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Glutathione S-transferase M1, T1, and P1 gene polymorphism in laryngeal squamous cell carcinoma

Glutathione S-transferase M1, T1, and P1 gene polymorphism in laryngeal squamous cell carcinoma
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  Glutathione S-transferase M1, T1 and P1 genepolymorphisms and the risk of developingtype 2 diabetes mellitus in Egyptian diabeticpatients with and without diabetic vascularcomplications Moyassar Ahmad Zaki  a, * , Thanaa Fathy Moghazy  a ,Mona Mohamed Kamal El-Deeb  a , Amira Hussein Mohamed  b ,Noha Arafa Ahmed Mohamed  a a Chemical Pathology Dept, Medical Research Institute, Alexandria University, Egypt b Nephrology Unit of the Internal Medicine Dept, Medical Research Institute, Alexandria University, Egypt Received 22 December 2013; accepted 22 March 2014 KEYWORDS Glutathione S-transferasegene polymorphisms;Oxidative stress;Type 2 diabetes mellitus Abstract  Background and aim of work:  Persistent oxidative stress is one of several factors thatparticipate in the pathogenesis of type 2 diabetes mellitus (T2DM). Glutathione S-transferases(GSTs) are a family of antioxidant enzymes that exert important antioxidant roles in the elimina-tion of reactive oxygen species. We aimed to assess the association of genetic polymorphisms in theGST isoenzymes M1, T1 and P1 with the risk of developing T2DM and its vascular related com-plications in Egyptian diabetic patients. Subjects and methods:  Fifty-four T2DM patients of whom twenty-seven were suffering from vas-cular complications were compared to fifty-one healthy volunteers. Null genotypes in the GSTM1 and T1 genes were screened using polymerase chain reaction (PCR). The A313G single nucle-otide polymorphism in the GSTP1 gene was detected using PCR–restriction fragment lengthpolymorphism. *Corresponding author. Address: Chemical Pathology Dept, Med-ical Research Institute, Alexandria University, PO Box 21561, 165El-Horreya Avenue, El-Hadara, Alexandria, Egypt. Tel.: +203 4282331/4285455; fax: +20 3 4283719.E-mail addresses:, moyassarmaz73@yahoo. com (M.A. Zaki).Peer review under responsibility of Alexandria University Faculty of Medicine. Production and hosting by Elsevier Alexandria Journal of Medicine (2014)  xxx , xxx  –  xxx Alexandria University Faculty of Medicine Alexandria Journal of Medicine www.sciencedirect.com2090-5068  ª  2014 Alexandria University Faculty of Medicine. Production and hosting by Elsevier B.V. All rights reserved. Please cite this article in press as: Zaki MA et al. Glutathione S-transferase M1, T1 and P1 gene polymorphisms and the risk of developing type 2diabetes mellitus in Egyptian diabetic patients with and without diabetic vascular complications,  Alex J Med   (2014), j.ajme.2014.03.003  Results:  No significant differences were noted between diabetic cases and control group regardingfrequencies of null genotypes of GSTM1 and GSTT1 genes ( v 2  p  = 0.631 and  v 2  p  = 0.832, respec-tively). Furthermore, both null genotypes were not associated with the risk of developing T2DM orits related vascular complications whether alone or in combination. The frequency of the heterozy-gous mutation (AG) in the A313G GSTP1 polymorphism among diabetic cases with and diabeticcases without vascular complications was significantly higher compared to the control group(  p  = 0.023). The risk of developing T2DM was significantly higher in cases presenting with com-bined heterozygous GSTP1 and null GSTM1 genotypes (Odds ratio = 6.285, 95% confidence inter-val = 1.184–33.347,  p  = 0.021). Conclusion:  Our results could point out to potential roles of GSTP1 polymorphism alone or com-bined with GSTM1 gene polymorphism in the pathogenesis of T2DM related oxidative stress.Screening for other functional GST gene polymorphisms is important to understand the impactof interaction of multiple genetic factors in the pathogenesis of T2DM. ª  2014 Alexandria University Faculty of Medicine. Production and hosting by Elsevier B.V. All rightsreserved. 