c Indian Academy of Sciences

ONLINE RESOURCES

Association of GSTP1 gene (I105V) polymorphism with acute leukaemia

NAGESWARA RAO DUNNA1 , SUGUNAKAR VUREE2 , SAILAJA KAGITA2 , D. SUREKHA2 , RAGHUNADHARAO DIGUMARTI3 , SENTHIL RAJAPPA3 and VISHNUPRIYA SATTI2
1

School of Chemical and Biotechnology, SASTRA University, Tirumalaisamudram 613 401, India 2 Department of Genetics, Osmania University, Hyderabad 500 007, India 3 Nizam Intitute of Medical Sciences, Panjagutta, Hyderabad 500 082, India

[Dunna N. R., Vuree S., Kagita S., Surekha D., Digumarti R., Rajappa S. and Satti V. 2012 Association of GSTP1 gene (I105V) polymorphism with acute leukaemia. J. Genet. 91, e60e63. Online only: http://www.ias.ac.in/jgenet/OnlineResources/91/e60.pdf]

Introduction
Glutathione S-transferase P1 (GSTP1) enzyme plays a key role in biotransformation and bioactivation of certain environmental pollutants such as benzo[a]pyrene-7, 8-diol-9, 10-epoxide (BPDE) and other diol epoxides of polycyclic aromatic hydrocarbons (Hengstler et al. 1998) and catalyses detoxication of base propanols that arise from DNA oxidation thus offering cellular protection against oxidative stress. GSTP1 gene belongs to the pi class gene family, located on chromosome 11q13 (Autrup 2000). It comprises of seven exons (Morrow et al. 1989; Bora et al. 1997) and codes for cytosolic GST enzyme (Fryer et al. 1986). The rst polymorphism identied is an AG polymorphism at nucleotide 313 in exon 5 of GSTP1 gene which leads to an amino acid substitution of isoleucine (IE) by valine (val) at 105 amino acid position (Ile105Val). This substitution results in three GSTP1 genotypes: they are isoleucine/isoleucine (Ile/Ile) homozygous wildtype, isoleucine/valine (Ile/Val) heterozygote and valine/valine (Val/Val) homozygous variant. GSTP1 codon 105 polymorphism might play an important role in leukaemiogenesis, as it potentially alters protein function, diminishing its detoxication ability for certain mutagens and carcinogens, which could result in increased DNA damage and mutation, and a greater risk of developing cancer. Biochemical studies indicated that GSTP1 Val105 allele has a lower thermal stability than GSTP1 Ile105 allele (Zimniak et al. 1994; Johanson et al. 1998), and Val homozygotes had a lower conjugating activity than Ile homozygotes, with heterozygotes displaying intermediate activity (Watson et al. 1998). Individuals with at least one Val allele at codon 105 of GSTP1 enzyme might have an underlying predisposition to cancer when exposed to environmentally derived
For correspondence. E-mail: vishnupriyamadam@gmail.com.

or endogenously formed GSTP1 substrates (Harries et al. 1997). Indeed, the GSTP1 codon 105Val allele was associated with a signicantly increased risk of lung, bladder, and testicular cancer (Harries et al. 1997; Ryberg et al. 1997). Individuals with at least one GSTP1 codon 105Val allele were signicantly overrepresented in therapy related acute myeloid leukaemia (t-AML) cases compared with de novo AML cases. These data suggested that inheritance of at least one Val allele at GSTP1 codon 105 confers a significantly increased risk of developing t-AML after cytotoxic chemotherapy, but not after radiotherapy (Allan et al. 2001). In view of this, we planned to identify the association of GSTP1 gene (Ile105Val) polymorphism with the development and progression of acute leukaemia.

