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Association of genetic polymorphism of glutathione S-transferase M1, T1, P1 and susceptibility to bladder cancer

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Association of genetic polymorphism of glutathione S-transferase M1, T1, P1 and susceptibility to bladder cancer
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  See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/7739879 Association of Genetic Polymorphism of Glutathione S-transferase M1, T1, P1 andSusceptibility to Bladder Cancer  Article   in  European Urology · September 2005 DOI: 10.1016/j.eururo.2005.02.007 · Source: PubMed CITATIONS 62 READS 61 6 authors , including: Some of the authors of this publication are also working on these related projects: Correlation of Natural Killer Cell counts in whole blood and Human Leukocyte Antigen-G expression inTumor tissue of Renal Cell Carcinoma Patients   View projectUnravel the potential therapeutic targets and novel molecular mechanism of Gastrointestinal (GI)motility disorders.   View projectDhruva Kumar MishraHouston Methodist Hospital 49   PUBLICATIONS   522   CITATIONS   SEE PROFILE Anil MandhaniSanjay Gandhi Post Graduate Institute of Me… 187   PUBLICATIONS   2,389   CITATIONS   SEE PROFILE Balraj MittalBabasaheb Bhimrao Ambedkar University 510   PUBLICATIONS   6,908   CITATIONS   SEE PROFILE All content following this page was uploaded by Daya Shankar Lal Srivastava on 26 April 2014. The user has requested enhancement of the downloaded file. All in-text references underlined in blue are added to the srcinal documentand are linked to publications on ResearchGate, letting you access and read them immediately.  TranslationalResearchFromLabtoClinic:Oncology AssociationofGeneticPolymorphismofGlutathioneS-transferaseM1,T1,P1andSusceptibilitytoBladderCancer Daya Shankar Lal Srivastava a, **, Dhruva Kumar Mishra a , Anil Mandhani a , Balraj Mittal b ,Anant Kumar a , Rama Devi Mittal a, * a  Department of Urology, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Raebareli Road, Lucknow-226014, Uttar Pradesh, India b  Department of Genetics, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Raebareli Road, Lucknow-226014, Uttar Pradesh, India Accepted 9 February 2005Available online 16 March 2005 Abstract Objective:  Glutathione- S  -transferases (GSTs) are active in the detoxification of wide variety of endogenous orexogenous carcinogens. We examined the association of the GST gene polymorphism with sporadic bladder cancerpatients in Northern India.  Material and methods:  The study constituted of 106 bladder cancer cases and 370 age-matched controls. TheGSTT1 and GSTM1 null genotypes were identified by multiplex PCR and GSTP1313 A/G by Polymerase ChainReaction/Restriction Fragment Length Polymorphism method (PCR/RFLP).  