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Association of glutathione S-transferase (GSTM1, T1 and P1) gene polymorphisms with type 2 diabetes mellitus in north Indian population HK Bid1, R Konwar1, M Saxena2, P Chaudhari1, CG Agrawal3, M Banerjee21 Endocrinology Division, CDRI, Lucknow, India 2 Molecular & Human Genetics Laboratory, Department of Zoology, University of Lucknow, Lucknow, India 3 Department of Medicine, CSMMU, Lucknow, India
Correspondence Address: DOI: 10.4103/0022-3859.68633 PMID: 20739761
Background: Diabetes mellitus is associated with an increased production of reactive oxygen species (ROS) and a reduction in antioxidant defense. The oxidative stress becomes evident as a result of accumulation of ROS in conditions of inflammation and Type 2 diabetes mellitus (T2DM). The genes involved in redox balance, which determines the susceptibility to T2DM remain unclear. In humans, the glutathione S-transferase (GST) family comprises several classes of GST isozymes, the polymorphic variants of GSTM1, T1 and P1 genes result in decreased or loss of enzyme activity. Aims: The present study evaluated the effect of genetic polymorphisms of the GST gene family on the risk of developing T2DM in the North Indian population. Settings and Design: GSTM1, T1 and P1 polymorphisms were genotyped in 100 T2DM patients and 200 healthy controls from North India to analyze their association with T2DM susceptibility. Materials and Methods: Analysis of GSTM1 and GSTT1 gene polymorphisms was performed by multiplex polymerase chain reaction (PCR) and GSTP1 by PCR-Restriction Fragment Length Polymorphism (RFLP). Statistical Analysis: Fisher's exact test and χ2 statistics using SPSS software (Version-15.0). Results: We observed significant association of GSTM1 null (P=0.004, OR= 2.042, 95%CI= 1.254-3.325) and GSTP1 (I/V) (P=0.001, OR= 0.397, 95%CI=0.225-0.701) with T2DM and no significant association with GSTT1 (P=0.493). The combined analysis of the three genotypes GSTM1 null, T1 present and P1 (I/I) demonstrated an increase in T2DM risk (P= 0.005, OR= 2.431 95% CI=1.315-4.496). Conclusions: This is the first study showing the association of a combined effect of GSTM1, T1 and P1 genotypes in a representative cohort of Indian patients with T2DM. Since significant association was seen in GSTM1 null and GSTP1 (I/V) and multiple association in GSTM1 null, T1 present and P1 (I/I), these polymorphisms can be screened in the population to determine the diabetic risk. Keywords: Genotypes, glutathione S-transferase, oxidative stress, PCR-RFLP, risk factors
The prevalence of Type 2 diabetes mellitus (T2DM) is increasing rapidly in India, evidence for which has come from recent population-based studies. [1],[2] According to a nationwide survey, nearly 12% of urban adults have T2DM. [3] It is estimated that the number of people with diabetes worldwide exceeds 200 million, most of them being patients with T2DM. [4],[5] T2DM is a complex disorder with polygenic inheritance, and multiple genes located on different loci contributing to its susceptibility. Analysis of genetic factors is further complicated by the fact that numerous environmental factors interact with the genes to produce T2DM. As Asian Indians have an greater susceptibility to diabetes and have increased insulin resistance, they are a unique population for carrying out genetic studies. [6] Oxygen free radicals and lipid peroxides have been implicated in the pathogenesis of a large number of diseases such as diabetes, cancer, rheumatoid arthritis, infectious diseases, atherosclerosis and aging. [7],[8],[9] Growing evidence indicates that oxidative stress is increased in diabetes due to overproduction of reactive oxygen species (ROS) and decreased efficiency of antioxidant defenses. [10] It has also been reported that defects in antioxidant defense against oxidative stress play an important role in the etiology of diabetic complications. [11],[12],[13] This initiated us to explore the antioxidant enzyme gene polymorphisms and their association with T2DM. The proposed work will add to the existing knowledge of understanding the genetic basis of T2DM in the North Indian population. The genotyping might help in early diagnosis and help in the management of the disease. Glutathione (GSH) also participates in the cellular defense system against oxidative stress by scavenging free radicals and reactive oxygen intermediates. Thus, a decrease in GSH level in diabetic patients increases the sensitivity of cells to oxidative stresses. [14] Kim et al.,[15] have recently reported that insulin and growth factors regulate drug-metabolizing enzyme gene and protein expression, including cytochromes P450 (CYP), glutathione S-transferases (GST) and microsomal epoxide hydrolase (mEH). A study from Japan has demonstrated that the GSTT1- and GSTT1-/GSTM1- genotypes are independent risk factors for development of T2DM. [16] To the best of our knowledge this is the first report on the association of combined polymorphisms of the GSTM1, T1 and P1 genes in patients with T2DM from a North Indian population. In the present study, an effort has been made to identify the clinical features associated with T2DM and determine the genotypic frequency of the GSTM1 null, GSTT1 null and GSTP1-313 A/G polymorphisms in order to understand their associations with the risk of developing T2DM in North India.
