Aomori Tohru, Yamamoto Koujirou, Oguchi-Katayama Atsuko, Kawai Yuki, Ishidao Takefumi, Mitani Yasumasa, Kogo Yasushi, Lezhava Alexander, Fujita Yukiyoshi, Obayashi Kyoko, Nakamura Katsunori, Kohnke Hugo, Wadelius Mia, Ekström Lena, Skogastierna Cristine, Rane Anders, Kurabayashi Masahiko, Murakami Masami, Cizdziel Paul E, Hayashizaki Yoshihide, Horiuchi Ryuya
Department of Clinical Pharmacology, Gunma University Graduate School of Medicine, Maebashi, Japan.
Clin Chem. 2009 Apr;55(4):804-12. doi: 10.1373/clinchem.2008.115295. Epub 2009 Jan 30.
Polymorphisms of the CYP2C9 (cytochrome P450, family 2, subfamily C, polypeptide 9) gene (CYP2C92, CYP2C93) 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.
We developed a single-nucleotide polymorphism (SNP)-detection assay based on the SMart Amplification Process version 2 (SMAP 2) to analyze CYP2C92, CYP2C93, 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.
We analyzed 125 blood samples by both SMAP 2 and PCR-RFLP methods. The results showed perfect concordance.
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.
细胞色素P450 2C9（CYP2C9）基因（CYP2C92、CYP2C93）和维生素K环氧化物还原酶复合体亚基1（VKORC1）基因（-1639G>A）的多态性极大地影响了华法林的维持剂量。在开药前对患者进行基因型预筛查有助于更快地个体化确定合适的维持剂量，将不良反应风险和血栓栓塞事件复发风险降至最低。采用当前方法，如果通过基因分型来确定负荷剂量，治疗可能会延迟数小时至1天。因此需要一种更简单、更快速的基因分型方法。
我们开发了一种基于第二代智能扩增技术（SMAP 2）的单核苷酸多态性（SNP）检测方法，以分析CYP2C92、CYP2C93和VKORC1 -1639G>A多态性。来自同意参与的受试者的血液直接用于封闭管实时检测，无需进行DNA纯化，在采血后1小时内即可获得结果。
我们采用SMAP 2和聚合酶链反应-限制性片段长度多态性（PCR-RFLP）方法对125份血样进行了分析。结果显示完全一致。
这些结果验证了SMAP 2在确定对华法林个体化治疗至关重要的SNP方面的准确性。SMAP 2具有速度快、样品制备简单、等温扩增方便以及检测设计灵活等优点，与传统基因分型技术相比具有显著优势。在这个例子以及其他需要进行基因检测以立即做出更明智治疗决策的临床场景中，基于SMAP 2的诊断方法具有关键优势。