Materials and methods

The present study includes 290 acute leukaemia cases comprising of 147 acute lymphocytic leukaemia (ALL), 143 acute myeloid leukaemia (AML), 248 healthy age and sex matched individuals without family history of leukaemia or any other cancers were selected to serve as control group. Five mL of blood sample were collected into EDTA vacutainers from acute leukaemia patients being reported at NIMS (Nizams Institute of Medical Sciences), Hyderabad, either before starting chemotherapy or after remission. The age and sex matched control samples were randomly selected from different areas of the local population. Patients clinical data like WBC count, blast%, platelet count, Hb, LDH, complete remission (CR) response to therapy and disease free survival (DFS) were noted from the tumour registry le with the help of medical oncologist. Genomic DNA was isolated using salting-out method (Nuremberg and Lahari 1991)

Keywords. glutathione S-transferase; acute myeloid leukaemia; acute lymphoblastic leukaemia; restriction fragment length polymorphism; human genetics.
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GSTP1 variants and leukaemia risk

Table 1. Genotype distribution of GSTP1 polymorphism in acute leukaemia and controls. Genotype frequency GSTP1 ALL AML Total Controls I/I no. (%) 56 (38.1) 52 (36.4) 108 (37.2) 140 (56.5) I/V no. (%) 75 (51.0) 64 (44.8) 139 (47.9) 105 (42.3) V/V no. (%) 16 (10.9) 27 (18.9) 43 (14.8) 3 (1.2) Total 147 143 290 248 Allele frequency I 0.64 0.59 0.61 0.78 V 0.36 0.41 0.39 0.22

ALL versus controls 2 -25.753; df-2, P-0.000*; AML versus controls 2 -44.492; df-2, P-0.000*; cases versus controls 2 -40.618; df-2, P-0.000*. Hardy-Weinberg equilibrium for i) ALL cases 2 -1.5; ii) AML cases 2 -0.84; iii) total cases 2 -0; controls 2 -11.86* OR, odds ratios; ALL, disease versus controls I/I versus I/V (OR) 0.562 95% CI (0.368 to 0.859)*; I/V versus V/V (OR) 0.1817, 95% CI (0.0705 to 0.4681)*; I/I versus V/V (OR) 0.0832, 95% CI (0.392 to 0.225)*; AML disease versus controls: I/I versus I/V (OR) 0.6106, 95% CI (0.392 to 0.950)*; I/V versus V/V (OR) 0.123, 95%) CI (0.056 to 0.269*); I/I versus V/V (OR) 0.065, 95% CI (0.029 to 0.145)*. Table 2. GSTP1 polymorphism and sex in AML group. Genotype frequency AML Males Females I/I no. (%) 35 (39.8) 16 (30.2) I/V no. (%) 39 (44.3) 24 (45.3) V/V no. (%) 14 (15.9) 13 (24.5) Total 88 53 Allele frequency I 0.619 0.528 V 0.381 0.472

2 -2.130; df-2, (P-0.345); OR (CI 95%), II versus IV : 1.3462 (from 0.6171 to 2.9368); OR (CI 95%), IV versus VV : 1.5089 (from 0.6072 to 3.7494); OR (CI 95%), II versus VV : 2.0313 (from 0.7785 to 5.3001). Table 3. GSTP1 polymorphism and age at onset in AML group. Genotype frequency AML < 20 2030 > 30 I/I no. (%) 5 (19.2) 27 (50.9) 19 (30.6) I/V no. (%) 14 (53.8) 17 (32.1) 32 (51.6) V/V no. (%) 7 (26.9) 9 (17.0) 11 (17.7) Total 26 53 62 Allele frequency I 0.462 0.670 0.565 V 0.538 0.330 0.435

2 -9.839; df-4, (P-0.05).

Table 4. Mean values of clinical variables with respect to GSTP1 polymorphism in ALL group. I/I Clinical variables Mean age Mean WBC (thousand) Mean blast% Mean platelet count (lakhs) Mean HB Mean LDH Mean DFS *P < 0.05 is signicant.
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I/V n 56 56 56 56 56 56 51 Mean SE 15.33 1.14 52.75 8.75 50.31 3.87 0.87 0.08 8.77 0.29 804.71 75.77 26.35 1.91 n 75 75 75 75 75 75 68

V/V Mean SE 13.69 2.18 * 97.35 28.44 58.44 7.76 0.80 0.16 8.66 .76 913.81 179.4 24.25 5.51 n 16 16 16 16 16 16 16 Total 147 147 147 147 147 147 135