Results:  We observed non-significant association in null alleles of the GSTM1 (  p  = 0.611, OR = 1.12, 95%CI = 0.72–1.74 and GSTT1 (  p  = 0.135, OR = 1.45, 95% CI = 0.89–2.37) with risk of bladder cancer. However, theG/G genotype of the GSTP1 gene polymorphism was highly significant when compared to controls (  p  = 0.000,OR = 7.12,95% CI = 3.14–16.16).The combinedanalysis of thethree risk genotypesdemonstrated further increasein the risk of bladder cancer (  p  = 0.000, OR = 7.29 95% CI = 2.81–18.93). Conclusion:  Our study demonstrated that GSTP1313 G/G polymorphism is a strong predisposing risk factor forbladder cancer. Combination of three GST genotypes association exhibiting gene-gene interaction further sub-stantiates the increased risk of bladder cancer. # 2005 Elsevier B.V. All rights reserved. Keywords:  Glutathione  S  -transferase; Genetic polymorphism; Detoxification; Gene-gene interaction; Bladdercancer 1. Introduction Agreatmajorityofchemicalcarcinogenincapableof producing DNA damage requires activation by enzymesystem (such as Cytochrome P450) in order to becomeactive carcinogen, for tumor initiation [1]. ConverselyphaseIImetabolizingenzymesuchasGST, NAT, epox-ide hydroxylase and sulfotransferase are involved indetoxificationofchemicalcarcinogenandsubsequentlytheir role is expected to be protective [2]. Exposure totobacco smoke and other environmental and occupa-tionalchemicalshavebeen described asarisk factor forbladdercancersuchas2-nepthylamine,benzidineand4-aminobiphenyl[3].Geneticdifferenceinthemetabolism ofthesechemicalshasbeenrecentlysuggestedasmodi-fiers of individual susceptibility to environmentallyinduced bladder cancer.Cytosolic glutathione  S  -transferases (GSTs) are afamily of related isozymes that catalyze the conjuga-tion of reduced glutathione to a wide range of electro-phillic substrate [4]. Among them, most extensivelystudied are the GSTM1 null, the GSTP1313 A/G EuropeanUrology  European Urology 48 (2005) 339–344* Corresponding author. Tel. +91 522 26680 048x2116;Fax: +91 522 2668 017. E-mail addresses:  dshankarpgi@yahoo.com (D.S.L. Srivastava),rmittal@sgpgi.ac.in, ramamittal@yahoo.com (R.D. Mittal).** Co-corresponding author: Tel. +91 522 2668 700;Fax: +91 522 2668 017. 0302-2838/$ – see front matter # 2005 Elsevier B.V. All rights reserved.doi:10.1016/j.eururo.2005.02.007  substitution and the GSTT1 null polymorphism. TheGSTP1313 A/G polymorphism at the nucleotidelevel leads to an amino acid variation of isoleucine/ valine at codon 105 in the protein. Valine aminoacid results in decreased enzymes activity [5]. Toruner et al (2001), reported that GSTP1313A/G or G/Ggenotype increases 1.75 fold of bladder cancer sus-ceptibility [6].Individuals with GSTT1 null genotype showreduced ability to detoxify metabolites of 1, 3 buta-diene and ethylene oxide [7,8]. GSTM1 de fi ciencysuspected to increased risk of bladder cancer was  fi rstreported by Bell et al, 1993 [9], a number of studies appearedwithsupporting fi nding[10,11].Incontrastto GSTM1, potential role of GSTT1 genotype in indivi-dual susceptibility to bladder cancer have beenreported with inconsistent results. A few studies exhib-ited non-signi fi cant decreased risk of bladder cancerwith GSTT1 null genotype [12,13] however; someother studies exhibited increased risk of bladder cancerwithGSTT1nullgenotype[14,15]. Inconsistentresults have also emerged from recent studies exploring thecombined effect of GSTM1 and GSTT1 genotype inthe development of malignancy [14,16]. Although promising data from these studies are accumulatingat aremarkable pace, they are still too sparse to supporta role for a speci fi c gene in bladder cancer risk. Recentstudy suggests that the frequencies of some poly-morphisms in certain genes differ among differentracial and ethnic groups [17]. Whether these geneticvariantscanhelpexplainpartofthelargedifferencesinbladder cancer risk between populations await furtherclari fi cation.In the present study, we determined the genotypicfrequency oftheGSTM1 null, GSTT1nullpolymorph-ism and GSTP1313 A/G in bladder cancer patients tosubstantiate its association with bladder cancer in thenorth Indian population. 