1. Introduction Diabetes mellitus (DM) represents a group of metabolic dis-eases characterized by hyperglycemia resulting from defectsin pancreatic insulin secretion, insulin action, or both. Thechronic hyperglycemia of diabetes is associated with long-termdamage, dysfunction, and failure of different organs, especiallythe eyes, kidneys, nerves, heart, and blood vessels. 1 Diabetesremains a major public health issue. In 2010, it was estimatedthat 4.787 million Egyptians suffer from diabetes, particularlytype 2 (T2DM), and that diabetes will increase to 8.615 millionEgyptians by the year 2030. 2,3 Oxidative stress is one of several mechanisms that contrib-ute in the pathogenesis of T2DM and its related vascular com-plications. It represents a state of imbalance between pro-oxidants and antioxidant defense system. The hyperglycemiainduced overproduction of reactive oxygen species (ROS) suchas superoxide, hydrogen peroxide and hydroxyl radical, alongwith reactive nitrogen species (RNS) such as nitric oxide causesoxidation of DNA, proteins and other cellular componentsleading to their damage. 4,5 The metabolic abnormalities of dia-betes cause increased mitochondrial superoxide overproduc-tion in endothelial cells of both large and small vessels, aswell as the myocardium. This causes the activation of majorpathways which increase intracellular ROS. 6,7 Studies have shown that individuals with loweredantioxidant capacity are at increased risk of T2DM. 8,9 Alterations in the endogenous ROS scavenging defense mech-anisms may lead to ineffective scavenging of ROS, resultingin oxidative damage and tissue injury. 3 Pancreatic  b -cellshave emerged as a putative target of oxidative stress-inducedtissue damage being sensitive to cytotoxic stress because of their little expression of antioxidant enzymes. This seems toexplain in part the progressive deterioration of   b -cell functionin T2DM. 10 Different families have been identified in detoxification orreduction of ROS production. Glutathione S-transferases(GSTs) are the most important family of phase II isoenzymesknown to detoxify a variety of electrophilic compounds,including carcinogens, chemotherapeutic drugs, environmentaltoxins, and DNA products generated by ROS damage to intra-cellular molecules. Detoxification via GSTs is achieved by con- jugating them with glutathione. GSTs thus play a major role ascellular antimutagen and in antioxidant defense mechanisms. 11 Two distinct superfamilies of GST isoenzymes exist; one fam-ily comprises cytosolic, soluble dimeric enzymes, 12 and theother superfamily is composed of membrane bound trimericproteins named the membrane associated proteins in eicosa-noid and glutathione (MAPEG) metabolism. 13 Human soluble GSTs collectively account for 4% of totalsoluble proteins in the liver. They exist as 50 KDa dimeric pro-teins with both subunits being from the same class of GSTs. 14 Based on sequence similarity, at least eight members of thecytosolic family have been identified in humans named Mu(M), Kappa (K), Alpha (A), Pi (P), Omega (O), Theta (T),Zeta (Z), and Sigma (S). 15 Among candidate genes related to oxidative stress, genesfor cytosolic GSTs, particularly GSTM1, GSTT1 and GSTP1were intensively studied in different disease states owing totheir potential modulating roles in individual susceptibility toenvironmentally induced diseases. GSTM1 and GSTT1 genesare polymorphic in humans and the null genotypes are accom-panied by lack of enzyme activity. 16,17 On the other hand, theGSTP1 single nucleotide polymorphism (SNP) present onexon 5 is characterized by guanine replacing adenine base atposition 313 (A313G) of the gene nucleotides. This results invaline replacing isoleucine amino acid at position 105 in theGSTP1 isoenzyme protein. Such replacement results in theappearance of a new allele with alteration in specific activityfor substrate compared to wild-type allele. 