Combined glutathione S transferase M1/T1 null genotypes is associated with type 2 diabetes mellitus.

BACKGROUND: Due to new genetic insights, a considerably large number of genes and polymorphic gene variants are screened and linked with the complex pathogenesis of type 2 diabetes (DM). Our study aimed to investigate the association between the two isoforms of the glutathione S-transferase genes (Glutathione S transferase isoemzyme type M1- GSTM1 and Glutathione S transferase isoemzyme type T1-GSTT1) and the prevalence of DM in the Northern Romanian population.
METHODS: We conducted a cross-sectional, randomized, case-control study evaluating the frequency of GSTM1 and GSTT1 null alleles in patients diagnosed with DM. A total of 106 patients diagnosed with DM and 124 healthy controls were included in the study. GSTM1 and GSTT1 null alleles genotyping was carried out using Multiplex PCR amplification of relevant gene fragments, followed by gel electrophoresis analysis of the resulting amplicons.
RESULTS: Molecular analysis did not reveal an increased frequency of the null GSTM1 and GSTT1 alleles (mutant genotypes) respectively in the DM group compared to controls (p=0.171, OR=1.444 CI=0.852-2.447; p=0.647, OR=0.854, CI=0.436-1.673). Nevertheless, the combined GSTM1/GSTT1 null genotypes were statistically significantly higher in DM patients compared to control subjects (p=0.0021, OR=0.313, CI=0.149-0.655).
CONCLUSIONS: The main finding of our study is that the combined, double GSTM1/GSTT1 null genotypes are to be considered among the polymorphic genetic risk factors for type 2 DM.

Introduction

Type 2 diabetes mellitus (DM), is a multifactorial, chronic adult-onset condition characterized by insufficient insulin secretion and inefficient insulin action, thus presenting the common manifestation of hyperglycemia [1].

According to WHO (2015), 374 million people around the world suffer from a form of diabetes, of which 90% have DM and estimates that diabetes related deaths will double between 2005 and 2030, therefore this condition and all its comorbidities are an important global health issue [2]. In 2014 Romania reported 9.3 prevalence for type 2 DM, with a total of 1.5 million diagnosed individuals [3].

Several studies have shown that individuals with a decreased antioxidant capacity are at higher risk of developing DM [4,5].

Glutathione S-transferases (GSTs) are the primary cellular defense mechanism against reactive oxygen species as phase II detoxifying isoenzymes known to metabolize a variety of electrophilic compounds, including drugs, carcinogens, toxins and ROS generated DNA products. Eight members of soluble GSTs have been identified in humans, named Mu (M), Kappa (K), Alpha (A), Pi, accounting for 4% of the total cytosolic proteins in the liver [6,7]. Among the numerous candidate genes involved in DM pathogenesis, the GST isoemzyme family has been intensely studied in all types of diabetes due to its potential modulating roles in individual susceptibility to multifactorial, environmentally induced disorders. Among these, glutathione S transferase isoemzyme type M1 (GSTM1) and glutathione S transferase isoemzyme type T1 (GSTT1) genes are extremely polymorphic in humans and the null genotypes resulting from loss of function mutations are accompanied by lack of enzyme activity [8,9].

Several studies investigated the possible relation between GSTM1 and GSTT1 null alleles and T2DM; Turkish, North Indian and Southern Iran populations highlighted an association between GSTM1 deletion and DM whereas Egyptian, Chinese, and Brazilian population reported significant association between GSTM1/GSTT1 null genotypes and type 2 diabetes [10-15].

In a recent studies and meta-analysis, GSTM1 and GSTT1 null genotypes have been associated with a higher risk of developing DM in Japanese and South Indian and African population [16-19].

The aim of the present study is to determine the possible association between the incidence of type 2 DM and gene polymorphisms of glutathione S-transferase GSTM1 and GSTT1 in a population group of Northern Romania and to see if GSTM1/GSTT1 null genotypes may be considered a genetic risk factor for DM.

Materials and methods

Patients and controls

This is a molecular epidemiologic case-controlled study, in which we included a group of 106 patients diagnosed with type 2 DM (patients of Diabetes Clinic of the Emergency County Hospital of Cluj) and 124 healthy controls (non-diabetic patients recruited from the Internal Medicine Department of the Emergency County Hospital Cluj). In order to obtain homogeneity regarding proportion and the range of ages and gender we selected and approximate similar age/gender matched subject for each of the subject, respectively control groups.

