Development of denaturing high performance liquid chromatography (Dhplc) in detection of common polymorphisms in the multi-drug resistance (MDR1) gene in Malay patients with leukemia
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Date
2006
Authors
Yahaya, Badrul Hisham
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Abstract
Although the use of chemotherapy has evolved remarkably during the past 30 years for treatment in leukemia patients, it remains a difficult disease to treat where the main problem is drug resistance. This is due to over expression of multi-drug resistance gene (MDR1) and it’s over expression has been described in many tumors resistant to cytotoxic drugs including leukemias. This gene is polymorphic and studies in different ethnic groups have indicated that the three most frequent single nucleotide polymorphisms (SNPs) are located in exons 12, 21 and 26 of the MDR1 gene. Denaturing high performance liquid chromatography (dHPLC) has been proven to be a feasible method in detecting target DNA for presence of mutations and polymorphisms including in the MDR1 gene. Studies on polymorphisms of the MDR1 gene, however has not been carried out in patients in this institution. Thus, the main objectives of this study were to develop and establish dHPLC technique for screening of these three most frequent polymorphisms located in exons 12, 21 and 26 of the MDR1 gene, to investigate the distribution of these polymorphisms of the MDR1 gene and to compare and combine the results from dHPLC technique with restriction fragment length polymorphism (RFLP) in detection of these polymorphisms of the MDR1 gene in Malay patients with leukemia in this institution. A total of hundred and one DNA samples from Malay patients diagnosed as leukemia were studied and subjected to dHPLC analysis and RFLP analysis. Statistical analysis was done using chi-square (χ2) and Fisher Exact’s test. Out of these, there were 35 (34.7%) patients diagnosed as acute myeloid leukemia (AML), 48 (47.5%) as acute lymphoblastic leukemia (ALL) and 18 (17.8%) as chronic myeloid leukemia (CML). There were 58 (57.4%) males and 43 (42.6%) females. From the results, if all of the three exons were combined, 25 (25.7%) of the patients showed heterozygous mutation, 7 (6.7%) of them had wild type, while 2 (1.9%) showed homozygous mutation. In all of these patients, there was a significant difference in the distribution of polymorphisms between exons 21 and 26 with a p<0.05. PCR-RFLP assays had been performed to confirm the results of dHPLC technique and there were 93.2%, 96.1% and 100% of common polymorphisms detected in exons 12, 21 and 26, respectively. In this study dHPLC was successfully developed for detection of common polymorphisms in the MDR1 gene in Malay patients with leukemia in this institution. The clinical implications of the distribution of these polymorphisms especially in the response of treatment in these patients should be studied for the future.
Description
Master
Keywords
Biological science , Liquid chromatography , Polymorphisms , Leukemia