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Association of an MDR1 Gene (C3435T) Polymorphism with Acute Leukemia RESEARCH COMMUNICATION Association of an MDR1 Gene (C3435T) Polymorphism with Acute Leukemia in India D Nageswara Rao1, C Anuradha1, S Vishnupriya1*, K Sailaja1, D Surekha1, D Raghunadharao2, Senthil Rajappa2 Abstract The multidrug resistance (MDR1) gene product P-glycoprotein is a membrane bound protein that functions as an ATP-dependent efflux pump, transporting exogenous and endogenous substrates from the cells. Since it plays an important role in chemotherapy, there is an increasing interest in the possible significance of genetic variation in MDR1. Our main objective was to study the MDR1gene polymorphism at C3435T with reference to development and progression of acute leukemia. The present study included 290 acute leukemia cases, comprising of 147 acute lymphocytic leukemia (ALL), 143 acute myeloid leukemia and 249 age-sex matched control samples for the analysis of MDR1 C3435T polymorphism, by the PCR-RFLP method. The MDR1 genotype distribution revealed an elevated frequency of the TT genotype in ALL cases (51.7%) as compared to controls (28.9%), whereas AML group did not show any association. The mean white blood cell count, blast% and LDH levels were increased in ALL patients with the CC genotype. No deviation was observed with respect to haemoglobin, platelet count and disease free survival in ALL patients. The association of CC genotype with clinical variables in ALL indicated that the CC genotype with high expression might be eliminating antileukemic drugs (anthracyclines, Daunorubicin, Vincristeine, Mitoxanthrone) which are P-gp substrates, leading to lower intra cellular drug concentrations and a poor prognosis. Such an association with the CC genotype was not observed in AML. In conclusion, these results suggested that the MDR1 TT genotype might influence risk of development of acute lympoblastic leukemia and the CC genotype might be linked to a poor prognosis of ALL. Keywords: MDR1 gene - ALL - AML - C3435T polymorphism - PCR-RFLP Asian Pacific J Cancer Prev, 11, 1063-1066 Introduction The human multi drug resistance gene (MDR1), located on chromosome 7q21, comprises 28 exons and encodes a 170 KD integral membrane protein product P-glycoprotein (Pgp), which belongs to the ATP binding cassette (ABC) super family of transporters resides in the plasma membrane and functions as an efflux transporter of a wide variety of natural compounds and lipophilic xenobiotics (Lin et al., 2003). P-gp seemed to have a role in transporting phospholipids across the cell membrane and might function as an antiapoptotic molecule. P-gp could inhibit the activation of down stream caspases 8 and 3 conferring resistance to apoptosis. Moreover P-gp conferred resistance to cell lysis induced by activated compliment factor (Johonstone et al., 2000). Over expression of Pgp in cancer cells causes a decrease in drug accumulation, thereby mediating cellular resistance to a variety of anti-cancer agents, whereas reduced expression leads to accumulation of carcinogens. P-gp drug substrates include anthracyclines and epidophyllotoxins that had been shown to be efficacious in the treatment of AML (Bradshaw et al., 1998). A reduced intra cellular concentration of cytotoxic drugs attributable to the action of P-gp in AML blasts may therefore be related to resistant disease and failure of AML therapy. Indeed 30% of younger patients (<60 years) and 50% of older patients (>60 years) suffering from AML have been shown to express readily detectable levels of P-gp on their blast cells (Leith et al., 1994; Chauncey et al., 2000). Since P-gp plays pivotal role in protecting cells from harmful chemicals and active metabolites, it might be plausible that this