Ethical issues

The current study was approved by the Local Ethics Committee. The study was conducted according to the Ethical Principles for Medical Research Involving Human Subjects of the World Medical Association Declaration of Helsinki. Written informed consent was obtained from each participant after a brief and clarified explanation about the survey.

(Protocol adapted after Bid HK et al.) [20]

Genotyping for GSTM1 and GSTT1 alleles was carried out using a Multiplex PCR protocol (18).

A total volume of 25 μl reaction commercial mixture containing 12.5 μl 2xPCR Master Mix (Thermo Fischer Scientific Inc., MA, USA), 25 μl of free nuclease water and 10 μm of each specific forward and reverse primers.

GSTM1 and GSTT1 null allele identification required the use of 3 pairs of primers

(5′-GAACTCCCTGAAAAGCTAAAGC-3′; 5′-GTTGGGCTCAAATATAGGGTGG-3′ and

5′-TTCCTTACTGGTCCTCACATCTC-3′; 5′-TCACCGGATCATGGCCAGCA-3′ (Eurogentec Bg.)

As an internal standard amplification marker, β Globin was co-amplified using another pair of primers (5′-CAACTTCATCCACGTTCACC-3′ and 5′-GAAGAGCCAAGGACAGGTAC-3′ (Eurogentec, Bg.) Termocycling conditions consisted of an initial denaturation at 94°C for 5 minutes followed by 35 repetitive cycles each with a DNA denaturation at 94°C for 1 minute, primers annealing for 1 minute at 58°C, polymerization at 72°C for 1 minute and a final elongation for 10 minutes at 72°C. (Mastercycler Gradient®, Eppendorf, Germany). The resulted amplified DNA products were then analyzed by agarose 2% gel electrophoresis (MetaPhor® Agarose, Cambrex Bio Science Inc.), obtaining 3 different fragments, one of 215 bp (for GSTM1gene), one of 480 bp (for GSTT1 gene) and a 268 bp fragment (for the β Globin gene control fragment). The absence of the amplification specific products revealed the presence of the mutant, null genotypes. We must specify that the current protocol easily identifies the GSTT1 and GSTM1 homozygous null genotypes but cannot distinguish between GSTM1 and GSTT1 homozygous and heterozygous positive genotypes (Fig. 1).

Figure 1

Electrophoretic analysis for GSTM1 and GSTT1 null genotypes (Multiplex PCR).

Lanes 1, 2, 6: GSTT1 positive genotype, GSTM1 null genotype, Lanes 3, 4: GSTT1/GSTM1 null genotypes; Lane 5: GSTM1/GSTT1 positive genotypes Lane 7: GSTM1 positive, GSTT1 null genotype, Lane L: 50 bp DNA ladder marker.

Statistical analysis

Statistical analysis used a licensed Graphpad software. Odds ration (OR) with 95% confidence limits calculated by logistic regression. GSTM1 and GSTT1 genotypes were classified as either null (homozygous deletion) or wild type, non-deleted. For a more accurate risk evaluation, Z and P values were also calculated.

Results

A total of 106 subjects with DM and 124 controls were investigated in the current study. The proportions of genders in case and control group proved not significantly different (Z-statistics=1.515, p-value=0.1298). The mean age of subjects with DM was 64.43±9.72 years old and of 65.31±5.92 years old for controls with no statistically significant differences (t-statistics=−0.80, p-value=0.4220) (Table 1). The studied genotype followed Hardy-Weinberg equilibrium of genetic distribution (Chi-square test p=0.08).

Table I

Parameters of study groups.

	Type 2 DM Group	Control Group	p-value
Number (M/F)	106 (52/54)	124 (62/62)	0.1298
Age (years)	64.43±9.72	65.31±5.92	0.4220
Weight (kg)	85.4 ±13.2	81.72± 12.7	0.2456
Body mass index (kg/m2)	27.84±2.1	25.1 ± 5.1	0.3561
HbA1c (%)	7.6±1.3	NA	NA

Statistical analysis did not reveal an increased frequency of GSTM1, respectively GSTT1 null genotypes (mutant alleles) in the DM group compared to controls (p=0.171, OR=1.444 CI=0.852–2.447; p=0.647, OR=0.854, CI=0.436–1.673). Nevertheless, the combined GSTM1/GSTT1 null genotypes were statistically significant higher in DM patients compared to control subjects (p=0.0021, OR=0.313, CI=0.149–0.655) (Table II).

