Molecular Study of BCR-ABLGene and Genetic Variations in Chronic Myeloid Leukemia Patients in Iraq

number: 
2715
إنجليزية
Degree: 
Imprint: 
Biotechnology
Author: 
Yasameen Ali Hadi AL-Yasiri
Supervisor: 
Dr. Saleh Ahmed wohaieb
Dr. Bilal Kamil Sulaiman
year: 
2011
Abstract:

This study is conducted to investigate the possible antileuckemic activity of ethanolic olive leaves extract against a group of patients with chronic myeloid leukemia (CML), diagnosed using Real Time-PCR. The response of those individuals to the treatment with Imatinib and the response of selected individuals to the treatment with olive leaves ethanolic extract were followed up using same method. Selected individuals of the chronic myeloid leukemia patients ere investigated for the genetic instability using Random Amplified Polymorphisum DNA-Polymerase chain reaction. Blood samples collected from 48 CML patients and 12 apparent healthy individual. Mononuclear cells (MNCs) isolated from both patients and controls using ficoll, while RNA was extracted using the method of Trizol. The RNA quantity and quality have been detected using spectrophotometer and a garose gel electrophoresis, respectively. Extracted RNA was used for synthesis of cDNA, to be used in the Real Time –PCR. Real Time-PCR results showed the presence of BCR-ABL fusion gene forming the P210 protein in 43 out of 48 CML patients. The other 5 patients may have expressed another type of fusion genes. The active compounds in the olive leaves ethanolic extract were detected and analyzed using chemical methods and results showed the presence of flavonoids, terrpens, glycosides and steroids while no alkaloid appeared in the extract. Three different concentrations (800, 1200, and 1450) μg/ml of the olive leaves extract applied on the MNCs of CML patients and controls, and incubated at 37˚C for 48 hr. Then the treated cells were recollected and used for the RNA extraction followed by Real Time-PCR to verify the presence of any change in the quantity of the expressed fusion gene in comparison with values obtained prior to treatment with OLE. Results showed no significant differences between the expression levels of the treated cells with that measured before the treatment for the three concentrations. Because genetic instability is a known phenomenon associated with the development of cancer, RAPD-PCR is used in the study to investigate the presence of any genetic instability for group of CML patients and group of normal controls. Twelve universal primers selected for the above mentioned purpose, 5 of them were already selected based on preliminary experiments. The results of RAPD-PCR revealed that some primers produced distinguished patterns in a group of patients with unique bands. This finding deserves further studies to assess its clinical significance, and whether or not they can be validated as a potential genetic marker for diagnosis of CML, or to assess the CML patient's response to antileukemic therapy.