Identification of the GST-T1 and GST-M1 Null Genotypes Using High Resolution Melting Analysis

Abstract

Glutathione S-transferases, including GST-T1 and GST-M1, are known to be involved in the phase II detoxification pathways for xenobiotics as well as in the metabolism of endogenous compounds. Polymorphisms in these genes have been linked to an increased susceptibility to carcinogenesis and associated with risk factors that predispose to certain inflammatory diseases. In addition, GST-T1 and GST-M1 null genotypes have been shown to be responsible for interindividual variations in metabolism of arsenic, a known human carcinogen. To assess the specific GST genotypes in the Mexican population chronically exposed to arsenic, we have developed a multiplex High Resolution Melting PCR (HRM-PCR) analysis using LightCycler480 instrument. This method is based on analysis of the PCR product melting curve that discriminates PCR products according to their lengths and base sequences. Three pairs of primers that specifically recognize GST-T1, GST-M1, and β-globin, an internal control, to produce amplicons of different length were designed and combined with LightCycler480 High Resolution Melting Master Mix containing ResoLight, a completely saturating DNA dye. Data collected from melting curve analysis were evaluated using LightCycler480 software to determine specific melting temperatures of individual melting curves representing target genes. Using this newly developed multiplex HRM-PCR analysis we evaluated GST-T1 and GST-M1 genotypes in 504 DNA samples isolated from blood of individuals residing in Zimapan, Lagunera, and Chihuahua regions in Mexico. We found that Zimapan and Lagunera populations have similar GST-T1 and GST-M1 genotype frequencies which differ from Chihuahua population. In addition, 14 individuals have been identified as carriers of double null genotype, i.e. null genotypes in both GST-T1 and GST-M1 genes. Although this procedure does not distinguish between biallelic (+/+) and monoallelic (+/−) genotypes it can be used in an automated workflow as a simple, sensitive, time and money saving procedure for rapid identification of the GST-T1 and GST-M1 positive or null genotypes.

Additive and Interactive Associations of Environmental and Sociodemographic Factors with the Genotypes of Three Glutathione S-Transferase Genes in Relation to the Blood Arsenic Concentrations of Children in Jamaica

“…Human GST genes, including glutathione-S-transferase pi 1 ( GSTP1 ), glutathione-S-transferase mu 1 ( GSTM1 ), and glutathione-S-transferase theta 1 ( GSTT1 ), play a crucial role in As metabolism [ 30 ]. These GST genes encode a family of phase II enzymes called glutathione transferases that facilitate the excretion of xenobiotics (e.g., As) by catalyzing their conjugation with the reduced form of glutathione (GSH) [ 31 , 32 ].…”

“…GST genes are highly polymorphic [ 36 ]; for example, null alleles of GST genes such as GSTT1 and GSTM1 can lead to a lack of functional enzymes and result in a decrease in detoxification capacity and cell protection from oxidative stress [ 37 , 38 ]. The null genotypes for GSTT1 and GSTM1 have been suggested as genetic factors responsible for inter-individual differences in As metabolism [ 30 ]. For example, previous studies have shown that the GSTM1 null genotype impedes As methylation, which results in increased As retention [ 12 ].…”

Review GST genes polymorphisms and the risk of colorectal cancer development

“…Proteins of the GST family (glutathione S-transferases) are enzymes detoxifying a wide range of hazardous substances, such as reactive oxygen species (ROS) or xenobionts [ 3 ]. These enzymes catalyse the reaction of the conjugation of chemical compounds, of both exogenous and endogenous origin, with glutathione [ 4 , 5 ]. Substances detoxified in this way include lipid peroxidation products, prostaglandins, various types of chemotherapeutics, and the majority of substances belonging to environmental carcinogens, such as heterocyclic aromatic amines [ 5 ].…”

“…These enzymes catalyse the reaction of the conjugation of chemical compounds, of both exogenous and endogenous origin, with glutathione [ 4 , 5 ]. Substances detoxified in this way include lipid peroxidation products, prostaglandins, various types of chemotherapeutics, and the majority of substances belonging to environmental carcinogens, such as heterocyclic aromatic amines [ 5 ]. The conjugation of electrophilic reagents with reduced glutathione, catalysed by GST enzymes, is aimed at limiting the possibilities of hazardous effects of these strongly reactive compounds on vitally important cell components, such as proteins or nucleic acids.…”