Table II

Distribution of single GSTM1, GSTT1 and combined GSTM1/GSTT1 null genotypes among patients with type 2 DM and controls.

Genotype	p	Odd ratios	95% IC
			Inferior limit	Superior limit
GSTM1 null	0.171	1.444	0.852	2.447
GSTT1 null	0.647	0.854	0.436	1.673
GSTM1/GSTT1 null	0.0021*	0.313	0.149	0.655

Significant statistical association for p<0.050.

The genotype-gender associated statistical data revealed a higher frequency for the combined GSTM1/GSTT1 null genotypes in women with diabetes compared to male subjects (p=0.037, OR=0.245, CI=0.065–0.918). There was also an increased frequency of the GSTT1 null genotypes in males diabetics compared to controls (p=0.021, OR=0.343. CI=0.138–0.851).

In cases of the GSTM1-null genotype and combined GSTM1/GSTT1 null genotypes there were higher levels of HbA1c compared to GSTM1/GSTT1 positive genotypes. (p=0.028, OR=0.271; CI=0.124–0.742) (Table III).

Table III

Significant Gender and HbA1C, single GSTM1, GSTT1 and combined GSTM1/GSTT1 null genotypes associations.

GENOTYPE T2DM	Gender		HbA1C >7%
	Male	Female	Male	Female
			P=0.028*
GSTM1null	-	-	OR=0.271	-
			CI=0.124–0.742
	P=0.021*
GSTT1 null	OR=0.343	-	-	-
	IC=0.138–0.851
		P=0.037*	P= 0.013*
GSTM1/GSTT1 null	-	OR=0.245	OR=0.436
		IC=0.065–0.918	IC=0.279–0.862

Significant statistical association for p<0.050.

HbA1C – Glycosylated haemoglobin

DM- Type 2 Diabetes Mellitus

Discussion

Type 2 DM is one of the most common chronic diseases in modern countries and represents a health problem and a major epidemic during the past decades. [21,22]

Many studies have evaluated the association between GST polymorphisms and to our knowledge this is the first study evaluating the association of GSTM1 and GSTT1 null genotypes and type 2 DM in the Romanian population.

We hypothesized that the genetic variability of GST detoxifying enzymes regulating oxidative stress could be involved in development of DM.

The GSTT1 and GSTM1 combined deletion polymorphisms (null alleles), which associate abolished enzyme activity, have been associated with type 2 DM when compared to control subjects. However, single GSTM1 and GSTT1 mutant variants did not have a higher frequency in diabetic patients compared to controls [23].

Our results come in agreement with other studies that highlighted the same results, placing combined GSTM1/GSTT1 deleted genotypes among the inheritable risk factor for DM development [19-22,24,25].

Another finding of the current study is that a genotype-gender statistical association revealed a higher frequency for the combined GSTM1/GSTT1 null genotypes in women with diabetes compared to male subjects. Also, there was an increased frequency of the GSTT1 null genotypes in males diabetics compared to controls. There are only a few studies that evaluate the GST mutant genotype – gender in DM.

In conclusion, GSTM1 and GSTT1 mutant variants individually are not associated with DM; however, combined GSTM1/GSTT1 null genotypes could represent independent risk factor for DM. Additionally, GSTT1 null genotypes and combined GSTM1/GSTT1 null genotypes could be considered genetic risk factor in type 2 DM in males and women respectively. Testing for genetic markers in genes encoding antioxidant enzymes like GSTs, either individually or in combination with other polymorphic genetic variants possibly involved in DM pathogen, could be added to a genetic panel for identifying patients at higher risk for developing diabetes in clinical practice.

Our study has a series of limitations. First, the small number of subjects represents a major limitation, therefore the resulting data of the study may not be representative for the general population. For this purpose, future studies with larger number of subjects with different population ethnicity are required and a longitudinal design is also necessary. Another limitation is the lack of correlation with clinical parameters associated with type 2 DM (lifestyle, medication, associated pathology, complications, etc.).