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 the 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 a 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 the blood of individuals residing in Zimapan, Lagunera, and Chihuahua regions in Mexico. We found that the Zimapan and Lagunera populations have similar GST-T1 and GST-M1 genotype frequencies which differ from those of the Chihuahua population. In addition, 14 individuals have been identified as carriers of the 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, and time and money saving procedure for rapid identification of the GST-T1 and GST-M1 positive or null genotypes.

Fig. 1. GST-M1 and GST-T1 genotypes in primary human hepatocytes

Results of the conventional singleplex PCR analysis of GST-M1 (A) and GST-T1 (B) for seven donors of primary human hepatocytes: 1 – Hu228; 2 – Hu229; 3 - Hu-230; 4 - Hu0503; 5 – Hu0507; 6 – Hu0531; 7-Hu0534; 8 – Negative Control (no DNA template).

Fig. 2. The conventional singleplex and multiplex PCR

GST-M1 and GST-T1 genotype in Hu0503 (A), Hu230 (B), and Hu0534 (C) primary human hepatocytes are shown as examples. The conventional PCR was performed using the following sets of primers with sequences described in Material and Methods and summarized in Table 2: 1 – β-globin; 2 – GST-M1; 3 – GST-T1; 4 - β-globin + GST-M1 ; 5 – β-globin + GST-T1; 6 – β-globin + GST-M1 + GST-T1; 7 - Negative Control (no DNA template).

Fig. 3. The singleplex and multiplex High Resolution Melting analysis

Melting peak profiles and melting temperatures (T_m) as determined by “T_m calling” analysis of melting curve for seven donors of primary human hepatocytes. Left side; Singleplex HRM-PCR: (A) – Melting peaks for GST-T1 with T_m = 79.8°C (N=5); (B) - melting peaks for GST-M1 with T_m = 82.8°C (N=3); (C) - melting peaks for β-globin with T_m = 87.3°C (N=7). Right side; Multiplex HRM-PCR: (D) – melting peak profiles for Hu0503 donor; (E) - melting peak profiles for Hu230 donor; (F) - melting peak profiles for Hu0534 donor.

Fig. 4. Representative melting peak profiles for the multiplex HRM-PCR analysis of GST-T1 and GST-M1 genotypes in DNA samples collected from the Mexican study subjects

(A) Results for 10 randomly selected samples representing GST-M1 and GST-T1 positive genotypes and internal control (β-globin); (B) Results for 6 randomly selected samples representing GST-M1 positive and GST-T1 null genotype plus internal control (β-globin); (C) Results for 10 randomly selected samples representing GST-T1 positive and GST-M1 null genotype and internal control (β-globin); (D) Results for 7 randomly selected samples representing GST-T1 and GST-M1 null genotype and internal control (β-globin).