18 Several investigators have determined the clinical or geneticfactors associated with T2DM with interests to detoxificationagents. As regards GSTM1, T1 and P1 isoenzymes, studieson Egyptian, 3 Chinese, 19 and Brazilian 20 populations reporteda significant association of the null mutation of GSTT1 geneand T2DM, whereas in studies involving Turkish, 21,22 NorthIndian, 23 and Southern Iran 24 populations this associationwas observed between GSTM1 deletion and T2DM. Recently,studies conducted on Japanese 25 and South Indian popula-tion 26 as well as another meta-analysis study involving Asian,European and African diabetic populations 27 reported theassociation of both GSTM1 and GSTT1 null genotypes withthe risk of developing T2DM. The North Indian 23 and anotherEgyptian study conducted in T2DM 28 were the only ones dem-onstrating a significant association of the GSTP1 SNP(A313G) with T2DM.2 M.A. Zaki et al. Please cite this article in press as: Zaki MA et al. Glutathione S-transferase M1, T1 and P1 gene polymorphisms and the risk of developing type 2diabetes mellitus in Egyptian diabetic patients with and without diabetic vascular complications,  Alex J Med   (2014), j.ajme.2014.03.003  The different ethnic backgrounds creating such a contro-versy in results and scarcity of GST genetic studies conductedamong Egyptian T2DM patients invited us to carry out thiscase-control study to assess the frequency of GSTM1, GSTT1and GSTP1 genotypes in T2DM and explore any possible rela-tion(s) between the genotypes and the risk of developingT2DM and its related vascular complications. To the best of our knowledge, the current study is one of few studies address-ing the GSTP1 point mutation as well as the GSTM1 andGSTT1 gene polymorphisms among Egyptian T2DM patients. 2. Subjects Informed consents were obtained from the one hundred andfive individuals enrolled in this study. The Research EthicsCommittee of the Medical Research Institute approved thestudy protocol. Fifty-four diabetic patients were selected fromthe Internal Medicine department of the Institute, with twenty-seven of them suffering from T2DM related vascular complica-tions at the time of the study. Fifty-one apparently healthy vol-unteers obtained from the outpatient clinics of the Instituteserved as a control group. Cases suffering from any type of malignancy as well as bronchial asthma, hypertension preced-ing T2DM, cardiac, primary renal and liver diseases wereexcluded from this study. 3. Methods 3.1. Clinical examination and anthropometric measurements To all the studied subjects, a thorough history was taken withstress on the duration of diabetes, as well as T2DM related vas-cular complications. Physical examination was done with stresson diabetes related vascular complications. Blood pressure wasrecorded. Ultrasonographic evaluation of the liver and kidneyswas done. A slit lamp fundus examination to document reti-nopathyanda12leadstandardelectrocardiogramtodocumentdiabeticischemicchangesweredone.Anthropometricmeasure-ments, namely body weight and height along with the calcula-tion of body mass index (BMI) were done. Ultrasonographicdeterminationoftherightandleftcarotidarteriesintimamediathickness (CIMT) was done using a  b -mode ultrasound todetect peripheral atherosclerotic changes. 29 3.2. Laboratory investigations3.2.1. Biochemical analysis Following a twelve hour fasting period, concomitant venousblood samples and early morning midstream urine specimenswere obtained from every participant. Fasting serum sampleswere used for the determination of concentrations of glucose,creatinine, total cholesterol and its high density fraction, tri-glycerides, and activity of alanine aminotransferase enzyme.Determination of urinary albumin and creatinine concentra-tions were done in the urine sample. Biochemical analysiswas conducted on the Olympus AU400 clinical chemistry ana-lyzer (Beckman Coulter Inc, Brea CA, USA). Calculations of serum low density lipoprotein fraction using Friedwald’s for-mula and urinary albumin to creatinine ratio (ACR) weredone. 30,31 Whole blood percent glycated hemoglobin (HbA 1c )value was determined using an ion exchange column chro-matographic technique (Biosystems SA, Barcelona, Spain)according to the manufacturer’s instructions. 