Assessment of Exon 2 and 3 Mutations in NPM1 Gene and Studying the Gene Expression of MDR1Gene in some Acute Myeloid Leukemia Patients in Iraq

Mutations in NPM1 gene ranked within the most frequent known genetic marker in AML. In order to detect the frequency of NPM1 mutations within the region of exon 2 and exon 3 in Iraqi AML patients using conventional polymerized chain reaction technique, the region from 2380 bp to 4140 bp of NPM1 gene,which represents the region flanking both exon 2 and 3, was amplified by using two specific primers in a thermal cycler. Results showed that the concentration of genomic DNA after extraction was ranged between (52-93) µg/ml with purity of (1.8-1.92). On the other hand results of amplification showed that an amplified product of NPMX1 primer was 1150 bp while the second primer NPMX2 was 1337 bp, both were represented the complete fragment of  NPM1  gene within exon 2 and 3. To investigate the possible mutations in NPM1 gene, the nucleotide sequence of the amplified product was determined. Results showed that three single nucleotide polymorphism SNP 1  T G/A792, G/A794 and G/A797 were detected
within intron region in 90% of patients and 70% of healthy volunteers.1T During the analysis of NPM1 gene sequencing, single nucleotide variant was identify in exon 3 only in 70% of AML patients A/G 1275 (rs 753788683). In addition SNP was observed in two sites G/A635 and G/A660 within intron region of  80% patients 1T but not in healthy controls.1T No genetic variation observed in  exon 2 during
sequences analysis of amplified NPM1 gene. The correlation between MDR1 gene over expression and resistance to chemotherapy treatment in acute leukemia was investigated. Blood samples were
collected from 40 AML patients and 10 healthy volunteers. The RNA was extracted from all samples and used as template for c DNA synthesis. The result showed that the concentration was ranged from (2.02-2.13) ng/ul and the purity from (1.90-1.95), the extracted RNA was reverse transcripted into cDNA. The  40 AML patients were categorized dependent on their chemotherapy induction, newly
diagnosed (No treatment started), first course chemotherapy induction, second chemotherapy induction and patients in consolidation. Their results showed that no significant differences in gene expression occurred for the newly diagnosed patients and first course induction patients as compared with controls. Nevertheless significant decreases in CT value were recorded for both the second induction AML group and AML consolidation group with p value of  0.0001and 0.0001, respectively, as compared with healthy controls, indicating the induction for higher expression of MDR1 gene by increasing the challenge of AML patients with chemotherapy regime.