2. Materialsandmethods 2.1. Patients The study group consisted of 106 bladder cancer patients (allwere transitional cell carcinoma; out of them 60% were stage Itumors, 28% were stage II, 3% were stage III and 9% were stage IVtumors) with mean age 55.5  1.38 and 370 normal healthyindividuals (controls) with mean age of 51.8  0.59. Ethnic srcinof cases and controls were same. Criteria for the patient selectionwere based on a questionnaire covering medical, pathological, andhisto-pathological records from the outpatient department of San- jay Gandhi postgraduate Institute of Medical Science, Lucknow.The inclusion criteria for the controls were absence of prior historyof cancer or pre-cancerous lesions. 2.2. DNA extraction and genotyping Five ml of blood was collected in EDTA vials from cases andcontrols. DNA was extracted from blood lymphocytes using theProteinase K and phenol chloroform extraction procedure [18].Analysis for GSTM1 and GSTT1 gene polymorphism was done bymultiplex PCR as described by Rehman et al. [19]. Isolated DNA (100 – 150 ng) was ampli fi ed in a total volume of 25 m l reactionmixture containing 20 pmol of each of the following primers.GSTM1:Forward, 5 0 -GAACTCCCTGAAAAGCTAAAGC-3 0 andReverse, 5 0 -GTTGGGCTCAAATATACGGTGG-3 0 ;GSTT1:Forward, 5 0 -TTCCTTACTGGTCCTCACATCTC-3 0 ;Reverse, 5 0 -TCACGGGATCATGGCCAG CA-3 0 .Exon 7 of CYP1A1 genes were co-ampli fi ed and used as aninternal control using following primers:Forward, 5 0 -GAACTGCCACTTCAGCTGTCT-3 0 andReverse, 5 0 CAGCTGCATTTGGAAGTGCTC-3 0 .Each set of reaction included both positive and negative con-trols.The multiplexPCR method was usedtodetect the presence orabsence of the GSTT1 and GSTM1 genes in the genomic DNAsamples, simultaneously in the same tube. The reaction mixturewassubjectedtoinitialdenaturationat94  8 Cfor2 min,followedby35 cycles of 94  8 C for 2 min, 59  8 C for 1 min and 72  8 C for 1 min.The  fi nal extension was done at 72  8 C for 10 min. The PCRproducts were eletrophoresed in 2% agarose gels, and visualizedby ethidium bromide staining. DNA from samples positive forGSTM1 and GSTT1 genotypes yielded bands of 215 bp and480 bprespectively while the internal positive control (CYP1A1) PCRproduct corresponded to 312 bp.GSTP1A313G polymorphism was analyzed using a previouslydescribed PCR-RFLP method [20]. Brie fl y, ampli fi cation wasconceded using primers:Forward, 5 0 -ACCCCAGGGCTCTATGGGAA-3 0 andReverse, 5 0 -TGAGGGCACAAGAAGCCCCT-3 0 .The 176-bp ampli fi ed product was digested with Alw261 andeletrophoresed on 3% Agarose gel. Presence of restriction siteresulting in only two fragments (91 bp and 85 bp) indicated mutantallele (G/G), and if A/G polymorphism incurred then it resulted inthree fragments of 176 bp, 91 bp and 85 bp. 2.3. Statistical analysis Statistical analysis was done with SPSS software 11.5 version.Binary Logistic Regression Model (BLRM) assessed difference ingenotype prevalence and association between case and controlgroup. Multivariate analysis, correlation coef  fi cient, odds ratio(OR) and its 95% con fi dence interval (CI) were used to describethe strength of association.  