Development of multiplex PCR method for the analysis of glutathione s-transferase polymorphism

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).