|Year : 2017 | Volume
| Issue : 6 | Page : 1479-1484
|Significant difference between the frequency of glutathione-S-transferase M1, glutathione-S-transferase T1 and glutathione-S-transferase P1 polymorphisms in type 1 diabetes patients and controls
Arvand Akbari1, Zivar Salehi2, Shahin Koohmanai3
1 Department of Biology, Faculty of Science, Fars Science and Research Branch, Islamic Azad University; Department of Biology, Faculty of Science, Marvdasht Branch, Islamic Azad University, Marvdasht, Iran
2 Department of Biology, Faculty of Sciences, University of Guilan, Rasht, Iran
3 Department of Pediatrics, Guilan University of Medical Sciences, Rasht, Iran
Click here for correspondence address and email
|Date of Web Publication||11-Jan-2018|
| Abstract|| |
Background and Aim: Oxidative damage can lead cells to apoptosis which is believed to be the main cause of pancreatic β-cell death and eventually end up to the development of type 1 diabetes (T1D). Glutathione S-transferase enzymes (GSTs) play a crucial role in counteracting reactive oxygen species (ROS). In this study, we evaluated the association of three well-known polymorphisms of GSTM1, GSTT1 and GSTP1 in the pathogenesis of T1D which are whole gene deletions in GSTM1 and GSTT1 and a single nucleotide polymorphism (SNP) in GSTP1 known as Ile105Val. Materials and Methods: Samples were collected from 159 patients diagnosed with T1D and 210 control subjects. Genotyping for GSTM1 and GSTT1 was performed by Multiplex Polymerase Chain Reaction (PCR) and by PCR-Restriction Fragment Length Polymorphism (PCR-RFLP) for GSTP1. Results: The GSTM1 and GSTT1 null genotypes were present at frequencies of 54 % and 59.1 % in T1D cases, whereas in controls the frequencies were 41.4 % and 43.3 %, respectively. Double-null genotype was found to be elevated among T1D patients (Odds Ratio [OR], 2.04; 95 % Confidence Interval [CI], 1.08-3.85; P = 0.027). No effect of any genotype for GSTP1 on T1D susceptibility was detected. Individuals with both the double-null and GSTP1 Ile/Val genotype combined appeared to be at increased risk of T1D (OR, 4.95; 95% CI, 2.11-11.6; P = 0.0002). Conclusion: This is the first study conducted on Iranian children with T1D. The absence of GSTM1 and/or GSTT1 may be important risk factor for T1D. Furthermore, presence of Val allele for GSTP1 could strengthen this risk.
Keywords: Deletion, Ile105Val, GSTM1, GSTT1, GSTP1, oxidative stress, polymorphism, type 1 diabetes
|How to cite this article:|
Akbari A, Salehi Z, Koohmanai S. Significant difference between the frequency of glutathione-S-transferase M1, glutathione-S-transferase T1 and glutathione-S-transferase P1 polymorphisms in type 1 diabetes patients and controls. Ann Trop Med Public Health 2017;10:1479-84
|How to cite this URL:|
Akbari A, Salehi Z, Koohmanai S. Significant difference between the frequency of glutathione-S-transferase M1, glutathione-S-transferase T1 and glutathione-S-transferase P1 polymorphisms in type 1 diabetes patients and controls. Ann Trop Med Public Health [serial online] 2017 [cited 2019 Mar 19];10:1479-84. Available from: http://www.atmph.org/text.asp?2017/10/6/1479/222653
| Introduction|| |
Type 1 diabetes (T1D) is a condition in which pancreatic β-cell destruction leads to absolute insulin deficiency. It is known as the most common severe chronic illness in children affecting at least 1 in 300 and probably more in adults.
