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.
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.
Aomori
Tohru
T
Department of Clinical Pharmacology, Gunma University Graduate School of Medicine, Maebashi, Japan.
Yamamoto
Koujirou
K
Oguchi-Katayama
Atsuko
A
Kawai
Yuki
Y
Ishidao
Takefumi
T
Mitani
Yasumasa
Y
Kogo
Yasushi
Y
Lezhava
Alexander
A
Fujita
Yukiyoshi
Y
Obayashi
Kyoko
K
Nakamura
Katsunori
K
Kohnke
Hugo
H
Wadelius
Mia
M
Ekström
Lena
L
Skogastierna
Cristine
C
Rane
Anders
A
Kurabayashi
Masahiko
M
Murakami
Masami
M
Cizdziel
Paul E
PE
Hayashizaki
Yoshihide
Y
Horiuchi
Ryuya
R
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*
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