Department of Pediatrics, Cancer Research Institute, Seoul National University College of Medicine, South Korea.
Mol Diagn Ther. 2011 Oct 1;15(5):285-92. doi: 10.1007/BF03256420.
Busulfan is a key compound in myeloablative chemotherapy before hematopoietic stem-cell transplantation in children. Genetic polymorphisms of glutathione S-transferase (GST), which is involved in the metabolism of busulfan, have been implicated in interindividual variability in busulfan pharmacokinetics. Development of a rapid and simplified method for polygenic analysis of GST may facilitate large pharmacogenetic studies and clinical application of individualized busulfan dose adjustment. We previously introduced an effective PCR method for analyzing multiple genes using a small amount of DNA, termed 'TotalPlex amplification'.
The aim of this study was to extend the application of the TotalPlex method to the specific GST gene families (A1, P1, M1, and T1) that are related to busulfan metabolism, and thereby facilitate pharmacogenetic analysis of GST polymorphisms.
Seven genetic polymorphisms (GSTA1 promoter -52G>A, -69C>T, -567T>G, and -631T>G; GSTP1 313A>G; GSTM1 deletion; and GSTT1 deletion) were analyzed by multiplex PCR and genotyping, and the genotyping results from TotalPlex were verified with those from uniplex PCR.
Using five pairs of specific bulging-specific primers, seven specific gene fragments were successfully amplified by multiplex amplification coupled to a multiplexed bead array detection system, with a smaller amount of DNA and a shorter process time than is needed for the conventional approach. The genotypes of seven loci from 30 different genomic DNA samples derived using the multiplex system were consistent with the results of standard genotyping methods.
Our multiplex system provides a fast, inexpensive, and accurate method of detecting multiple GST polymorphisms (GSTA1, GSTP1, GSTM1, and GSTT1).
在儿童造血干细胞移植前的骨髓清除性化疗中，白消安是一种关键化合物。谷胱甘肽 S-转移酶（GST）的遗传多态性与白消安的代谢有关，它与白消安药代动力学的个体间变异性有关。开发一种快速简便的 GST 多基因分析方法可能有助于大型遗传药理学研究和个体化白消安剂量调整的临床应用。我们之前介绍了一种使用少量 DNA 分析多个基因的有效 PCR 方法，称为“TotalPlex 扩增”。
本研究的目的是将 TotalPlex 方法扩展应用于与白消安代谢相关的特定 GST 基因家族（A1、P1、M1 和 T1），从而促进 GST 多态性的遗传药理学分析。
通过多重 PCR 和基因分型分析了 7 种遗传多态性（GSTA1 启动子-52G>A、-69C>T、-567T>G 和-631T>G；GSTP1 313A>G；GSTM1 缺失；和 GSTT1 缺失），并通过验证 TotalPlex 的基因分型结果与单重 PCR 的结果。
使用 5 对特定的凸起特异性引物，通过多重扩增与多重珠阵列检测系统相结合，成功地扩增了 7 个特定的基因片段，所需的 DNA 量和处理时间比传统方法都要少。使用多重系统从 30 个不同基因组 DNA 样本中扩增的 7 个基因座的基因型与标准基因分型方法的结果一致。
我们的多重系统提供了一种快速、经济、准确的方法来检测多个 GST 多态性（GSTA1、GSTP1、GSTM1 和 GSTT1）。
