; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D001189:Aryl Hydrocarbon Hydroxylases; C578523:VKORC1 protein, human; D064417:Vitamin K Epoxide Reductases