Mean SE 16.52 1.37 46.06 6.39 51.07 4.44 0.87 0.09 9.04 0.37 773.48 99.11 29.27 3.06

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Nageswara Rao Dunna et al. and used for genotyping of GSTP1 Ile105Val polymorphism through PCR-RFLP analysis.
Genotyping of GSTP1 polymorphism (Ile105Val) Table 6. GSTP1 polymorphism and complete remission rates in ALL and AML groups. GSTP1 IL/IL n ALL CR + VE CR VE AML CR + VE CR VE % n IL/Val % Val/Val n % Total

Genomic DNA was used to amplify 436-bp fragment using specic primer sequences: forward: 5 -GTA TTT TGC CCA AGG TCA AG-3 reverse: 5 -AGC CAC CTG AGG GGT AAG-3 . The 25 L PCR reaction mixture consisted of approximately 100150 ng of genomic DNA, 15 pmol/L of each primer, 200 mol/L of dNTPs, 20 mmol/L of Tris HCl, 50 mM of KCl, 2.5 mmol/L of MgCl2 , 0.5 U of Taq DNA polymerase. The PCR cycling conditions include initial denaturation at 94 C for 5 min followed by 35 cycles of denaturation at 94 C for 1 min, annealing at 60 C for 2 min, extension at 72 C for 3 min and nal extension at 72 C for 5 min. After amplication, PCR products were subjected to restriction digestion using of BsmA1enzyme (MBI Fermentas, Glen Burnie, Maryland, USA). The samples were genotyped on 3% agarose gel electrophoresis. Isoleucine variant at 105 position produced two fragments (329 and 107 bp) and valine variant at 105 position produced three fragments (216, 113 and 107 bp).
Statistical analysis

51 3

38.1 60.0

68 1

50.7 20.0

15 1

11.2 20.0

134 5

2 -1.840; df-2, (P-0.399) 27 14 42.9 35.9 27 18 42.9 46.2 9 7 14.3 17.9 63 39

2 -0.556; df-2, (P-0.757)

Statistical package for social sciences v15.0 was used for data analysis SPSS (IBM, SPSS Inc., Chicago, USA). The allele and genotype frequencies were calculated by direct counting. Differences in genotype frequency distribution between patients and controls were studied using 22 contingency 2 test, and 2 test for heterogeneity. Students t-test was performed to test the signicance of association of clinical variables with the risk of acute leukaemia. All the P values were two sided and the level of signicance was taken as P < 0.05.

Results and discussion

GSTP1 was the rst isoenzyme shown to inhibit the c-Jun NH2 -terminal kinase (JNK) complex and to play a role in

the damage induced by reactive oxygen species due to oxidative or chemical stress (Alder et al. 1999). In our study, signicant elevation of GSTP1 Val/Val genotype frequency was observed in both ALL and AML patients, compared to controls (table 1), indicating that this genotype might confer risk to develop acute leukaemia (P < 0.001). Val/Val genotype is known to be associated with defective detoxication of base propanols that arise from DNA oxidation thus interfering with cellular protection against oxidative stress. Several studies had also reported signicant association of valine allele with susceptibility to develop tumours of bladder, breast, lung, and multiple myeloma (Ryberg et al. 1997; Helzlsouer et al. 1998; Maggini et al. 2008). Previous studies showed that GSTP1 105 val genotype had been associated with favourable prognosis following chemotherapy with drugs known to be GSTP1 substrates in a variety of malignancies such as paediatric acute lymphoblastic leukaemia, breast and colon cancers (Stanulla et al. 2000; Sweeney et al. 2000; Dasgupta et al. 2003). With respect to sex of the proband GSTP1 Val/Val genotype frequency was increased in female AML patients as compared to male patients, whereas sex association was not observed in ALL patients (table 2). Val/Val genotype was also associated with early onset of AML (<20 years)

Table 5. Mean values of clinical variables with respect to GSTP1 polymorphism in AML group. I/I Clinical variables Mean age Mean WBC (thousand) Mean blast% Mean platelet count (lakhs) Mean HB Mean LDH Mean DFS *P < 0.05 is signicant.
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I/V n 51 51 51 51 51 51 34 Mean SE 34.11 2.15 53.09 9.11 58.83 3.39 0.80 0.14 8.31 0.31 476.38 40.96 11.43 1.50 n 63 63 63 63 63 63 35