Patients with T2DM were enrolled from the outpatients Diabetes Clinic of Chhatrapati Shahuji Maharaj Medical University (CSMMU), Lucknow, India from March to October 2007. Out of 183 subjects screened, 100 patients (age 22-77 years) were included in this study. Most of the patients were from the eastern part of Uttar Pradesh in North India. Subjects were categorized in the diabetic group if they had a fasting glucose concentration ≥126 mg/dl or glucose concentration ≥200 mg/dl 2 h after a 75-g oral glucose tolerance test. All clinical details of individuals were recorded as per a self-administered questionnaire which included clinical history of diabetes, family history and associated complications such as hypertension. [17] All patients were on oral hypoglycemic agents. Medical records of these patients were reviewed to ascertain diabetes-associated complications. Two hundred age/sex-matched normal healthy controls were screened from healthy staff members of the Institute and University with normal oral glucose tolerance test. The normal healthy controls were also matched with waist hip ratio (WHR), body mass index (BMI), lipid levels, blood pressure, economic status and ethnicity [Table 1]. Those having a history of coronary artery disease or other metabolic disorders (including obesity, nephropathy, neuropathy and other complications) were excluded from both the groups. The study was approved by the Institutional Ethics Committee (No. 3742/R.Cell-08 dated 24 th Oct, 2008).
Height and weight of patients were measured and BMI was defined as weight (in kilograms) divided by square of height (in meters). Abdominal obesity was measured by waist-hip ratio, >0.9 in men and >0.8 in women. Fasting and post-prandial glucose, total cholesterol, high-density lipoprotein (HDL), triglyceride, low-density lipoprotein (LDL), very low-density lipoprotein (VLDL), blood pressure (systolic/diastolic) were also determined [Table 1]. After a written, informed consent, a 5-ml blood sample was taken in Ethyl Diamine Tetra Acetic acid (EDTA) vials from both the groups. Genomic DNA was extracted from peripheral blood leucocytes using the standard salting out method. [18] Amplification was performed in a 20-΅l reaction mixture containing genomic DNA (100-150 ng), 2-20 pmol of each primer set, 200 mM dNTPs, and 0·5U of Taq DNA polymerase (MBI-Fermentas, USA) per tube using a programmed thermal cycler (Master cycler gradient ep, Eppendorf, USA). Analysis of GSTM1 and GSTT1 gene polymorphisms was performed by multiplex polymerase chain reaction (PCR), as described by Abdel-Rahman et al. [19] GSTM1: Forward 5′-GAACTCCCTGAAAAGCTAAAGC-3' Reverse 5′-GTTGGGCTCAAATATACGGTGG-3' GSTT1: Forward 5'-TTCCTTACTGGTCCTCACATCTC-3' Reverse 5'-TCACGGGATCATGGCCAGCA-3' Exon-7 of CYP1A1 gene was co-amplified and used as an internal control using the following primers: Forward 5'-GAACTGCCACTTCAGCTGTCT-3' Reverse 5'-CAGCTGCATTTGGAAGTGCTC-3' Each set of reactions included both positive and negative controls. The multiplex PCR method was used to detect the presence or absence of GSTM1 and GSTT1 genes simultaneously in the same tube. The reaction mixture was subjected to initial denaturation at 94°C for 2 min, followed by 35 cycles at 94°C for 2 min, 59°C for 1 min and 72°C for 1 min. The final extension was carried out at 72°C for 10 min. The PCR products were electrophoresed in a 2% agarose gel and visualized by ethidium bromide (Et-Br) staining. DNA from samples positive for GSTM1 and GSTT1 genotypes yielded bands of 215 bp and 480 bp, respectively, while the internal positive control (CYP1A1) PCR product corresponded to 312 bp. GSTP1: The A313G polymorphism of GSTP1 was analyzed using a previously described PCR-Restriction Fragment Length Polymorphism (RFLP) method. [20] Briefly, amplification was carried out using the following primers: Forward 5'-ACCCCAGGGCTCTATGGGAA-3' Reverse 5'-TGAGGGCACAAGAAGCCCCT-3' The 176-bp amplified product was digested with Alw261 and electrophoresed in a 3% agarose gel. The presence of a restriction site resulted in two fragments (91 and 85 bp) indicating G allele and three fragments (176, 91 and 85 bp) for A/G polymorphism. Allele and genotype frequencies in both groups were compared using a 2x2 contingency table by Fisher's exact test. The Hardy-Weinberg equilibrium at individual loci was assessed by χ2 statistics using SPSS software (Version 15.0). All P values were two-sided and differences were considered statistically significant for P<0.05. Odds ratio (OR) at 95% confidence intervals (CI) was determined to describe the strength of association by Logistic Regression Model. Logistic regression was used to analyze sex, age, systolic blood pressure (SBP), diastolic blood pressure (DBP), body mass index (BMI), waist circumference (WC), waist:hip ratio (WHR), triglycerides (TG), total cholesterol (CH), high-density lipoprotein (HDL) and low-density lipoprotein (LDL).