Apoptosis is probably the main form of β-cell death leading to both types of diabetes. There are different mechanisms by which apoptosis is triggered; Cytokines such as Interleukin-1 beta, tumor necrosis factor-alpha and interferon-gamma which are released by immune system cells can induce apoptosis through the activation of a network of genes. Another known mechanism is oxidative stress caused by overproduction of reactive oxygen species (ROS). Increased oxidative stress and impaired antioxidant defenses are widely accepted participants in the development and progression of diabetes.,
ROS in a low level are crucial for several cellular processes, including intracellular signaling, regulatory mechanisms, defense against pathogens, etc., while a high level of free radicals, a state known as oxidative stress, are considered to be toxic and cause damage through fatty acid and lipid peroxidation, oxidative damage to proteins, mutations in DNA and activation of pro-cell death factors leading to apoptosis. Several biologically important compounds have been reported to have antioxidant functions; such as Vitamins A, E, and C, superoxide dismutase (SOD), glutathione peroxidase, catalase (CAT) and Glutathione S-transferases (GSTs).
GST enzymes catalyze the nucleophilic attack by glutathione on nonpolar compounds that contain an electrophilic carbon, nitrogen, or sulfur atom. This family is made up of seven classes: alpha, Mu, Omega, Theta, Zeta, Pi, and Sigma. GSTM1 gene (GenBank: 2944) is part of the Mu-class GST gene cluster at 1p13.3; it is enclosed in a region with extensive homologies and flanked by two almost identical 4.2 kb regions. The GSM1 null allele forms when a homologous recombination of these two 4.2 kb repeats happens. It results in a 16 kb deletion containing the GSTM1 gene thoroughly.
GSTT1 gene (GenBank: 2952) is part of the Theta-class GST gene cluster at 22q11.2. It is also embedded in a region with huge homologies and sided by two 18 kb regions, known as HA3 and HA5, which are > 90% homologous. In their central portions, HA3 and HA5 share a 403 bp sequence with 100% identity. The GSTT1 null allele comes up when a homologous recombination of the left and right 403 bp repeats takes place. This results in a 54 kb deletion containing the entire GSTT1 gene.
GSTP1 gene (GenBank: 2950) is the only gene in the GSTπ subfamily. It is located at 11q13 with a length of 2.8 kb and containing seven exons., Two common nonsynonymous single nucleotide polymorphisms (SNPs) have been reported regarding this gene; an A to G and another C to T substitution resulting in amino acid substitutions in codons 105 (Ile → Val) and 113 (Ala → Val) in exons 5 and 6, respectively. These amino acids both affect substrate specification in the matter of telling the difference between planar and nonplanar substrates.,
Since GST enzymes play a key role in GSH activity in defense against oxidative stress, their numerous polymorphisms could affect their catalytic activity and prove useful in association studies considering oxidative stress-related diseases., The aim of this study was to find out whether GSTM1, GSTT1, and GSTP1 polymorphism could have been influential in developing T1D in northern Iranian children. We studied GSTM1 and GSTT1 whole gene deletions and also Ile105Val polymorphism in the GSTP1 gene in controls and patients.
| Materials and Methods|| |
The study population included 159 patients diagnosed with T1D by an endocrinologist based on the criteria issued by the American Diabetes Association. Patients were all northern Iranians and in the range of 1–14 years old. We double-checked that they were natives of northern Iran by speaking to their parents personally and we did not include those with roots from other regions in the present study. Control group comprised of 210 healthy unrelated subjects who matched the patients in age and sex. We received a written and signed consent letter from each participant's parents. This study was conducted in accordance with the Declaration of Helsinki regarding the use of human samples.
Blood sample (1 ml) was collected by venipuncture and drawn into tubes containing ethylene diamine tetraacetic acid to avoid blood from clotting (Venoject, Belgium). Genomic DNA was isolated from blood cells through GPP™ solution kit (Gen Pajoohan Pouya Co. Tehran, Iran) following standard protocol. DNA with an optimal density ratio 260/280 of 1.8 or more was used. The extracted DNA was stored at − 20°C.