V/V Mean SE 28.81 2.61 74.36 17.47* 70.67 4.29* 1.48 0.31* 8.16 0.52 501.74 54.71 7.29 1.51* n 27 27 27 27 27 27 14 Total 141 141 141 141 141 141 83

Mean SE 30.88 2.00 37.37 9.85 56.08 4.01 1.05 0.17 8.41 0.33 472.65 57.55 13.79 1.73

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GSTP1 variants and leukaemia risk (table 3). The mean WBC count and LDH levels were substantially increased in ALL and AML with Val/Val genotype which was also associated with reduction in DFS (tables 4 and 5). There was no association between GSTP1 polymorphism with the rate of complete remission failure indicating lack of genotype effect on response to remission induction therapy (table 6). In conclusion, Val/Val genotype might be considered as risk genotype for developing ALL and AML and was associated with poor prognosis.
Acknowledgement This work was supported by Department of Medical Oncology, Nizams Institute of Medical Sciences, Hyderabad, India. to bladder, testicular and prostate cancer. Carcinogenesis 18, 641644. Helzlsouer K. J., Selmin O., Huang H.-Y., Strickland P. T., Hoffman S., Alberg A. J. et al. 1998 Association between gilutathione S-transferase M1,P1, and T1 genetic polymorphisms and development of breast cancer 1. J. Natl. Cancer Inst. 90, 512518. Hengstler J. G., Arand M., Herrero M. E. and Oesch F. 1998 Polymorphism of N-acetyltransferases, glutathione S-transferases, microsomal epoxide hydrolase and sulfotransferases: inuence on cancer susceptibility. Recent Results Cancer Res. 154, 4785. Johanson A. S., Stenberg G., Widersten M. and Mannervik B. 1998 Structure-activity relationships and thermal stability of human glutathione S-tranferase P1-1 governed by the H-site residue 105. Mol. Biol. 278, 687698. Maggini V., Buda G., Galimberti S., Martino A., Orciuolo E., Marabito F. et al. 2008 Lack of association of NQO1 and GSTP1 polymorphisams with multiple myeloma risk. Leuk. Res. 32, 988990. Morrow C. S., Cowan K. H. and Goldsmith M. E. 1989 Structure of the human genomic glutathione S-transferase-pi gene. Gene 75, 311. Nuremberg J. and Lahari Jr D. K. 1991 A rapid non-enzymatic method for the preparation of HMW from blood RFLP studies. Nucleic Acid Res. 19, 5444. Ryberg D., Skaug V., Hewer A., Phillips D. H., Harries L. W., Wolf C. R. et al. 1997 Genotypes of glutathione transferase M1 and P1 and their signicance for lung DNA adduct levels and cancer risk. Carcinogenesis 18, 12851289. Stanulla M., Schrappe M., Brechlin A. M., Zimmermann M. and Welte K. 2000 Polymorphism with in glutathione S-trasferase genes (GSTM1,GSTT1GSTP1) and risk of relapse in childhood B-cell precursor acute lymphoblastic leukemia: A case controle study. Blood 95, 12221228. Sweeney C., McClure G. Y., Fares M. Y., Stone A., Coles B. F., Thompson P. A. et al. 2000 Association between survival after treatment for breast cancer and glutathione S-transferase P1 Ile105Val polymorphism. Cancer Res. 60, 56215624. Watson M. A., Stewart R. K., Smith G. B. J., Massey T. E. and Bell D. A. 1998 Human gluthione S-transferase P1 polymorphisms:relationship to lung tissue enzsyme activity and population frequency distribution. Carcinogenesis 19, 275280. Zimniak P., Nanduri B., Pilula S. and Bandorowiczpikula J. 1994 Naturally occurring human glutathione S-transferase GSTP1.1 isoforms with isoleucine and valine at position 104 differ in enzymatic properties. Eur. J. Biochem. 224, 893899.

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Received 5 February 2011, in nal revised form 22 December 2011; accepted 2 January 2012 Published on the Web: 17 April 2012

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