Deletion of GSTM 1 and T 1 Genes as a Risk Factor for Development of Acute Leukemia

The glutathione S-transferases (GSTs) are a family of enzymes involved in the detoxification of a wide range of chemicals, including important environmental carcinogens, as well as chemotherapeutic agents. In the present study 294 acute leukemia cases, comprising 152 of acute lymphocytic leukemia (ALL) and 142 of acute myeloid leukemia, and 251 control samples were analyzed for GSTM1 and GSTT1 polymorphisms through multiplex PCR methods. Significantly increased frequencies of GSTM1 null genotype (M0), GSTT1 null genotype (T0) and GST double null genotype (T0M0) were observed in the both ALL and AML cases as compared to controls. When data were analyzed with respect to clinical variables, increased mean levels of WBC, Blast %, LDH and significant reduction in DFS were observed in both ALL and AML cases with T0 genotype. In conclusion, absence of both GST M and GST T might confer increased risk of developing ALL or AML. The absence of GST enzyme might lead to oxidative stress and subsequent DNA damage resulting in genomic instability, a hallmark of acute leukemia. The GST enzyme deficiency might also exert impact on clinical prognosis leading to poorer DFS. Hence GST genotyping can be made mandatory in management of acute leukemia so that more aggressive therapy such as allogenic stem cell transplantation may be planned in the case of patients with a null genotype.


Introduction
Glutathione S transferases (GSTs), super family of dimeric phase II metabolizing enzymes, play an important role in the cellular defense system.GST enzymes catalyze the conjugation of toxic and carcinogenic electrophilic molecules with glutathione and thereby protect cellular macromolecules from damage (Boyer et al., 1985).Thus GST enzymes regulate cytotoxicity of a variety of chemotherapeutic drugs (Hoban et al., 1992).Glutathione S-transferases (GSTs) constitute a family of enzymes encoded by five gene families μ, θ, π, α, σ which are involved in phase II metabolism and implicated in the detoxification of a broad range of compounds, including xenobiotics, pesticides, environmental carcinogens, PAH, and some chemotherapeutic drugs (including alkylating agents, Doxorubicin, and Vincristein).Functional polymorphisms have been reported in at least three of the genes that code for GSTs including GSTM1, GSTT1, and GSTP.Both GSTT1, and GSTM1 genes, exhibited a greater degree of polymorphism, one of them being the complete deletion of the gene that causes the loss of enzymatic activity (Alves et al., 2002).20-50 % of individuals do not express the enzyme due to homozygous deletion and are more susceptible to DNA damage caused 1 School of Chemical & Biotechnology, SASTRA University, Thanjavur, 2 Department of Genetics, Osmania University, 3 Department of Medical Oncology, Nizams Institute of Medical Sciences, Hyderabad, India *For correspondence: sattivishnupriya@gmail.com

Deletion of GSTM1 and T1 Genes as a Risk Factor for Development of Acute Leukemia
Nageswara Rao Dunna 1 , Sugunakar Vure 2 , K Sailaja 2 , D Surekha 2 , D Raghunadharao 3 , Senthil Rajappa 3 , S Vishnupriya 2 * by PAH and other mutagens (Strange et al., 2001).The GST gene family might modulate leukemia risk via two potential mechanisms either by mediating the metabolism of specific leukemogens or by directly affecting the redox potential within the cell, protecting DNA from free radicalinduced damage.
Polymorphisms within the GST genes were found to be associated with susceptibility to non malignant and malignant diseases including AML, (Alves et al., 2002).Patients with a GSTs null genotype were believed to exhibit impaired detoxification of environmental genotoxic agents and chemotherapeutic drugs leading to an increased risk of developing primary and secondary cancers and treatment related complications indicating GST polymorphism might contribute to the susceptibility to t-AML/t-MDS.Children carrying the GSTM1 null genotype were reported to be at increased risk of developing ALL (Krajinovic et al., 1999;Saadat et al., 2000).Crump et al. (2000) reported no association between the GSTT1, GSTM1 gene deletions and AML.Patients with secondary AML had a slightly higher prevalence of the GSTT1 and GSTM1 gene deletions compared with denovo AML patients.Over representation of GSTM1 null homozygous genotype in the ALL samples (68.1%) was observed when compared to the control population (49%).The GSTM1 null genotype was found to be correlated with an increased risk of malignancy (Alves et al., 2002).The null GSTM1 genotype could be associated with increased risk of acute leukemia.Furthermore, GSTM1 and GSTT1 null genotypes were apparently related to response, drug side effects and prognosis of patients with AML.The present study attempts to identify the role of GSTM1, T1 null genotypes in the development of acute leukemia.

Materials and Methods
294 primary acute leukemia cases comprising of 152 acute lymphocytic leukemia (ALL), 142 acute myeloid leukemia (AML) being treated at NIMS (Nizams Institute of Medical Sciences), Hyderabad were selected for the present study.The age and sex matched control samples were randomly selected from different locations in Hyderabad.Patient's clinical data like WBC count, blast%, platelet count, Hb, LDH, complete remission rate (CR) and disease free survival rate (DFS) was noted from the tumor registry file with the help of medical oncologist.Blood samples from both patients and control group were collected into EDTA vacutainers.Genomic DNA was isolated by using salting-out method (Nuremberg and Lahari, 1991).