Genotyping for GSTP1
Section of the gene including the SNP was determined through ncbi SNP search; primers for this part were designed using Oligo primer analysis software (Version 7.54, Molecular Biology Insights Inc., Cascade, Co, USA) as follows: 5'-TCCTTCCACGCACATCCTCT-3' as forward primer and 5'-AGCCCCTTTCTTTGTTCAGC-3' as reverse primer and the amplicon was 290 bp long. Designed oligonucleotides were manufactured by Bioneer. Each polymerase chain reaction (PCR) mixture (25 μl) contained 30 ng of DNA template, ×1 PCR buffer, 1.5 mM of MgCl2, 0.2 mM of deoxyribonucleotide triphosphate mixture mix), 0.5 μM of each primer, and 1.5 U of Taq DNA polymerase (Bioflux, Japan). Samples were initially denatured at 94°C for 4 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 64°C for 30 s, extension at 72°C for 30 s, and a final extension at 72°C for 7 min. PCR products were electrophoresed using 2% agarose gel stained with ethidium bromide and visualized under ultraviolet (UV) illumination. Furthermore, PCR was repeated for any sample with ambiguous results.
Later on, the corresponding PCR products were digested with BsmAI to assess the polymorphism of GSTP1 at position 313 of exon 5. The lack of the polymorphic site within the GSTP1 gene generated a single product of 290 bp (Ile allele). The presence of a BsmAI site was indicated by the cleavage of 290 bp amplified product to yield fragments of 220 and 70 bp (Val allele).
Genotyping for GSTM1 and GSTT1 deletions
Multiplex PCR was performed to detect GSTM1 and GSTT1 null genotypes. GSTM1 was co-amplified with GSTP1 and primers 5'-AGGCAATATTCATAGCTACCTCC-3' as forward and 5'-CGGGCATTTCAACATATACTCC-3' as reverse were used to amplify a 143 bp long amplicon. For GSTT1, primers 5'-GCAGGACTTCAGCAACTAGCC-3' as forward and 5'-ACATCTCCTTAGCTGACCTCGT-3' as reverse were used and a 107 bp long product was amplified. In addition, another pair which was previously designed for MMP9 gene was used as internal control, with 5'-ACTTATTACGGTGCTTGACACA-3' as forward and 5'-TCACTCCTTTCTTCCTAGCCA-3' as reverse primers and a 690 bp long amplicon. All of the mentioned oligonucleotides were ordered to be manufactured by Bioneer.
In PCR reaction for GSTM1, initially, samples were denatured at 94°C for 5 min, followed by 35 cycles of denaturation at 94°C for 40 s, annealing at 62°C for 1 min, extension at 72°C for 1 min, and a final extension at 72° for 10 min. PCR products were electrophoresed on a 2% agarose gel stained with ethidium bromide and visualized under UV illumination.
PCR conditions for GSTT1 were denaturation at 95°C for 4 min, followed by 35 cycles of denaturation at 95°C for 30 s, annealing at 69°C for 2 min, extension at 72 for 2 min, and a final extension at 72°C for 10 min. PCR products were electrophoresed on a 2% agarose gel stained with ethidium bromide and visualized under UV illumination.
To evaluate the contribution of different allele, genotype and phenotype variants in developing type I diabetes, P values were calculated by Chi-squared tests; furthermore, odds ratios (OR) were computed in 95% confidence intervals to ensure the differences were significant. MedCalc software (version 22.214.171.124, Mariakerke, Belgium) was used to carry out the analyses. The value of P < 0.05 was considered statistically significant.
| Results|| |
Genotypes were identified in a control group including 105 females and 105 males and a patient group with 82 males and 77 females. There was no significant difference between age and sex distribution between these two groups (P > 0.05). PCR products for GSTM1 are demonstrated in [Figure 1]. Genotype frequencies for GSTM1 were 58.6% for GSTM1 present and 41.4% for GSTM1 null in control group, and 46% for GSTM1 present and 54% for GSTM1 null among patients. There was a significant difference in genotype distributions between patients and controls (OR 1.66; 95% confidence interval [CI], 1.0–2.5; P = 0.016). PCR products for GSTT1 are displayed in [Figure 2]. Genotype frequencies for GSTT1 were 56.7% for GSTT1 present and 43.3% for GSTT1 null in control group; and 40.9% for GSTT1 present and 59.1% for GSTT1 null in patients. Distributions of GSTT1 were statistically different between patients and controls (OR 1.89; 95% CI, 1.2–2.8; P = 0.002). GSTM1/GSTT1 combined analyses presented that individuals with GSTM1/GSTT1 null genotype were at a higher 2.04-fold risk to develop T1D (95% CI, 1.08–3.85; P = 0.027).
