Evaluation of C 3435 T MDR 1 Gene Polymorphism in Adult Patient with Acute Lymphoblastic Leukemia

Background: P-glycoprotein (P-gp) is a transmembrane pump encoded by MDR1 gene. It contributes in protection of the cells against xenobiotic and toxic compounds. P-gp also contributes in multidrug resistance in acute lymphoblastic leukemia (ALL). Human MDR1 polymorphism include C to T exchange at position 3435, individuals with TT polymorphism have lower expression of P-gp than the others with CC genotypes. Accumulation of xenobiotics in the cells can cause some diseases like cancers. Materials and Methods: To evaluate MDR1C3435T gene polymorphism in adult patients with ALL, 54 patients and 96 healthy controls were involved in our survey. Genotyping of ALL patients and healthy controls was performed by Polymerase Chain Reaction – Restriction FragmentLength Polymorphism (PCR-RFLP). Data analysis was done by SPSS software through T-test and ChiSquare. Results: No significant difference was found between C3435T MDR1 gene polymorphisms and incidence of ALL in adult patients. Also there was not any significant difference between T and C alleles and incidence of ALL. Conclusion: C3435T MDR1 polymorphism is not associated with the incidence of ALL in the population studied. Keyword: P-gp, C3435T, MDR1, ALL, polymorphism 1. Department of Biochemistry, Fasa University of Medical Sciences 2. Department of Pathology, Fasa University of Medical Sciences 3. Department of Biochemistry, Fasa University of Medical Sciences 4. Department of Pathology, Fasa University of Medical Sciences Correspondence: Behnoosh Miladpoor, Dept. of Biochemistry, Fasa University of Medical Sciences. Email: behnooshmiladpoor@gmail.com Tel: 00987312220994, Fax: 00987312216300 resistance – 1 (MDR1) gene, also known as adenosine triphosphate – binding cassette subfamily B transporter – 1 (ABCB1), is one of the most interesting candidates for pharmacogenetic studies.6 MDR1 gene encodes P – glycoprotein (P –gp), which belong to the family of ABC transporter proteins. P–gp is a membrane-associated protein, which acts as an ATP – dependent pump taking part in the transmembrane transport of many xenobiotics, including steroid, anthracyclines and Vinca alkaloids that are important in chemotherapy of adult ALL.7,8 The functional role of P – gp as an efflux pump has been well established, relating to drug transport across cellular membranes, thus preventing their intracellular accumulation. Differences in the P –gp activity may reflect genetic polymorphism.7,8 Human MDR1 polymorphism has been described and correlated with potential clinical effects. This polymorphism is a C to T exchange at position 3435 T polymorphism significantly correlated with changes in the expression and activity of P – gp.9 Individuals with the T/T genotype had significantly lower duodenal MDR1 expression than those with the C/C genotype. The difference in P–gp levels between two groups was greater than 65–fold, and affected the pharmacokinetics of the glycoprotein substrate, i.e. digoxin.9 Previous studies showed a predisposing role for MDR1 C3435T gene polymorphism in children with ALL.10,11 It is supposed that the lower expression of P-gp can cause accumulation of xenobiotics and toxic compounds in the cell and results in predisposition to diseases such as cancers. On the other hand higher expression of P-gp, in the patients, may worsen their prognosis because of its functional effects in effluxing chemotherapeutic agents from the cell membrane. Accordingly, we tried to evaluate C3435T MDR1gene polymorphisms in adult patients with ALL. Materials and Methods 54 patients with ALL and 96 healthy people comprising the control group -of Iranian originparticipated in our study. All patients were referred to Vali-e-Asr Hospital, affiliated to Fasa University of Medical Sciences, Fasa, Fars, Iran. After explaining the objectives of the research program to the subjects and obtained written permission from them, we took the subjects’ demographic information including name, age, sex, living area, family history of cancer (first and second generation). The patients with other types of cancer and those below the age limit (≥ 20 years) of the study were excluded. The control group was chosen from healthy people with corresponding demographic information were referred to the hospital lab for the check up. Then, they were asked if there was any history of family cancer in their first and second generation. If they had, they would have been excluded from the study. Three ml whole blood was collected from the enrolling subjects and genotyping of ALL patients and healthy controls was performed by Polymerase Chain Reaction – Restriction FragmentLength Polymorphism (PCR-RFLP). DNA was isolated from peripheral blood cells. For a 50μl PCR, the reaction contained 200 ng genomic DNA, 200 mmol/ l of each of dNTPs (dATP, dCTP, dGTP and dTTP), 250 ng of each primer [5'-ACT CTT GTT TTC AGC TGC TTG-3' and 5'AGA GAC TTA ACT TAG GCA GTG ACT-3', (Microsynth, Swiss)], 1.5 mmol/l magnesium chloride and 1 U Taq DNA polymerase (Fermentas, Litwini). The primer design was based on published sequences for genotyping procedure of MDR1 polymorphism using genomic DNA (10). PCR amplification consisted of an initial 5 min denaturation at 94oC, followed by 35 cycles of denaturation at 94oC for90 S, annealing at 60oC for 30 S, and extension at 72oC for 30 S. Amplified DNA fragments (206 bp) were digested by MboI enzyme (Fermentas, USA) for 24h in 37oC. After digestion DNA fragments of 130 and 76 bp for homozygous wild type C allele or one 206 bp band for homozygous mutant T allele, and three bands of 206, 130 and 76 bp for heterozygous CT genotype were expected. Statistical analysis of the data was performed by the SPSS software.The Chi-square tests, odd ratio (OR) with 95% CI was calculated when P < 0.05. Results: 54 patients with ALL of both sexes (29 male, 25 female) and 96 healthy controls (49 male, 47 female) of Iranian origin participated in our study. The mean age in the two groups was almost the same (41±6.22 for patients and 44±5.85 for the control group). The clinical factor like white blood cell count (WBC ≥30000, <30000) at diagnosis were measured. We found 18.5% of TT genotype and 59.30% of TC genotype and 22.20% of CC genotype among patients with ALL. However, the control group showed 16.7% of TT genotype and 62.5% of TC genotype and 20.80% of CC genotype (Table-I). DNA was digested with MboI enzyme and DNA fragments detected after electrophoresis. No significant difference was found among different genotypes in groups after PCR-RFLP (Table-II). We found no significant difference between two groups alleles (P = 0.969). Moreover, the association of MDR1 gene C3435T polymorphism with parameters included WBC at diagnosis and sex were evaluated. However, no significant difference was found between these parameters and different genotypes (P = 0.39, P = 0.99 respectively). Table-I The genotypes frequency distribution in groups Groups Genotypes Alleles TT TC CC Total C T Total N (%) N (%) N (%) N (%) N (%) N (%) N (%) Patient 10(18.50) 32(59.30) 12(22.20) 54(100) 56 52 108 Control 16(16.70) 60(62.5) 20(20.80) 96(100) 100 92 192 Total 144 156 150(100) 32(21.30) 92(61.30) 26(17.30) According to chi-square test there was not any significant difference between T and C alleles in cases and controls (p = 0.969) Evaluation of C3435T MDR1 Gene Polymorphism in Adult Patient with Acute Lymphoblastic Leukemia JM Vol. 12, No. 1