Genotyping of GSTM1 and GSTT1 polymorphism
PCR was performed using 150-200ng of genomic DNA, 20 pmol/l of each primer (see Table 1), 200µmol/l of dNTPs, 20 mmol/l of Tris HCl, 50 mM of KCl, 2.5 mmol/l of MgCl2, 1U of Taq DNA polymerase.The PCR cycling conditions consisted of initial denaturation at 94 0 C for 3 minutes followed by 35 cycles of denaturation at 94 0 C for1minute, annealing at 60 0 C for 1 minute, extension at 72 0 C for 2 minutes and final extension at 72 0 C for 5 minutes.Based on the presence or absence of 219bp and 480bp (see Figure 1), the genotypes were determined as M1T1, M0T0, M1T0 and M0T1.

Statistical analysis
All the statistical analyses were performed with Statistical Package for the Social Science (SPSS) 15.0.Chi square test was calculated to test the significance of genotype association with the occurrence of acute leukemia and its prognosis.t-test was done to test the significance of association of clinical variables All the p values were two sided and the level of significance was taken as P<0.05.

Results
In the present study, significantly increased frequencies of GST M and T null genotypes were observed in the both ALL and AML patients as compared to controls (Tables 2  and 3) which indicated that GST null genotypes confer risk to develop acute leukemia This could be due to inefficient detoxification polycyclic aromatic compounds (PAH), environmental pollutants and other mutagens leading to DNA damage (Norappa et al., 2004).GSTT1 null status was linked to an increased frequency of diepoxy butane induced sister chromatid exchange in culture lymphocytes (Wiencke et al., 1995).The genotype frequencies of GST M0, T0 and M0T0 did not show association with the sex of the proband in both ALL and AML (Table 4).
With respect to age at onset, increase in the frequency of M0 null genotype was observed in ALL patients with late age at onset of >20 years (67.4%) and in AML patients with early onset <30 years as compared to corresponding age groups.However, double null genotype frequency (M0T0) was elevated in ALL females as compared to ALL males.There was no significant variation in clinical variables of ALL and AML patients with M0 genotype.But patients with T0 genotype had significant leukocytosis, increased blast % and reduction in mean DFS.When both patients with deletion of both GST M and T were analyzed (GSTM0T0) for various clinical parameters, the results were similar to those observed with respect of T0 genotype indicating that absence GST is associated with poor prognosis.This might be due to inefficient metabolism of chemotherapeutic agents leading to lack of drug response.Further, it may be observed that patients with both M and T alleles (M1T1) exhibited favorable clinical parameters when compared to those with M0T0 genotype.The deletion of M or T genes is significantly associated with reduction in disease free survival rate indicating the importance of GST enzymes in the metabolism of chemotherapy agents.The data on CR failed to reveal any significant contribution with GST gene deletion which could be due to limited available data on CR.
It was reported that the adult AML patients with GSTM null genotype had a trend towards a poorer survival than those with M1 allele, but no such effects for GSTT1 and GSTP genotypes were reported (Autrup et al., 2002).Barragan et al. (2007) reported the probability of DFS was significantly diminished in patients with GSTM null genotype compared to patients with undeleted GSTM1.The absence of GSTM enzyme (GSTM0) might predispose to leukemia and also influence the clinical variables specially associated with reduced disease survival.Zhijin et al. (2008) reported that AML Patients with deletions of GSTM1 or GSTT or both had a lower probability to achieve CR on induction therapy and shorter survival as compared to patients with intact GST genes.In a systemic review and Meta analysis of 30 published case control studies, it was suggested that GSTM1 and GSTT, polymorphism appeared to be associated with a modest increase the risk of acute lymphoblastic leukemia (Zhang et al., 2005).Voso et al. (2009) also reported that GSTT1 null genotype and GSTM1 null genotype predict or poor response chemotherapy and in consequently to shorter overall survival (OS) in adult AML patients.

Discussion
In conclusion, absence of both GST M & GST T might confer risk to develop ALL or AML.The absence of GST enzyme might lead to oxidative stress and subsequently DNA damage resulting in genomic instability, the hall mark of acute leukemia.The GST enzyme deficiencies might also exert impact on clinical prognosis leading to poorer DFS.Hence the GST genotyping can be made mandatory in management of acute leukemia so that more aggressive therapy such as allogenic stem cell transplantation can be planned in the case of patients null genotype.

Table 3 . Genotype Distribution of GST M0 T0 Polymorphism and Sex and GST M0T0 polymorphism and Age at onset in Acute Leukemia
*p<0.05 is significant Figure 1.Gel Photograph of GST1M1 Polymorphism Asian Pacific Journal of Cancer Prevention, Vol 14, 2013 2223