|Figure 1: Agarose gel electrophoresis manifesting GSTM1 genotypes. Co-amplification of internal control (GSTP1, product size 290 bp) approves the polymerase chain reaction reaction. (M) 50 bp DNA marker. (1–3) GSTM1 present genotypes. (4–6) GSTM1 homozygous null genotypes|
Click here to view
|Figure 2: Agarose gel electrophoresis representing GSTT1 genotypes. In each reaction, co-amplification of internal control (MMP9 gene, product size 690 bp) proves the existence of genomic DNA. (M) 50 bp DNA marker. (1–3) GSTT1 peresent genotypes. (4–6) Homozygous GSTT1 null genotypes|
Click here to view
PCR products for GSTP1 are shown in [Figure 3]. Genotype frequencies for GSTP1 were 51.9% for Ile/Ile and 48.1% for Ile/Val in control group, and 52.2% for Ile/Ile and 47.8% for Ile/Val among patients. There was not a participant with Val/Val genotype in patients or control subjects [Figure 4].
|Figure 3: Agarose gel electrophoresis displaying GSTP1 amplicons. 50 bp DNA marker (M) and five 290 bp long polymerase chain reaction products (1–5)|
Click here to view
|Figure 4: Agarose gel electrophoresis of polymerase chain reaction fragments after BsmAI digestion. (M) 50 bp DNA marker. (1–3) Ile/Val genotypes produced three fragments; 70 bp, 220 bp, and 290 bp. (4–6) Ile/Ile genotypes failed to be cleaved by BsmAI|
Click here to view
No significant differences were clarified in genotype distributions of GSTP1 polymorphism between patients and controls (P = 0.955). Furthermore, there was not a significant increase in Val allele frequency among the patients (P = 0.962). The results for GSTM1, GSTT1 and GSTP1 genotype and allele frequencies are summarized in [Table 1]. Combined analyses of GSTM1/GSTT1/GSTP1 suggested that individuals with GSTM1 null/GSTT1 null/Val105Val were at a higher 4.95-fold risk of developing T1D (95% CI, 2.11–11.6, P = 0.0002). Combined analyses are summarized in [Table 2].
|Table 1: GSTM1, GSTT1 and GSTP1 genotype and allele frequencies and their association with the risk of type 1 diabetes|
Click here to view
|Table 2: Combined analyses of GSTM1, GSTT1 and GSTP1 genotype frequencies and their association with the risk of type 1 diabetes|
Click here to view
| Discussion|| |
Increased oxidative stress is generally accepted to participate in the development and progression of diabetes and its complications. While Diabetes itself causes increased production of free radicals and impaired antioxidant defenses, it is also accompanied by hyperglycemia which is found to promote lipid peroxidation of low-density lipoproteins by a superoxide-dependent pathway resulting in the generation of even more free radicals and damaging endogenous antioxidant defense. Genes belonging to GST superfamily are of great importance in the metabolism and detoxification of ROS, xenobiotics and carcinogens. Epidemiological studies confirm that the GSTM1 and GSTT1 null genotypes which result in a lack of functional proteins are correlated with an increased susceptibility to diseases in relation to oxidative stress.
