Received on September 3, 2008
Accepted on December 16, 2008

Point-of-Care Testing

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

¹ Department of Clinical Pharmacology, Gunma University Graduate School of Medicine, Maebashi, Japan, and Department of Pharmacy, Gunma University Hospital, Maebashi, Japan
² Genome Exploration Research Group (Genome Network Project Core Group), RIKEN Genomic Sciences Center (GSC), RIKEN Yokohama Institute, Yokohama, Japan
³ Genome Exploration Research Group (Genome Network Project Core Group), RIKEN Genomic Sciences Center (GSC), RIKEN Yokohama Institute, Yokohama, Japan, and K.K. DNAFORM, Yokohama, Japan
⁴ Department of Pharmacy, Gunma University Hospital, Maebashi, Japan
⁵ Department of Clinical Pharmacology, Gunma University Graduate School of Medicine, Maebashi, Japan
⁶ Department of Medical Sciences, Clinical Pharmacology, Uppsala University Hospital, Uppsala, Sweden
⁷ Karolinska Institute, Department of Clinical Pharmacology, Karolinska University Hospital, Stockholm, Sweden
⁸ Department of Medicine and Biological Science, Gunma University Graduate School of Medicine, Maebashi, Japan
⁹ Department of Clinical Laboratory Medicine, Gunma University Graduate School of Medicine, Maebashi, Japan
¹⁰ Genome Exploration Research Group (Genome Network Project Core Group), RIKEN Genomic Sciences Center (GSC), RIKEN Yokohama Institute, Yokohama, Japan, and Genome Science Laboratory, Discovery Research Institute, RIKEN Wako Institute, Wako, Japan

^* To whom correspondence should be addressed. E-mail: koujirou{at}med.gunma-u.ac.jp.

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 of 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.