3.2.2. Genomic analysis Whole EDTA blood was used for genomic DNA extractionfrom peripheral mononuclear cells using a GeneJET  columnbased genomic DNA purification kit (Fermentas, ThermoFischer Scientific Inc., USA) according to the manufacturer’sinstructions. The integrity of the extracted DNA was assessedqualitatively by electrophoresis on a 1% agarose gel. Quantita-tive determination of concentration and purity of DNA wasdone using the NanoDrop  1000 Spectrophotometer (ThermoFischer Scientific, Wilmington, Delaware USA).The A313G SNP of the GSTP1 gene was determined usinga PCR – restriction fragment length polymorphism (RFLP)according to the method described by Harries LW et al.(1997). 32 Briefly, 12  l L (50–150 ng) of genomic DNA weremixed in a 0.2 mL sterile eppendorf tube with 0.2  l L of eachforward (5 0 -ACCCCAGGGCTCTATGGGAA-3 0 ) and reverse(5 0 -TGAGGGCACAAGAAGCCCCT-3 0 ) primers (Fermentas – Thermo Fischer Scientific Inc., USA) in concentrations of 5 pmol per reaction tube, 12.5  l L DreamTaq   Green PCRMaster Mix (2 · ) (Fermentas – Thermo Fischer ScientificInc., USA), and completed to a final reaction volume of 25  l L using nuclease free sterile water. The PCR thermalcycler (Quanta Biotech, UK) conditions were as follows; a5 min initial denaturation phase at 95  C, followed by 30 cyclesof denaturation (94  C, 30 s), annealing (55  C, 30 s), andextension (72  C, 30 s), and a final elongation step of 5 minat 72  C. The resulting 176-bp fragment, generated by PCR,was electrophoretically separated on a 2% agarose gel andvisualized by ethidium bromide staining to confirm its pres-ence. The PCR product was subjected to an RFLP using anAlw261 restriction endonuclease (Fermentas – Thermo FischerScientific Inc., USA). The digestion reaction was carried out ina 1.5 mL sterile eppendorf tube, where 5  l L of PCR productwere mixed with 0.5  l L enzyme, 1  l L enzyme buffer, and com-pleted to a final reaction volume of 15  l L with nuclease freesterile water. The mixture was incubated at 37  C for one hourusing a thermomixer (Eppendorf AG Hamburg, Germany).The digestion products electrophoretically separated on a2% agarose gel revealed one of three possibilities; a singleundigested band at 176 base pairs indicating the presence of a homozygote AA allele (wild type), the presence of a restric-tion site resulting in two fragments (91 and 85 base pairs) indi-cating the presence of a GG homozygote mutant allele, andlastly three bands (176, 91 and 85 base pairs) indicating thepresence of an A/G heterozygote mutant allele.Screening for deletions in the GSTM1 and GSTT1 geneswas done using PCR according to the method described byBid HK et al. (2010) for both genes. 23 Briefly, 12  l L of geno-mic DNA (50–150 ng) was mixed in a 0.2 mL sterile eppren-dorf tube with 0.2  l L of forward and reverse primers(Fermentas – Thermo Fischer Scientific Inc., USA) in concen-trations of 5 pmol per reaction tube, 12.5  l L DreamTaq  Green PCR Master Mix (2 · ) (Fermentas – Thermo FischerScientific Inc., USA), and completed to a final reaction volumeof 25  l L using nuclease free sterile water. An internal controlwas used with every reaction in a multiplex manner to verifythe successfulness of PCR composed of forward and reverseGene polymorphisms and risk of type 2 diabetes mellitus 3 Please cite this article in press as: Zaki MA et al. Glutathione S-transferase M1, T1 and P1 gene polymorphisms and the risk of developing type 2diabetes mellitus in Egyptian diabetic patients with and without diabetic vascular complications,  Alex J Med   (2014), j.ajme.2014.03.003  primers that amplify exon-7 of the CYP1A1 gene. Details of primer sequences and thermocycler (S96 Quanta Biotech,UK) conditions are available in Table 1. The PCR productswere visualized using electrophoretic separation on a 2% aga-rose gel. PCR products representing GSTM1 and GSTT1 posi-tive genotypes yielded bands of 215 and 480 bp, respectively,while the internal positive control (CYP1A1) PCR productband corresponded to 312 bp. Such genotyping approach didnot allow for detecting heterozygous carriers of GSTM1 orGSTT1 deletion; hence, the GSTM1-0 or GSTT1-0 genotypegroupincludedonly patientshomozygous forGSTM1or GSTT1deletion. The GSTM1-1 or GSTT1-1 genotype group includedhomozygous and heterozygous carriers of the functional allele. 