expression might contribute significantly in cellular response to stress stimuli. In the promoter region of MDR1 gene, the putative binding sites for various transcription factors were identified. Hence, these sites may be targets of environmental signaling. These evidences clearly point out the possibilities of MDR1 gene regulation by a myriad of environmental signals. Further evidence had indicated that protein kinase C activation which increases P-gp activity also exhibit positive impact on MDR1 gene expression. The three most frequent SNPs in the Caucasian population observed were in exons 12, 21 and 26 at nucleotide positions 1236, 2677, 1Department of Genetics, Osmania University, 2Department of Medical Oncology, Nizams Institute of Medical Sciences, Hyderabad, India. * For correspondence : sattivishnupriya@gmail.com Asian Pacific Journal of Cancer Prevention, Vol 11, 2010 1063 D Nageswara Rao et al and 3435. Moreover, it was shown in healthy volunteers that these changes were in linkage disequilibrium and might therefore be associated with transcriptional regulation of MDR1 mRNA (Kim et al., 2001). The MDR1 gene polymorphism in exon 26 at 3435 C>T position is a synonymous polymorphism located near nucleotide binding site of transmembrane P-gp. This synonymous polymorphism (C3435) could affect the function of the P-gp protein by various mechanisms like alterations in MDR1 mRNA folding and stability. Materials and Methods The present study includes 290 acute leukemia cases comprising of 147 acute lymphocytic leukemia (ALL), 143 acute myeloid leukemia (AML) and 249 control samples. Blood samples were collected from freshly diagnosed patients with acute leukemia who were reporting at NIMS (Nizams Institute of Medical Sciences), Hyderabad. The age and sex matched control samples were randomly selected from different areas of Andhra pradesh. Patient's clinical data like WBC count, blast%, platelet count, Hb, LDH, complete remission (CR) response to therapy and period of disease free survival (DFS) was noted from the tumor registry files with the help of medical oncologist during followup. Genomic DNA was isolated by using salting-out method (Lahiri and Nurnberger, 1991) and used for genotyping of MDR C3435T polymorphism through PCR-RFLP analysis. The present study has been planned to identify the association of MDR1 C3435T polymorphism with the development of ALL, AML and their progression. Genotyping of the MDR1 C3435T polymorphism MDR1 C3435T polymorphism was analyzed through PCR-RFLP (restriction fragment polymorphism length polymorphism) method. 244bp fragment was amplified using specific primer sequences: Forward: 5'-GAT CTG TGA ACT CTT GTT TTC A-3''Reverse: 5'- GAA GAG AGA CTT ACA TTA GGC-3' The 25l PCR reaction mixture consists of approximately 100-150ng of genomic DNA, 15pmol/l of each primer, 200mol/l of dNTPs, 20 mmol/l of Tris HCl, 50 mM of KCl, 2.5mmol/l of MgCl2, 1 U of Taq DNA polymerase and deionized water (varied). The PCR Cycling Conditions include initial denaturation at 94C for 5 minutes followed by 35 cycles of denaturation at 94C for 30 seconds, annealing at 55C for 30 seconds, extension at 72C for 1 minute and final Table 1. Estimation of Relative Risk for ALL and AML Associated with MDR1 C3435T Genotype Genotype Control (n=249) ALL Cases Value (n=147) 2 P Frequency Genotype (n, %) CC 49 (19.7) CT 128 (51.4) TT 72 (28.9) Allele C 0.45 T 0.55 18 (12.2) 53 (36.1) 76 (51.7) 0.3 0.7 20.624# 0.00 Odds Ratios ORs (95%CI) CC vs CT CT vs TT CC vs TT 0.88 0.39* 0.37* 0.47-1.65 0.25-0.62 0.21-0.66 # 2 value significant; *Odds ratios significant AML Cases Value (n=143) 32 (22.4) 73 (51.0) 38 (26.6) 0.48 0.52 ORs 1.14 1.08 1.24 2 0.550 (95%CI) 0.67-1.95 0.67-0.18 0.68-2.24 P 0.779 Table 2. MDR1 C3435T Polymorphism with Respect to