Comparison of cytochrome P4502C9 genotyping methods and implications for the clinical laboratory
Comparison of cytochrome P4502C9 genotyping methods and implications for the clinical laboratory
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DOI：
10.1592/phco.24.8.720.36074
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发表时间：
2004-06-01
期刊：
影响因子：
4.1
通讯作者：
Johnson, JA
中科院分区：
文献类型：
--
作者：
Aquilante, CL;Lobmeyer, MT;Johnson, JA
研究目的。比较细胞色素P450（CYP）2C 9代谢酶基因中已知变异体基因分型的两种方法的准确性、速度和成本。比较研究。大学研究中心。样品。15毫升漱口水样本采集自253名参与华法林药物基因组学研究的受试者。采用焦磷酸测序和聚合酶链反应（PCR）-限制性片段长度多态性（RFLP）方法对所有253例样本进行了359位密码子异亮氨酸-亮氨酸突变（Ile（359）Leu [*3]多态性）的基因分型。通过焦磷酸测序法对所有样本进行144位密码子的精氨酸-半胱氨酸突变（Arg（144）Cys [*2]多态性）的基因分型，并通过PCR-RFLP法对136个样本的随机子集进行基因分型。在每个变异位点进行焦磷酸测序和PCR-RFLP的基因分型成功率、时间效率和成本分析的比较。焦磷酸测序和PCR-RFLP在Arg（144）Cys变体（分别为93.3%和90.4%）和Ile（359）Leu变体（分别为83.8%和79.1%）的首次基因分型尝试中产生了相似的成功率。使用焦磷酸测序，可以在1小时内对96个样品进行任一多态性的基因分型。相比之下，通过RFLP对96个样品进行基因分型，Arg（144）Cys变体需要10小时，Ile（359）Leu变体需要20小时。Arg（144）Cys基因分型的总成本/样本为1.90美元，PCR-RFLP为3.14美元，Ile（359）Leu基因分型的总成本/样本为1.88美元，PCR-RFLP为10.18美元。与RFLP相比，焦磷酸测序的基因型测定是一种更省时、更经济、更可靠的CYP 2C 9基因分型方法。由于其广泛的适用性和易用性，焦磷酸测序是未来药物基因组学研究的一项有前途的技术。
Study Objective. To compare the accuracy, speed, and cost of two methodologies used for genotyping known variants in the cytochrome P450 (CYP) 2C9 metabolizing enzyme gene.Design. Comparative study.Setting. University research center.Samples. Fifteen-milliliter mouthwash samples collected from 253 subjects participating in a warfarin pharmacogenomic study.Intervention. Genotyping for the isoleucine-to-leucine change at codon 359 (Ile(359)Leu [*3] polymorphism) was performed by using the Pyrosequencing and polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) methods in all 253 samples. Genotyping for the arginine-to-cysteine change at codon 144 (Arg(144)Cys [*2] polymorphism) was performed by using Pyrosequencing in all samples and by PCR-RFLP in a random subset of 136 samples.Measurements and Main Results. Comparisons of genotyping success rates, time efficiency, and cost analyses were conducted for Pyrosequencing and PCR-RFLP at each variant site. Pyrosequencing and PCR-RFLP produced similar success rates on the first genotyping attempt for the Arg(144)Cys variant (93.3% vs 90.4%, respectively) and the Ile(359)Leu variant (83.8% vs 79.1%, respectively). With Pyrosequencing, genotyping 96 samples for either polymorphism could be performed in I hour. In contrast, genotyping 96 samples by RFLP took 10 hours for the Arg(144)Cys variant and 20 hours for the Ile(359)Leu variant. Total cost/sample for Arg(144)Cys genotyping was $1.90 with PCR-Pyrosequencing and $3.14 with PCR-RFLR Total cost/sample for Ile(359)Leu genotyping was $1.88 with PCR-Pyrosequencing and $10.18 with PCR-RFLPConclusion. Compared with RFLP, genotype determination by Pyrosequencing is a more time-efficient, cost-effective, and robust method for CYP2C9 genotyping. Because of its wide applicability and ease of use, Pyrosequencing is a promising technology for future pharmacogenomic investigations.