Comparison of clinical characteristics of T2DM patients (n=100) and controls (n=200) are summarized in [Table 1]. Three biochemical parameters viz. BMI, fasting glucose and VLDL-cholesterol in T2DM patients showed a significant association when compared to controls. [Table 2] gives the frequency distribution of GSTM1, T1 and P1 alleles and genotypes along with their double and triple combinations in both cases and controls. Genotype distributions in controls were in agreement with the Hardy-Weinberg equilibrium. In the control samples, the frequency of GSTM1 null and GSTT1 null was 36.5 and 14.0% respectively while in T2DM patients, the frequency of GSTM1 null and GSTT1 null was 54.0 and 17.0% respectively. The GSTP1 Ile allele in controls was present as homozygous (I/I) in 59.5%, homozygous Val allele (V/V) in 3.0% and heterozygous (I/V) condition in the remaining 37.5%. In case of patients, GSTP1 Ile allele was present as I/I in 80%, Val allele (V/V) in 0% and the remaining 20% were heterozygous (I/V). We observed a significant association of null genotype of GSTM1(P=0.004) and heterozygous genotype (A/G, I/V) (P=0.001) of GSTP1 gene with the risk of developing T2DM [Table 2]. The combination of the two high-risk genotypes either GSTM1 null/GSTT1 null, GSTM1 null and GSTP1 (I/I) showed that the risk of developing T2DM increased up to 2.0 times (P=0.016 and 0.021 respectively). However, in individuals with GSTP1 (I/V) genotype in combination with either GSTM1 (+/+) or GSTT1 (+/+), the risk was only 0.3 times (P=0.007, 0.001) [Table 2].
We further investigated the risk associated with all the three high-risk GST genotypes compared to non-risk genotypes (positive genotypes of GSTM1 and GSTT1 and 313 A/A genotype (I/I) of GSTP1 were designated as the reference group). The risk for M1 (-/-), T1 (+/+) and P1 (I/I) genotypes was 2.431 folds higher (P = 0.005, OR=2.431 95% CI = 1.315-4.496). We also compared the GSTM1, GSTT1 and GSTP1 genotypes with different clinical parameters but did not find any significant association (P>0.05) [Table 3].
India is facing a major healthcare burden due to the high prevalence of T2DM and its related complications. [21] The epidemic of T2DM observed in recent years is a clear indication of the importance of environmental factors in diabetes onset; in particular, obesity and physical inactivity. Several genes and their variants involved in various biochemical pathways are shown to be associated with Type 2 diabetes. [22],[23],[24],[25] One of the adverse forces in T2DM is increased production of reactive oxygen species and reduction in antioxidant defense leading to oxidative stress. [26],[27] Glutathione S-transferases (GSTs) belong to a group of multigene and multifunctional detoxification enzymes, which defend cells from oxidative stress usually observed in diabetes. In humans, the various subfamilies of GSTs are coded by genes which exhibit polymorphisms resulting in individual variations in clearance of toxic intermediates and susceptibility to damage from oxidative stress. [28] In the present study we investigated the three major GSTs i.e. M1, T1 and P1 gene polymorphisms in T2DM patients and healthy controls. We also attempted to evaluate the association of these polymorphic genes with different clinical parameters in T2DM patients. The results of our present investigation showed a significant association of the frequency of GSTM1 genotype in T2DM patients, similar to a study in Turkish diabetes patients. [29] Type 2 diabetic patients in the North Indian population had higher prevalence of GSTM1 null genotype than controls (54 vs. 36.5%). The presence of GSTM1 null genotype seems to increase the risk of having T2DM almost twofold, that is, the individuals are not capable of detoxifying the products of oxidative stress. However, a study has reported a protective association against alcohol-induced pancreatitis with the GSTM1 null genotype. [30] The GSTT1 genotype did not show any significant association with the disease in our study population. The frequencies of GSTT1-positive (83.0% and 86.0%) and -negative (17.0% and 14%) individuals were similar in T2DM patients and controls respectively. On the contrary, studies in other populations showed an association of GSTT1 null genotype and not GSTM1 genotype with the risk of developing T2DM. [28],[31],[32] GSTT1- and GSTT1-/GSTM1- genotypes have been convincingly proved to be independent risk factors for development of T2DM as reported