p -value  < 0.05 was considered asstatistically signi fi cant for the study.  D.S.L. Srivastava et al./European Urology 48 (2005) 339–344 340  3. Results Table 1: (see also Figs. 1 and 2) presents the frequencies of GSTM1, GSTT1 and GSTP1 allelesand genotypes by case-control status and the associa-tion of GSTs genotypes with bladder cancer risk. Inthe control samples, frequency of GSTM1 nulland GSTT1 null was 36.9 and 20.7 respectively.In GSTP1 polymorphism A allele (51.6%) while Gallele was 3.5%; homozygous and the remaining44.9% were heterozygous. Genotype distributions incontrols were in agreement with Hardy  – WeinbergEquilibrium.We observed non-signi fi cant association with nullgenotype of GSTM1 (OR = 1.12, 95% CI = 0.72 – 1.74,  p  = 0.611) and GSTT1 (OR = 1.45, 95% CI = 0.89 – 2.37,  p  = 0.135). However, the A/G genotype(OR = 1.99, 95% CI = 1.23 – 3.20,  p  = 0.005) and G/ G genotype (OR = 7.12, 95% CI = 3.14 – 16.16,  p  = 0.000) of the GSTP1 gene polymorphism werehighly signi fi cant as compared to the controls(Table 1).Combination of the two high-risk genotypesGSTM1 null and GSTT1 null or G allele of GSTP1genotype revealed that the risk increased up to 2.63times (OR = 2.63, 95% CI: 1.39 – 4.98;  p  = 0.003) forGSTP1and GSTM1 and1.9 times(OR = 1.90,95% CI:0.98 – 3.69;  p  = 0.059) for GSTM1 and GSTT1 geno-type. However, risk increased more than 2.91 folds(OR = 2.91, 95% CI: 1.41 – 6.01;  p  = 0.004) for GSTT1and GSTP1 genotype when compared with non-risk genotypes (Table 2).We further investigated the risk associated with allthe three high-risk GST genotypes compared to non-risk genotypes (positive genotypes of GSTM1 andGSTT1 and 313 A/A genotype of GSTP1 were desig-nated as the reference group). The OR for the threehigh-risk genotypes verses non-risk genotypes was7.29 folds higher (  p  = 0.000, OR = 7.29 95%CI = 2.81 – 18.93) (Table 3, Figs. 1 and 2). Theclinicalstageoftumorwascategorizedintofourgroups:T1(Ta + T1), T2,T3andT4.Weobserved non-signi fi cant association ( P > 0.05) for the GSTs geno-types with stage of bladder tumor (Fig. 3).  D.S.L. Srivastava et al./European Urology 48 (2005) 339  –  344  341 Table1 Distribution of GST genotypes among bladder cancer patients and controlGenotype Controls  n  = 370 Patients (Ca-B)  n  = 106  p -value OR (95% CI)GSTM1Present 230 (62.2%) 63 (59.4%) 1.0 (Ref.)Null 140 (37.8%) 43 (40.6%) 0.611 1.12 (0.72 – 1.74)GSTT1Present 291 (78.6%) 78 (73.6%) 1.0 (Ref.)Null 79 (21.4%) 28 (26.4%) 0.135 1.45 (0.89 – 2.37)GSTP1Ile/Ile 191 (51.6%) 33 (31.1%) 1.0 (Ref.)Ile/Val 166 (44.9%) 58 (54.7%) 0.005 1.98 (1.23 – 3.20)Val/Val 13 (3.5%) 15 (14.2%) 0.000 7.12 (3.14 – 16.16) Table2 Distribution of doubles GST genotypes among bladder cancer patients and controlsGenotypes Controls  n  = 370 Patients (Ca-B)  n  = 106  p -value OR (95% CI)GSTM1and GSTT1Both Present 182 (49.2%) 51 (48.1%) 1.09 (Ref.)Either Null 156 (42.2%) 38 (35.9%) 0.560 0.86 (0.54 – 1.39)Both Null 32 (8.6%) 17 (16.0%) 0.059 1.90 (0.98 – 3.69)GSTM1 and GSTP1M1 (+/+)&P1 (Ile/Ile) 123 (33.2%) 20 (18.8%) 1.0 (Ref.)M1 (+/+)&P1 (Ile/Val/or Val/Val) 109 (29.5%) 43 (40.6%) 0.003 2.43 (1.34 – 4.37)M1 (   /   )&P1 (Ile/Ile) 68 (18.4%) 13 (12.3%) 0.676 1.18 (0.55 – 2.51)M1 (   /   )&P1 (Ile/Val/or Val/Val) 70 (18.9%) 30 (28.3%) 0.003 2.63 (1.39- 4.98)GSTT1 and GSTP1T1 (+/+)&P1 (Ile/Ile) 151 (40.7%) 21 (19.8%) 1.0 (Ref.)T1 (+/+)&P1 (Ile/Val/or Val/Val) 139 (37.6%) 56 (52.8%) 0.000 2.89 (1.67- 5.03)T1 (   /   )&P1 (Ile/Ile) 38 (10.3%) 12 (11.3%) 0.043 2.27 (1.03- 5.02)T1 (   /   )&P1 (Ile/Val/or Val/Val) 42 (11.4%) 17 (16.1%) 0.004 2.91 (1.41-6.01)  We also classi fi ed pathological  fi ndings of gradeinto three groups: well differentiated (Grade1), mod-erately differentiated (Grade2) and poorly differen-tiated (Grade3). Correlations of GSTs genotypeswith tumor grade of the disease were also insigni fi cantfor the risk of bladder cancer (Fig. 4). 4. Discussion We observed an increased risk for bladder cancerassociatedwith theGSTP1A/Gor G/G polymorphism.Our observation is in accord with  fi ndings in Turkish[6] and British population [20], where they observed signi fi cant increased risk of bladder cancer for GSTP1A/G or G/G polymorphism. However, Steinhoff et al.[21] in German and Katoh et al. [22] in Japanese population observed no association for GSTP1 A/Gor G/G genotype with susceptibility to bladder cancer.The present study indicates that polymorphic GSTP1locus may be an important factor for genetic suscept-ibility to bladder cancer. Mechanism behind this asso-ciation suggests that person having G allele of GSTP1undergo lower activity towards detoxi fi cation of car-cinogeniccompoundsduetoinactiveformofenzymes.Inthepresentstudy,atendencyofincreasedriskwasobserved with GSTT1 null genotypes, although thisdifference was not statistically signi fi cant for bladdercancer when compared with the controls. Our  fi ndings  D.S.L. Srivastava et al./European Urology 48 (2005) 339  –  344 342 Table3 Distribution of triple GST genotypes among bladder cancer patients and controlsGenotypes (GSTM1, GSTT1 and GSTP1) Controls  n  = 370 Patients (Ca-B)  n  = 106  p -value OR (95% CI)M1& T1 (+/+)&P1 (Ile/Ile) 101 (27.3%) 12 (11.3%) 1.0M1& T1 (+/+)&P1 (Ile/Val/or Val/Val) 82 (22.2%) 40 (37.7%) 0.000 4.11 (2.02 – 8.33)M1 (   /   ), T1 (+/+)&P1 (Ile/Ile) 52 (14.1%) 9 (8.5%) 0.428 1.46 (0.58 – 3.68)M1 (   /   ), T1 (+/+)&P1 (Ile/Val/or Val/Val) 56 (15.1%) 16 (15.1%) 0.035 2.41 (1.06 – 5.94)M1 (+/+), T1 (   /   )&P1 (Ile/Ile) 21 (5.7%) 8 (7.5%) 0.024 3.21 (1.17 – 8.81)M1 (+/+), T1 (   /   )&P1 (Ile/Val/or Val/Val) 26 (7.0%) 4 (3.8%) 0.676 1.29 (0.39 – 4.35)M1 (   /   ), T1 (   /   )&P1 (Ile/Ile) 17 (4.6%) 4 (3.8%) 0.281 1.98 (0.57 – 6.86)M1 (   /   ), T1 (   /   ) &P1 (Ile/Val/or Val/Val) 15 (4.5%) 13 (12.3%) 0.000 7.29 (2.81 – 18.93)Fig. 1. Lane M: 100 bp ladder; lane 1: negative controls, lane 7 depicts M1and T1 null alleles; lane 2, 3, 9 signify M1 and T1 positive, lane 5 is M1positive and lanes 4, 6, 8 are T1 positive.Fig. 2. Lane M: 100 bp ladder; lane 1, 3 wild type (Ile/Ile); lanes 2, 4, 5, 6heterozygous (Ile/Val) and lane 7 indicate the mutant type (Val/Val)genotypes.Fig.3. S1 = stage1 (Ta and T1),S2 = stage2and S3 – S4 = stage 3and stage4 of bladder tumor.Fig. 4. G1 = grade1, G2 = grade 2 and G3 = grade 3 of bladder tumor.
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