To the best of our knowledge, this is the first study conducted on children with T1D in Iran. To this date, there has been only one published study conducted specifically on the association of polymorphisms of GST genes (GSTM1 and GSTT1) with T1D. In this study which was performed in Sweden, Bekris et al. claimed that presence of GSTM1 could boost the chances of developing T1D and that GSTM1 null genotype was to be considered protective against T1D. No association was reported for GSTT1. Their findings clearly contradict ours; however, conflicting results are common in polymorphism studies due to a variety of causes such as the ethnic structure of the studied population, quantity of cases involved in the study and the impact of other genetic and environmental factors. We observed an association between GSTM1 and GSTT1 null genotypes but not with the GSTP1 Ile105Val polymorphism in Northern Iranian children with T1D. This finding is consistent with some previous studies; Hovnik et al. suggested GSTM1 null genotype as a genetic marker for increased risk of diabetic neuropathy. Moreover, Datta et al. found GSTM1 and GSTT1 deletions singly or together in association with chronic diabetic kidney disease.
Conflicting results have been published about GSTP1 Ile105Val polymorphism as well; Ramprasath et al. stated that individuals with Ile/Val and Val/Val genotypes show a significant risk for T2D. On the contrary, Gönül et al. declared no statistical significance for GSTP1 Ile105Val polymorphism between T2D patients and control groups.
The results of this study support the idea that individuals with GSTM1 and/ or GSTT1 null genotypes could prove to have weaker antioxidant defense systems. In addition, GSTP1 Ile105Val genotype combined with GSTM1 and GSTT1 double null genotype could offer even weaker activitwy against oxidative stress. There are also other polymorphic sites in these genes which can be studied to reach a definitive conclusion about their involvement in T1D; such as, GSTP1 Ala114Val, GSTM1 Lys172Asn, and GSTT1 Thr104Pro which was reported not to have any detectable enzyme activity or immune-reactive protein in vivo.,
| Conclusion|| |
The current study, which is the first study conducted in Northern Iranian children with T1D, implies that GSTM1 and GSTT1 null genotypes alone, together or combined with GSTP1 Ile105Val genotype could be introduced as risk factors for developing T1D. According to the usual differences in results of polymorphism studies, we suggest further studies in different and larger populations for coming to a general and reliable understanding of the subject at hand.
This study was partly funded by Guilan University. We wish to express our gratitude to everyone who helped us make it happen.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
American Diabetes Association. Diagnosis and classification of diabetes mellitus. Diabetes Care 2009;32 Suppl 1:S62-7.
Wucherpfennig KW, Eisenbarth GS. Type 1 diabetes. Nat Immunol 2001;2:767-8.
Cnop M, Welsh N, Jonas JC, Jörns A, Lenzen S, Eizirik DL. Mechanisms of pancreatic β-Cell death in type 1 and type 2 diabetes many differences, few similarities. Diabetes 2005;54:97-107.
Ceriello A. Oxidative stress and glycemic regulation. Metabolism 2000;49:27-9.
Halliwell B, Gutteridge JM. Role of free radicals and catalytic metal ions in human disease: An overview. Methods Enzymol 1990;186:1-85.
Kangralkar V, Patil SD, Bandivadekar R. Oxidative stress and diabetes: A review. Int J Pharm Appl 2010;1:38-45.
Maritim A, Sanders R, Watkins 3rd
J. Diabetes, oxidative stress, and antioxidants: A review. J Biochem Toxicol 2003;17:24-38.
Mannervik B, Helena Danielson U, Ketterer B. Glutathione transferases-structure and catalytic activit. Crit Rev Biochem Mol Biol 1988;23:283-37.
Xu Sj, Wang Yp, Roe B, Pearson WR. Characterization of the human class mu glutathiones-transferase gene cluster and the GSTM1 deletion. J Biol Chem 1998;273:3517-27.
Parl FF. Glutathione S-transferase genotypes and cancer risk. Cancer Lett 2005;221:123-9.
Landi S. Mammalian class theta GST and differential susceptibility to carcinogens: A review. Mutat Res 2000;463:247-83.
Sprenger R, Schlagenhaufer R, Kerb R, Bruhn C, Brockmöller J, Roots I. et al
. Characterization of the glutathione S-transferase GSTT1 deletion: Discrimination of all genotypes by polymerase chain reaction indicates a trimodular genotype-phenotype correlation. Pharmacogenet Genomics 2000;10:557-65.
