Progonostic effect of GSTM1/GSTT1 polymorphism in determining cardiovascular diseases risk among type 2 diabetes patients in South Indian population

Cardiovascular disease (CVD) is a significant complication of type 2 diabetes mellitus (T2DM), with oxidative stress playing a significant role. Glutathione S-transferase (GST) polymorphisms - GSTM1, GSTT1 - have been linked to CVD and T2DM. The role of GSTM1 and GSTT1 in CVD development among T2DM patients in the South Indian population is investigated in this study. The volunteers were grouped as Group 1: control, Group 2: T2DM, Group 3: CVD, and Group 4: T2DM with CVD (n = 100 each). Blood glucose, lipid profile, plasma GST, MDA, and total antioxidants were measured. GSTM1 and GSTT1 were genotyped using PCR. GSTT1 plays a significant role in the development of T2DM and CVD [OR 2.96(1.64–5.33), < 0.001 and 3.05(1.67–5.58), < 0.001] while GSTM1 null genotype was not associated with disease development. Individuals with dual null GSTM1/GSTT1 genotype had the highest risk of developing CVD [3.70(1.50–9.11), 0.004]. Group 2 and 3 individuals showed higher lipid peroxidation and lower total antioxidant levels. Pathway analysis further indicated that GSTT1 significantly affects GST plasma levels. GSTT1 null genotype may be considered a contributing factor that increases the susceptibility and risk of CVD and T2DM in the South Indian population.


Introduction
Type 2 diabetes mellitus (T2DM) is a significant public health problem worldwide, with a higher prevalence in low and middle-income countries. T2DM has been linked to an increased risk of macrovascular complications such as Cardiovascular Disease (CVD) [1]. CVD, including coronary heart disease (CHD) and myocardial infarction (MI), accounts for one-third to one-half of all diabetes-related deaths, and people with diabetes are two to three times more likely to develop CVD [1,2]. CVD progression in T2DM patients is multifactorial, involving a variety of genetic and environmental factors [3]. Hyperglycemia has been linked to macrovascular complications by inducing polyol metabolism, the formation of Advanced Glycation End Products (AGEs), and the activation of Protein Kinase C (PKC), which increases the production of Reactive Oxygen Species (ROS) and thus oxidative stress. Oxidative stress plays a vital role in developing macrovascular complications such as CVD in T2DM patients [4]. Genetic variations in antioxidant enzyme genes have been linked to T2DM or CVD susceptibility in various populations [3], and thus genetic polymorphisms reducing antioxidant enzyme activity may alter CVD susceptibility in T2DM.
GSTs are essential phase II antioxidant genes that participate in ROS detoxification via glutathione conjugation and maintain redox homeostasis, implying a role in oxidative stress-related diseases [5]. GST genes are highly polymorphic, with GSTM1 and GSTT1 being the most extensively discussed. GSTM1 (GST) is found on chromosome 1p13.3, while GSTT1 is found on chromosome 22. The null polymorphism occurs in both GSTM1 and GSTT1 due to the deletion of the entire gene, resulting in loss of GST function, and has been linked to the progression and initiation of several diseases [6,7]. Because of the decreased antioxidant capacity, the null genotype polymorphism causes oxidative stress, which leads to inflammation and other cellular dysfunctions in chronic disorders [6]. GSTM1 and GSTT1 null genotypes are associated with an increased risk of developing T2DM [8], CVD [9], and CVD in T2DM patients [10]. However, the relationship between GSTM1 and GSTT1 null genotypes with the development of CVD in T2DM patients in the South Indian population remains uncertain. The previous study did not include CVD as a case group or the impact of combined GST polymorphism on CVD development in T2DM patients as individuals with GSTM1 and GSTT1-null genotypes have a higher risk of CVD [9,10].
Hence, the current case-control study aims to investigate the impact of GSTM1 and GSTT1 genetic variants on the development of CVD in T2DM patients in the South Indian population. The impact of GST polymorphism on plasma GST and antioxidant levels is also being studied.

Study population
A total of 400 volunteers visiting Chettinad Hospital and Research Institute in Chennai, India, were randomly selected and grouped as : Group 1: controls, Group 2: T2DM patients without comorbidities, Group 3: CVD patients, and Group 4: T2DM patients with CVD (n = 100 in each). The Institutional Human Ethics Committee (IHEC No.: 611/ IHEC/ [11][12][13][14][15][16][17][18][19] approved this study and informed consent was obtained from all participants. T2DM patients were identified according to the American Diabetes Association guidelines, CVD patients were diagnosed by cardiologist based on clinical symptoms and ECG, and healthy individuals without any history of diabetes or cardiac problems were chosen as controls. Cancer patients, individuals with liver, kidney, and lung diseases, pregnant women, HIV-positive patients, and patients with allergies were all excluded from the study.

Data collection
During blood sample collection, each patient provided baseline demographic information such as age, gender, height, weight, family history of diabetes/CVD, duration of T2DM/ CVD, smoking status, and alcohol consumption. Additionally, clinical characteristics such as blood glucose levels, HbA1c, and lipid profiles were obtained. The Castelli risk index I (CRI-I = [TC /HDL-C]), and Castelli risk index II (CRI-II = [LDL/HDL-C]) were calculated using established formulas to assess the risk of cardiovascular disease.