3.3. Statistical analysis Statisticalanalysisofdatawasperformedusingstatisticalpack-ages of Social Science (SPSS) version 20 (SPSS, Inc., Chicago,IL,USA). 33 DatawerecodedandfedtotheSPSSsoftwarepack-age.D’Agostino–Pearson K  -squaredtestfornormalitywasusedto test for the degree of deviation from normal distributionacross all quantitative variables in all groups and subgroups.Fornormallydistributedvariables,descriptivemeasuresnamelymean and standard deviation were applied and independentsamples  t -test for comparison between groups. For abnormallydistributed quantitative variables, descriptive measures namelymedian, and range were applied, and Mann–Whitney test forcomparison between groups. For comparing nominal clinicaldata variables between groups, Chi-square test with a MonteCarlo estimate of exact  p -values as well as Fisher’s exact testwere used depending on the expected frequencies. Being a casecontrol study, Odds ratio was used to measure the impact of GSTgenotypesorallelesontheriskofdevelopingtype2diabe-tesmellitus.Chi-squaretestforgoodnessoffitwasusedtocom-pare the observed frequencies of different GSTP1 genotypesamong all subjects to expected frequencies according toHardy–Weinberg equilibrium equation. 34 A  p -value less than0.05 was considered statistically significant. 4. Results A total number of 105 individuals (54 T2DM patients and 51healthy volunteers) were genotyped for the three members of the GST family. Screening for the GSTT1, M1 and P1 genepolymorphisms was done using multiplex PCR for the first 2isoenzymes and PCR-RFLP for the third one. The demo-graphical characteristics, namely age and sex, are summarizedin Table 2. The duration of disease in all diabetic cases variedfrom five to fifteen years. A significantly higher BMI meanvalue was demonstrated in all diabetic cases compared to thecontrol group (Table 2). Atherosclerosis in diabetics was evi-denced by a significantly higher median value of CIMT com-pared to the control group (Table 2). Also median values of both systolic and diastolic blood pressure were significantlyhigher in diabetic cases with vascular complications comparedto those without vascular complications and control group(Table 2).Poor glycemic control noted in diabetic cases was evidencedby the significantly higher mean values of whole blood glycat-ed hemoglobin, and serum fasting glucose compared to thecontrol group. Renal affection in cases suffering from diabeticnephropathy was noted by the significantly higher medianvalue of urinary albumin to creatinine ratio in diabetics withvascular complications compared to those without vascularcomplications and control group (Table 2). The hypertensivestate present in diabetics with vascular complications couldbe an aggravating factor. Furthermore the disturbed lipid pat-tern secondary to poor glycemic control was also noted in dia-betic cases (Table 2).As regards the GSTs gene polymorphisms, the GSTP1genotypes in controls and diabetic cases as well as the totalsubjects were in agreement with Hardy Weinberg equilibrium(Table 3). No significant differences were noted between dia-betic patients with and without vascular complications andcontrol group regarding GSTM1 and GSTT1 both insertedand deleted (  p  = 0.631 and  p  = 0.832, respectively) (Table 4).The only difference noted was in the GSTP1 SNP where dia-betic cases had a lower wild genotype (AA) and a higher het-erozygous (AG) genotype compared to control group with  p -valueapproaching statistical significance (  p  = 0.053) (Table 4).Furthermore, in cases with vascular complications the GSTP1wild (AA) genotype was significantly lower and the heterozy-gous (AG) genotype was significantly higher compared tocases without vascular complications and control group(  p  = 0.023) (Table 4). When diabetic cases with vascular com-plications were categorized according to the type of vascular Table 1  Primer sequences and cycler conditions for amplification of GSTM1 and GSTT1 genes. Genes Primers Cycler conditionsGSTM1 Forward GSTM1 Initial Denaturation 95  C–5 min5 0 -GAACTCCCTGAAAAGCTAAAGC-3 0 35 Cycles