Demography of Sex of the Proband with Acute Leukemia Genotype distribution with respect to Sex of the proband in ALL ALL Genotype Frequency CC CT TT n%n%n% Males 12 12.0 32 32.0 56 56.0 Females 6 12 21 44.7 20 42.6 2=2.56; df =2, p=0.27 OR (CI 95%) : CC vs CT : 1.3125 ( 0.4265 to 4.0388 ) OR (CI 95%) : CT Vs TT : 0.5442 (0.2569 to 1.153 ) OR (CI 95%) : CC Vs TT : 0.7143 ( 0.2366 to 2.1567 ) Total 100 47 Allele Frequency CT 0.28 0.72 0.35 0.65 Genotype distribution with respect to Sex of the proband in AML AML Genotype Frequency CC CT TT n%n%n% Males 18 20.5 43 48.9 27 30.7 Females 14 26.4 29 54.7 10 18.9 2=2.49; df=2, p=0.28 OR (CI 95%) : CC vs CT :0.8671 (0.3734 to 2.0133) OR (CI 95%) : CT Vs TT : 0.5492 (0.2312 to 1.3044) OR (CI 95%) : CC Vs TT : 0.4762 (0.1739 to 1.3037) 1064 Asian Pacific Journal of Cancer Prevention, Vol 11, 2010 Total 88 53 Allele Frequency CT 0.45 0.55 0.54 0.46 Association of an MDR1 Gene (C3435T) Polymorphism with Acute Leukemia Table 3. MDR1 C3435T Polymorphism with Respect to Demography of Age at Onset with Acute Leukemia Genotype distribution with respect to Age at onset in ALL ALL <10 10-20 >20 Genotype Frequency CC n% 6 14.3 6 9.5 6 14.3 CT TT n%n% 17 40.5 19 45.2 22 34.9 35 55.6 14 33.3 22 52.4 2=1.52; df=4, p=0.82 Total 42 63 42 Allele Frequency CT 0.35 0.65 0.27 0.73 0.31 0.69 Genotype distribution with respect to Age at onset in AML AML <20 20-30 >30 CC n% 4 16.7 13 23.6 15 24.2 Genotype Frequency CT TT n%n% 11 45.8 9 37.5 30 54.5 12 21.8 31 50.0 16 25.8 2=2.31; df=4, p=0.67 Total 24 55 62 Allele Frequency CT 0.39 0.61 0.51 0.49 0.49 0.51 Table 4. Mean Values of Clinical Variables with Respect to MDR1 C3435T Polymorphism in ALL and AML Cases Clinical Variable ALL AML Mean Age n MeanS.E n CC 18 18.893.37 32 CT 53 15.571.32 72 TT 76 15.121.01 37 147 141 Mean WBC(thousands) CC 18 74.2317.88 32 CT 53 53.8212.80 72 TT 76 49.096.11 37 147 141 Mean Blast% CC 18 63.896.74# 32 CT 53 47.324.56 72 TT 76 47.113.83 37 Mean Platelet(lakhs) CC CT 147 18 53 0.870.21 1.060.10 141 32 72 TT 76 0.680.06 37 147 141 Mean HB CC 18 8.600.71 32 CT 53 8.720.40 72 TT 76 8.980.25 37 147 141 Mean LDH CC 18 1007.6170.27* 32 CT 53 677.5351.15 72 TT 76 819.7580.97 37 147 141 Mean DFS CC 17 27.654.83 22 CT 47 26.132.34 36 TT 71 28.072.42 26 135 84 t test values: # as compared with CT or TT, p<0.05; * as compared with CT, p<0.05 Mean S.E 34.633.24 32.331.73 29.782.22 37.049.71 61.029.82 45.5312.50 59.255.05 59.533.20 66.733.69 0.980.23 0.850.13 1.390.25 8.420.37 8.200.28 8.590.47 431.8458.20 504.045.81 475.5747.5 11.591.60 11.941.78 10.921.56 extension at 72C for 5 minutes. After amplification, PCR products were subjected for restriction digestion using DpnII enzyme (New England Biolabs USA) for 1 hour at 55C. For 3435-C allele, two fragments of sizes 172 bp and 72 bp were obtained whereas a fragment of size 244 was observed for 3435Tallel. After digestion, the products were electrophoresed on a 3% agarose gel for genotyping. Statistical Analysis All the statistical analyses were performed with SPSS 15.0 (Statistical Package for the Social Science). Chi square test was done to test the significance of genotype association with the occurrence of acute leukemia and its prognosis. All the P values were two sided and the level Asian Pacific Journal of Cancer Prevention, Vol 11, 2010 1065 D Nageswara Rao et al of significance was taken as P <0.05. Results and Discussion In the present study 3435 TT genotype of MDR1 was significantly associated with ALL as compared to controls. The frequency of 3435 TT genotype was significantly elevated in ALL (51.7%) as compared to controls (28.9%). Similar significant association was not observed with AML (Table 1). This indicated that MDR1 C3435 TT polymorphism might confer an increased risk for developing acute lymphoblastic leukemia. 