Kano T, Sakai M, Muramatsu M. Structure and expression of a human class π glutathione S-transferase messenger RNA. Canc Res J 1987;47:5626-30.
Morrow CS, Cowan KH, Goldsmith ME. Structure of the human genomic glutathione S-transferase-π gene. Gene 1989;75:3-11.
Moyer AM, Salavaggione OE, Wu TY, Moon I, Eckloff BW, Hildebrandt MA, et al
. 2008. Glutathione S-transferase P1: gene sequence variation and functional genomic studies. Canc Res J 2008;68:4791-801.
Ali-Osman F, Akande O, Antoun G, Mao JX, Buolamwini J. Molecular cloning, characterization, and expression in Escherichia
coli of full-length cDNAs of three human glutathione S-transferase Pi gene variants. Evidence for differential catalytic activity of the encoded proteins. J Biol Chem 1997;272:10004-12.
Ji X, Blaszczyk J, Xiao B, O'Donnell R, Hu X, Herzog C, et al
. Structure and function of residue 104 and water molecules in the xenobiotic substrate-binding site in human glutathione S-transferase P1-1. Biochemistry 1999;38:10231-8.
Salagovic J, Kalina I, Stubna J, Habalova V, Hrivnak M, Valanský L, et al
. Genetic polymorphism of glutathione S-transferases M1 and T1 as a risk factor in lung and bladder cancers. Neoplasma 1998;45:312-7.
Tamer L, Calikoǧlu M, Ates NA, Yildirim H, Ercan B, Saritas E, et al
. Glutathione-S-transferase gene polymorphisms (GSTT1, GSTM1, GSTP1) as increased risk factors for asthma. Respirology 2004;9:493-8.
Mellitus D. Diagnosis and classification of diabetes mellitus. Diabetes care 2011;27:5-10.
Beckett GJ, Hayes JD. Glutathione S-transferases: Biomedical applications. Adv Clin Chem 1993;30:281-380.
Bekris LM, Shephard C, Peterson M, Hoehna J, Yserloo BV, Rutledge E, et al
. Glutathione-s-transferase M1 and T1 polymorphisms and associations with type 1 diabetes age-at-onset. Autoimmunity 2005;38:567-75.
Hovnik T, Dolzan V, Bratina NU, Podkrajsek KT, Battelino T. Genetic polymorphisms in genes encoding antioxidant enzymes are associated with diabetic retinopathy in type 1 diabetes. Diabetes Care 2009;32:2258-62.
Datta SK, Kumar V, Pathak R, Tripathi AK, Ahmed RS, Kalra OP, et al
. Association of glutathione S-transferase M1 and T1 gene polymorphism with oxidative stress in diabetic and nondiabetic chronic kidney disease. Ren Failure 2010;32:1189-95.
Ramprasath T, Senthil Murugan P, Prabakaran AD, Gomathi P, Rathinavel A, Selvam GS, et al.
Potential risk modifications of GSTT1, GSTM1 and GSTP1 (glutathione-S-transferases) variants and their association to CAD in patients with type-2 diabetes. Biochem Biophys Res Commun 2011;407:49-53.
Gönül N, Kadioglu E, Kocabaş NA, Özkaya M, Karakaya AE, Karahalil B. The role of GSTM1, GSTT1, GSTP1, and OGG1 polymorphisms in type 2 diabetes mellitus risk: A case control study in a Turkish population. Gene 2012;505:121-7.
Alexandrie AK, Rannug A, Juronen E, Tasa G, Warholm M. Detection and characterization of a novel functional polymorphism in the GSTT1 gene. Pharmacogenet Genomics 2002;12:613-9.
McIlwain CC, Townsend DM, Tew KD. Glutathione S-transferase polymorphisms: Cancer incidence and therapy. Oncogene 2006;25:1639-48.
Department of Biology, Faculty of Sciences, University of Guilan, Namjoo Street, PO Box 1914, Rasht
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2]
| Article Access Statistics|
| Viewed||998 |
| Printed||35 |
| Emailed||0 |
| PDF Downloaded||18 |
| Comments ||[Add] |