Rai Alex J, Udar Nitin, Saad Rana, Fleisher Martin
Departments of Clinical Laboratories and Pathology, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA.
Clin Chem. 2009 Apr;55(4):823-6. doi: 10.1373/clinchem.2008.118497. Epub 2009 Feb 20.
Patients differ in responses to warfarin, which is commonly prescribed to treat thromboembolic events. Genetic variations in the cytochrome P450, family 2, subfamily C, polypeptide 9 (CYP2C9), vitamin K epoxide reductase complex, subunit 1 (VKORC1), and gamma-glutamyl carboxylase (GGCX) genes have been shown to contribute to impaired metabolism of warfarin.
We designed a custom multiplex single-nucleotide polymorphism (SNP) panel to interrogate the CYP2C9 *2, *3, VKORC1 (-1639G-->A), and GGCX (1181T-->G) alleles simultaneously in a single sample by use of single-base extension and capillary electrophoresis after genomic DNA extraction and PCR amplification.
Our assay successfully detected various genotypes from known controls and 24 unknown samples. It was found to be 100% concordant with sequencing results.
Our multiplexed SNP panel can be successfully used in genotyping of patient blood samples. Results can be combined with other clinical parameters in an algorithm for warfarin dosing. These data provide a proof-in-principle of multiplexed SNP analysis using rapid single-base extension and capillary electrophoresis, and warrant additional validation using a larger cohort of patient samples.
华法林常用于治疗血栓栓塞事件，但不同患者对其反应各异。细胞色素P450 2C9（CYP2C9）、维生素K环氧化物还原酶复合体亚单位1（VKORC1）和γ-谷氨酰羧化酶（GGCX）基因的遗传变异已被证实与华法林代谢受损有关。
我们设计了一个定制的多重单核苷酸多态性（SNP）检测板，通过基因组DNA提取和PCR扩增后，利用单碱基延伸和毛细管电泳技术，在单个样本中同时检测CYP2C9 *2、*3、VKORC1（-1639G→A）和GGCX（1181T→G）等位基因。
我们的检测方法成功地从已知对照和24个未知样本中检测出了各种基因型。结果显示与测序结果100%一致。
我们的多重SNP检测板可成功用于患者血样的基因分型。检测结果可与其他临床参数相结合，用于华法林剂量算法。这些数据为使用快速单碱基延伸和毛细管电泳进行多重SNP分析提供了原理证明，并需要使用更大规模的患者样本队列进行进一步验证。
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