Denaturation 94  C–30 sReverse Annealing 55  C–30 s5 0 -GTTGGGCTCAAATATACGGTGG-3 0 Extension 72  C–30 sFinal elongation 72  C–10 minGSTT1 Forward GSTT1 Initial Denaturation 94  C–5 min5 0 -TTCCTTACTGGTCCTCACATCTC-3 0 35 Cycles Denaturation 94  C–1 minReverse Annealing 59  C–1 min5 0 -TCACGGGATCATGGCCAGCA-3 0 Extension 72  C–1 minFinal elongation 72  C–10 minCYP1A1 Forward(Exon-7) 5 0 -GAACTGCCACTTCAGCTGTCT-3 0 Reverse5 0 -CAGCTGCATTTGGAAGTGCTC-3 0 4 M.A. Zaki et al. Please cite this article in press as: Zaki MA et al. Glutathione S-transferase M1, T1 and P1 gene polymorphisms and the risk of developing type 2diabetes mellitus in Egyptian diabetic patients with and without diabetic vascular complications,  Alex J Med   (2014), j.ajme.2014.03.003  Table 2  Some demographical and clinical characteristics, anthropometric and radiological data, as well as biochemical parametersamong the studied groups. Items Controls ( n  = 51)mean/median ± SD/Min–MaxDiabetic patients( n  = 54)mean/median ± SD/Min–Max  p -value Gender Male  n  = 16 (31.4%)  n  = 20 (37.0%)  v 2  p  = 0.541Female  n  = 35 (68.6%)  n  = 34 (63.0%)Age (years) 43.37 ± 11.07 49.96 ± 8.99  t  p 1 = 0.001 * BMI (kg/m 2 ) 27.39 ± 2.8 30.56 ± 3.97  t  p 1 < 0.001 * CIMT (cm) 0.44 (0.3–0.62) 0.58 (0.40–2.30)  MW  p 1 < 0.001 * Duration of disease (years) – 10.5 ± 5.49 – Whole blood HbA 1c  (%) 4.84 ± 0.38 8.52 ± 2.25  t  p 1 < 0.001 * Serum (Fasting) Glucose (mg/dL) 90 (74–99) 168 (78–448)  MW  p 1 < 0.001 * Creatinine (mg/dL) 0.9 (0.6–1.2) 1.0 (0.7–5.1)  MW  p 1 < 0.001 * ALT (U/L) 15 ± 7 26 ± 12  t  p 1 < 0.001 * Total cholesterol (mg/dL) 174 ± 22 207 ± 12  t  p 1 < 0.001 * HDL-cholesterol (mg/dL) 55 ± 12 44 ± 13  t  p 1 < 0.001 * LDL-cholesterol (mg/dL) 100 ± 19 130 ± 45  t  p 1 < 0.001 * Triglycerides (mg/dL) 94 ± 31 172 ± 77  t  p 1 < 0.001 * Items Control group( n  = 51) T2DM without vascularcomplications( n  = 27)T2DM with vascularcomplications( n  = 27)  p 2/  p 3/  p 4 values Blood pressure Systolic (mmHg) 115 (90–135) 130 (90–135) 140 (110–180)  t  p 2 < 0.001 *t  p 3 < 0.001 * Diastolic (mmHg) 70 (60–85) 80 (60–85) 90 (60–110)  t  p 4 < 0.001 * Urine ACR (mg/gm) 11.0(2.0–28.5) 15.7 (2.3–29.6) 60.0 (1.5–1749.0)  t  p 2 = 0.153 t  p 3 < 0.001 *t  p 4 < 0.001 *  p 1-value describes the statistical difference between control group and whole diabetic cases.  p 2-value describes the statistical difference in urinary ACR between control group and diabetic cases without vascular complications.  p 3-value describes the statistical difference in urinary ACR between control group and diabetic cases with vascular complications.  p 4-value describes the statistical difference in urinary ACR between diabetic cases without and with vascular complications. Abbreviations:  ACR, Albumin to creatinine ratio; ALT, Alanine aminotransferase; BMI, body mass index; CIMT, Carotid intima mediathickness, HbA1c, Glycated hemoglobin, HDL-C, high density lipoprotein cholesterol, LDL-C, Low density lipoprotein cholesterol. * A  p -value less than 0.001 was considered highly significant. * A  p -value less than 0.05 was considered statistically significant. Table 3  The agreement of GSTP1 genotypes with Hardy Weinberg (HW) equilibrium. Genotype Observed HW expected  x 2  p -valueNumber % Number % Control group Wild (AA) 38 74.5 37.1 72.7 1.002 0.316Heterozygous mutant (AG) 11 21.6 12.8 25.1Homozygous mutant (GG) 2 3.9 1.1 2.2 Diabetic group Wild (AA) 29 53.7 30.4 56.3 0.995 0.318Heterozygous mutant (AG) 23 42.6 20.3 37.6Homozygous mutant (GG) 2 3.7 3.4 6.1 Total subjects Wild (AA) 67 63.8 67.2 64.0 0.014 0.902Heterozygous mutant (AG) 34 32.4 33.6 32.0Homozygous mutant (GG) 4 3.8 4.2 4.0A  p -value less than 0.05 was considered statistically significant. Gene polymorphisms and risk of type 2 diabetes mellitus 5 Please cite this article in press as: Zaki MA et al. Glutathione S-transferase M1, T1 and P1 gene polymorphisms and the risk of developing type 2diabetes mellitus in Egyptian diabetic patients with and without diabetic vascular complications,  Alex J Med   (2014), j.ajme.2014.03.003
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