by Hori et al., [16] The genetic absence of GSTT1 enzyme was also proved as an independent and powerful predictor of serious vascular complication in T2DM individuals. [33] Our study is probably the first to show that the GSTP1 heterozygous (I/V) genotype is significantly (P=0.001) associated with T2DM. There are reports on GSTP1 polymorphism in relation to other diseases such as skin lesions, docetaxel-induced peripheral neuropathy, higher risk of DNA damage in pesticide-exposed fruit growers, [34],[35],[36] and different cancers. [37],[38],[39] Extensive haplotype analyses of GSTM1, T1 and P1 genes showed some interesting results in our study population. The combination of the two high-risk genotypes either GSTM1 null/T1 null or GSTM1 null and P1 (I/I) showed that the risk of developing T2DM increased up to 2.0 times. The inherited homozygous deletions of GSTT1 or GSTM1 genes (null genotype) cause a complete absence of enzymatic activity. The GSTM1 class catalyses the detoxification of genotoxins, GSTT1 utilizes oxidized lipids and oxidized DNA while GSTP1 catalyses the detoxification of products that arise from DNA oxidation [19] and also utilizes smoke-derived carcinogens. [40] Two genetic polymorphisms have been identified in GSTP1 gene that is associated with reduced enzymatic activity and altered enzyme kinetics. In one the P1 variant allele contains an A313G change, leading to an Ile to Val substitution in amino acid 105 within the active site of the enzyme. The other P1 variant allele features an Ala to Val change at amino acid 114. [39],[40] The GSTP1 (I/I) allele in combination with GSTM1 null and T1 present increases the risk to more than 2.0 times (OR 2.431; 95% CI 1.315-4.496; P=0.005). The frequency of patients with this combination of genotype was higher compared to controls (39.0% vs. 17.5%). The wild genotype (I/I) is present in 80% diabetic cases in comparison to 59.5% controls. Although the GSTP1 (I/I) genotype corresponds to normal enzyme activity, it seems to have a major contribution in the development of T2DM in combination with other classes of GST i.e. GSTM1 null and presence of GSTT1. The presence of Val allele (I/V) affects the active site of GSTP1 resulting in reduced enzymatic activity, this is evident from our results as well, GSTP1 (I/V) along with M1 +/+ and T1 +/+ showed a significant association with the disease (P=0.007; 0.001). Our population showed a complete absence of the homozygous (V/V) genotype in T2DM patients which was similar to results seen in the Japanese population. [41] In the present study we also compared several biochemical parameters including age, duration of diabetes, BMI, WHR, blood glucose levels, lipid profile and blood pressure in diabetic subjects for GSTM1 (present, null), T1 (present, null) and P1 genotypes. It was observed that the diabetic patients with GSTM1 genotype alone showed significant association with total cholesterol and LDL-cholesterol, no other genotypes showed any significant change. The variation in the North Indian population from other world populations signifies the impact of ethnicity. It can be assumed that GSTM1 gene and GSTP1 (I/V) may be a useful marker for prediction of T2DM susceptibility. Moreover, the combination of two or three GST genotypes further increases the risk of T2DM in the Indian population. In our results we found that a combination of GSTM1 null (-/-), T1 (+/+) and P1 (I/I) showed 2.431 times increased risk of developing T2DM. However, the significance of GSTP1 (I/I) genotype in the susceptibility of the disease remains unexplained although it clearly increases T2DM risk in combination. Since very few studies have been reported to show the association of GST gene family variants with T2DM, we conducted this study in the north Indian population to observe the association of GSTM1, T1 and P1 polymorphisms with T2DM. This approach has the potential for identifying susceptible individuals. We strongly believe that further investigations in large-scale cohort studies in different populations may confirm the role of GSTM1, T1 and P1 gene polymorphisms in the pathogenesis of T2DM and its associated complications.
We gratefully thank the staff of the Diabetic Clinic at the Department of Medicine, CSMMU, Lucknow, India for their clinical support. We also thank the Department of Biotechnology, New Delhi, India for providing research funding.
[Table 1], [Table 2], [Table 3]
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