Clinical characteristics
After an overnight fast, 5 ml of blood was drawn from each study participant via venepuncture. Plasma glutathione S-transferase (GST), total antioxidant status (TAS), and lipid peroxidation levels were measured.

DNA extraction and genotype determination
GSTM1 and GSTT1 were genotyped using polymerase chain reaction (PCR) with β-globulin as an internal control. Under the conditions described in Table 1, a PCR reaction mixture of 100 ng of genomic DNA, 10 pM each primer and master mix containing 1.5 mM MgCl 2 , 0.2 mM dNTPs, and 0.5 U Taq polymerase made up to 25 µL with molecular grade water was amplified. 2% agarose gel electrophoresis was used to examine the amplified PCR products. The presence of a band indicated that GSTM1/GSTT1 was present, whereas the absence indicated the null polymorphism. Random genotyping of 10% of samples was conducted to confirm the validity of the genotyping method.

Statistical analysis
Continuous variables in the study were expressed as mean ± standard deviation (SD), whereas non-continuous variables were expressed as frequency (n%). To assess the distribution of data, a normality test was performed using the Shapiro-Wilk test. The chi-square goodness of fit test was used to calculate the frequency of genetic variations in GSTM1 and GSTT1. Multinomial logistic regression analysis was used to calculate the association of genetic variations with the risk of developing CVD using odds ratios (OR) and 95% confidence intervals (CI). The Student's t-test was used to calculate mean differences between 2 groups, and the oneway ANOVA with Turkey HSD post hoc test or the Kruskal-Wallis test was used to determine mean differences between more than 2 groups. SmartPLS 3.3.7 was used to calculate the path analysis between GST plasma levels, GST polymorphism, and CVD risk in T2DM patients. SPSS software version 22.0 was used for all statistical analyses, and a p-value of less than 0.05 was considered statistically significant.

Baseline characteristics
The average age of the participants in the four groups was 40 ± 12, 49 ± 12, 46 ± 12 and 50 ± 11 respectively. The baseline characteristics of the study population are described in Table 2.
Group 1 had a higher proportion of females, while the other groups had a higher proportion of males. Male participants were significantly more likely to develop T2DM and T2DM/CVD, as evidenced by odds ratios of 2.102 (1.189-3.719); P-value of 0.010 and 2.680 (1.510-4.756); P-value < 0.001, respectively. Furthermore, males were discovered to be three times more likely to develop CVD than females, with an odds ratio of 3.036 (1.705-5.405) and a P-value < 0.001. Individuals with a family history of CVD are three times more likely to develop the disease than those without, as evidenced by an odds ratio

Clinical characteristics
The clinical characteristics and antioxidant parameters of the four groups are shown in Table 3. When compared to the control, group 2 had significantly higher fasting blood glucose (FBP), postprandial glucose (PP), HbA1c, and Castelli Risk II levels. The increased Castelli risk indicates higher cardiovascular risk among T2DM individuals. Individuals with T2DM/CVD had significantly higher FBP, PP, HbA1c and CK-MB than controls, whereas in group 3, a lowered HDL, higher Castelli risk II, VLDL, lipase, CK-MB and Troponin differed from controls. When compared to the control groups, CVD patients had disrupted lipid metabolism. Regression analysis revealed that higher PP glucose levels increased the risk of T2DM and CVD. Lipase levels were significantly higher in the disease groups than in the control groups. Oxidative stress plays a crucial role in the pathophysiology of CVD in T2DM patients. The disease groups had significantly higher malondialdehyde (MDA) and lower total antioxidant status (TAS) levels when compared to the control. Individuals with T2DM/CVD had the lowest antioxidant levels and the highest lipid peroxidation levels, implying that oxidative stress raises the risk of CVD in hyperglycemia. GST polymorphism Table 4 shows the allelic frequency of GSTM1 and GSTT1 in the study population. The GSTM1 null genotype was more common in groups 4 and 3 than in the control group, but the difference was not statistically significant As shown in Table 5, GSTM1 null genotype was not associated with the development of T2DM or CVD, but males with GSTM1 null genotype had a significantly increased risk of T2DM, T2DM/CVD, and CVD    Table 6 depicts the relationship between GSTM1 null genotype and antioxidant parameter. Compared to GSTM1 null patients, the presence of GSTM1 was associated with increased lipid peroxidation and lower antioxidant status in group 3 patients. Other clinical parameters, such as FBG, PP, and lipid profile, did not differ significantly between GSTM1 null and GSTM1 present genotypes Table 4 shows that the frequency of the GSTT1 null genotype was significantly higher in disease groups than in the control group and was associated with the development of CVD  Other clinical and antioxidant parameters did not differ significantly between GSTT1 null and GSTT1 present genotypes. The combined GSTM1 and GSTT1 analysis revealed that the absence of both genotypes increased CVD risk significantly, as shown in Table 5. GSTT1 presence and absence of GSTM1 genotype showed a protective effect, indicating that GSTT1 has a protective effect. Table 7 depicts the relationship between GST plasma levels and the risk of CVD in T2DM patients with GSTM1 and GSTT1 polymorphisms. The findings show that GSTT1 significantly correlates with GST plasma levels, whereas GSTM1 has no significant correlation with either GST plasma levels or CVD risk as shown in Fig. 1. These findings support the genotype results suggesting that the GSTM1 and GSTT1 genotype may not affect CVD development in T2DM patients

