title: Rapid single-nucleotide polymorphism detection of cytochrome P450 (CYP2C9) and vitamin K epoxide reductase (VKORC1) genes for the warfarin dose adjustment by the SMart-amplification process version 2.
abstract: BACKGROUND
  Polymorphisms of the CYP2C9 (cytochrome P450, family 2, subfamily C, polypeptide 9) gene (CYP2C9*2, CYP2C9*3) and the VKORC1 (vitamin K epoxide reductase complex, subunit 1) gene (-1639G>A) greatly impact the maintenance dose for the drug warfarin. Prescreening patients for their genotypes before prescribing the drug facilitates a faster individualized determination of the proper maintenance dose, minimizing the risk for adverse reaction and reoccurrence of thromboembolic episodes. With current methodologies, therapy can be delayed by several hours to 1 day if genotyping is to determine the loading dose. A simpler and more rapid genotyping method is required.
  
  
  METHODS
  We developed a single-nucleotide polymorphism (SNP)-detection assay based on the SMart Amplification Process version 2 (SMAP 2) to analyze CYP2C9*2, CYP2C9*3, and VKORC1 -1639G>A polymorphisms. Blood from consenting participants was used directly in a closed-tube real-time assay without DNA purification to obtain results within 1 h after blood collection.
  
  
  RESULTS
  We analyzed 125 blood samples by both SMAP 2 and PCR-RFLP methods. The results showed perfect concordance.
  
  
  CONCLUSIONS
  The results validate the accuracy of the SMAP 2 for determination of SNPs critical to personalized warfarin therapy. SMAP 2 offers speed, simplicity of sample preparation, the convenience of isothermal amplification, and assay-design flexibility, which are significant advantages over conventional genotyping technologies. In this example and other clinical scenarios in which genetic testing is required for immediate and better-informed therapeutic decisions, SMAP 2-based diagnostics have key advantages.
authors.affiliation: Department of Clinical Pharmacology, Gunma University Graduate School of Medicine, Maebashi, Japan.
authors.forename: Tohru
authors.initials: T
authors.lastname: Aomori
authors.forename: Koujirou
authors.initials: K
authors.lastname: Yamamoto
authors.forename: Atsuko
authors.initials: A
authors.lastname: Oguchi-Katayama
authors.forename: Yuki
authors.initials: Y
authors.lastname: Kawai
authors.forename: Takefumi
authors.initials: T
authors.lastname: Ishidao
authors.forename: Yasumasa
authors.initials: Y
authors.lastname: Mitani
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Kogo
authors.forename: Alexander
authors.initials: A
authors.lastname: Lezhava
authors.forename: Yukiyoshi
authors.initials: Y
authors.lastname: Fujita
authors.forename: Kyoko
authors.initials: K
authors.lastname: Obayashi
authors.forename: Katsunori
authors.initials: K
authors.lastname: Nakamura
authors.forename: Hugo
authors.initials: H
authors.lastname: Kohnke
authors.forename: Mia
authors.initials: M
authors.lastname: Wadelius
authors.forename: Lena
authors.initials: L
authors.lastname: Ekström
authors.forename: Cristine
authors.initials: C
authors.lastname: Skogastierna
authors.forename: Anders
authors.initials: A
authors.lastname: Rane
authors.forename: Masahiko
authors.initials: M
authors.lastname: Kurabayashi
authors.forename: Masami
authors.initials: M
authors.lastname: Murakami
authors.forename: Paul E
authors.initials: PE
authors.lastname: Cizdziel
authors.forename: Yoshihide
authors.initials: Y
authors.lastname: Hayashizaki
authors.forename: Ryuya
authors.initials: R
authors.lastname: Horiuchi
mesh_terms: D001189:Aryl Hydrocarbon Hydroxylases / Q000032:analysis* / Q000145:classification / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D065729:Cytochrome P-450 CYP2C9; D004305:Dose-Response Relationship, Drug; D006801:Humans; D006899:Mixed Function Oxygenases / Q000032:analysis* / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D016415:Sequence Alignment; D013997:Time Factors; D064417:Vitamin K Epoxide Reductases; D014859:Warfarin / Q000494:pharmacology*
chemical_list: D014859:Warfarin; 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