3435 TT genotype with low P-gp expression levels had less efficiency to efflux the toxins, resulting in higher intracellular concentrations of mutagens or toxins leading to DNA damage and accumulation of mutations resulting in disease progression. Previous studies had also reported an increased risk of acute lymphoblastic leukemia (ALL) associated with TT genotype, possibly due to a quantitative change in the MDR1 gene expression resulting from genetic polymorphisms (Jamroziak et al., 2004; Hattori et al., 2007).When the distribution of genotype frequencies were analyzed with respect to sex of the proband, MDR1 TT genotype was associated with male sex in ALL and AML patients (56.0%, 30.7%) (Table 2). MDR1 TT genotype frequency was found to be increased with age at onset more than 10 yrs in ALL and with age at onset <20yrs in AML (Table 3). With respect to clinical variables Mean WBC, mean blast% and mean LDH levels were increased in ALL patients with CC genotype. The association of CC genotype with clinical variables in ALL indicated that CC genotype with high expression might be eliminating the antileukemic drugs (anthracyclines, Daunorubicin, Vincristeine, Mitoxanthrone) which are P-gp substrates more effectively leading to low intra cellular drug concentration and poor prognosis. No such association was observed in AML, indicating MDR1 C3435 polymorphism did not appear to have significant clinical implications in AML which was in accordance with earlier report (Hur et al., 2008). In conclusion these results suggested that MDR1 TT genotype might confer risk to development of acute lympoblastic leukemia and CC genotype with poor prognosis. a southwest oncology group. Leuk Res, 24, 567-74. Hattori H, Suminoe, Wada M, et al (2007). Regulatory polymorphisms of multidrug resistance 1 (MDR1) gene are associated with the development of childhood acute lymphoblastic leukemia. Leuk Res, 31, 1633-40. Hur E-H, Lee J-H, Lee MJ, et al (2008). C3435T polymorphism of the MDR1 gene is not associated with P-glycoprotein function of leukemic blasts and clinical outcome in patients with acute myeloid leukemia. Leuk Res, 32, 1601-4. Jamroziak K, Balcerczak E, Cebula B, et al (2006). No influence of 3435C>T ABCB1 (MDR1) gene polymorphism on risk of adult acute myeloid leukemia and P-glycoprotein expression in blast cells. Ther Drug Monit, 28, 707-11. Johnstone RW, Ruefli AA, Smyth MJ (2000). Multiple physiological functions for multidrug transporter P-glycoprotein?. Trends Biochem Sci, 25, 1-6. Kim RB, Leake BF, Choo EF, et al (2001). Identification of functionally variant MDR1 alleles among European Americans and African Americans. Clin Pharmacol Ther. 70, 189-99. Lahiri DK, Nurnberger JI (1991). A rapid non-enzymatic method for the preparation of HMW DNA from blood for RFLP studies. Nucleic Acids Res, 19, 5444 Lin JH, Yamazaki M (2003). Role of p-glycoprotein in pharmacokinetics: clinical implications. Clin Pharmacokinet, 42, 59-98. Leith CP, Kopecky KJ, Chen IM, et al (1999). Frequency and clinical significance of the expression of the multidrug resistance proteins MDR1/P-glycoprotein, MRP1, and LRP in acute myeloid leukemia: A southwest oncology group study. Blood, 94, 1086-99. Acknowledgements This work was supported by Department of Medical Oncology, Nizams Institute of Medical Sciences, Hyderabad, India. References Bradshaw DM, Arceci RJ (1998). Clinical relevance of transmembrane drug efflux as a mechanism of multidrug resistance. J Clin Oncol, 16, 3674-90. Chauncey TR, Rankin C, Anderson JE, et al (2000). A phase I study of induction chemotherapy for older patients with newly diagnosed acute myeloid leukemia (AML) using mitoxantrone, etoposide, and the MDR modulator PSC 833: 1066 Asian Pacific Journal of Cancer Prevention, Vol 11, 2010