Discussion
A significant association between Glutathione-S-Transferases Theta 1 (GSTT1) polymorphism and type 2 diabetes mellitus (T2DM) or cardiovascular diseases (CVD) development was found among the two GST polymorphisms investigated in this study. GSTM1 and GSTT1 did not significantly affect CVD susceptibility in T2DM patients.
It is well understood that oxidative stress is essential in the development of various conditions such as T2DM and CVD [11,12]. Because of the long-term presence of hyperglycemia and hyperinsulinemia, T2DM patients are prone to oxidative stress, which may contribute to the progression of atherosclerosis [13]. The loss of function polymorphism in GSTM1, GSTT1 and SNP in GSTP1 have been linked to an increased T2DM risk as GSTs are vital antioxidant enzymes that play an important role in the removal  of a variety of harmful compounds [14]. Variations in the genes that encode GSTM1/GSTT1 have been linked to an increased risk of developing cardiovascular disease in some studies [7,15]. However, there has been little research into the role of GSTM1/GSTT1 in the development of CVD in T2DM patients. The average age of the participants in the current study was 46 years, consistent with a previous study that found the highest occurrence of diagnosed CVD among individuals over 45 years of age in India [16]. Males had a higher risk of CVD than women, which is consistent with previous research that has shown that males have a higher incidence of CVD due to a combination of physiological, lifestyle, and societal factors and that the presence of diabetes in men increases their risk of developing CVD even further [17,18]. Individuals with a CVD family history were found to have an increased risk due to shared genetic, environmental, and behavioural factors, with genetics being the most common [19]. Furthermore, cigarette smoking and alcohol consumption have been linked to an increased risk of CVD via oxidative stress, mitochondrial dysfunction, and anatomical damage to the cardiovascular system [20,21]. Thus, quitting modifiable risk factors like smoking and drinking can significantly reduce the risk of CVD. Dyslipidemia is a major risk factor for T2DM and CVD development and progression [10]. In this study, T2DM patients had a higher Castelli risk, indicating atherogenic dyslipidemia, whereas CVD patients had lower HDL and a higher atherogenic index, with a higher Castelli risk indicating dyslipidemia [22,23]. The increase in Castelli risk among T2DM is a characteristic of dyslipidemia commonly found among the diabetic patients [24]. Oxidative stress is a well-known contributor to CVD development. The study's findings revealed elevated Malondialdehyde (MDA) levels and lower total antioxidant status (TAS) in T2DM and CVD patients, indicating the role of oxidative stress in the pathogenesis of these diseases. As a result, MDA and TAS can be considered as potential biomarkers for oxidative stress in people with T2DM and/or CVD. The association of GSTM1/ GSTT1 genotypes with the development of CVD in T2DM individuals in the South Indian population was investigated in this study. GSTM1 null genotype had no association with CVD development in T2DM individuals, which is consistent with the earlier study [10]. The findings revealed that the GSTM1 null genotype had higher lipid peroxidation levels but also higher total antioxidant status, which could be attributed to the interference of other antioxidants or GST genotypes such as GSTT1 and GSTP1. GSTT1 null genotype was associated with an increased risk of both T2DM and CVD, which was consistent with previous research [9,10].
However, there was no clear link between GSTM1/ GSTT1 genotypes and CVD development in T2DM patients. More research is needed to better understand the role of antioxidants and genetic variants in the development and progression of CVD in T2DM. Overall, the findings emphasise the importance of monitoring plasma GST, lipid peroxidation, and TAS levels in CVD and T2DM patients as potential biomarkers of oxidative stress. Despite the small sample size, the current study provides valuable insights into the complex interplay of factors contributing to CVD development in T2DM patients. This emphasise the need for more extensive and comprehensive studies to understand the individual susceptibility to CVD in this population fully. This includes exploring gene-gene and gene-environment interactions, as well as other factors like lifestyle, socioeconomic status, and cultural background. Further research may also aid in the identification of novel biomarkers for the early detection and effective management of CVD in T2DM patients.

Conclusion
This study supports the role of GSTT1 in predicting cardiovascular disease in the South Indian population. The study also emphasises the significance of oxidative stress and antioxidant levels in the development of cardiovascular disease. The findings suggest that variations in GSTT1 and the presence of oxidative stress can increase the risk of Fig. 1 The path analysis of a GSTM1 and b GSTT1 polymorphism with CVD risk in T2DM patients cardiovascular disease; however, larger sample size studies are needed to fully understand the relationship between GSTT1, oxidative stress, and cardiovascular disease in T2DM patients.