Genetic Mutation Assessment of NPM1 Gene and Gene Expression of MDR1 in Iraqi Patients with Acute Myeloid Leukemia

Corresponding Author: Ahmed Abduljabbar Suleiman Department of Biology, University of Anbar, College of Science, Iraq Tell: +964 7904774532 Email: ahmed.suleiman@uoanbar.edu.iq Abstract: Since its discovery, mutations in NPM1 have been frequently associated with a large number of Acute Myeloid Leukemia (AML) patients. The vast majority of genetic changes were previously detected in exon 12, however little information report mutations in Nuclear Export Signal region (NES) of NPM1. Sequencing analysis included exon 2 and 3 for 75 Iraqi AML patients showed three SNPs, G/A792, G/A794 and G/A797 were detected within intron region of 90% AML and 70% healthy subjects. Other SNPs were only detected in AML subjects in which single nucleotide variant was identified in exon 3 of 70% AML subjects (A/G1275 rs753788683) in addition, two SNPs (G/A635 and G/A660) within intron region of 80% of AML subjects were detected. No genetic variation observed inexon 2 of amplified NPM1 gene. The correlation between MDR1 gene over expression and resistance to chemotherapy treatment showed no significant differences in gene expression between newly diagnosed and first-course induction subjects. Nevertheless, significant decreases in CT value were recorded for both the second induction AML and AML consolidation patients with p value of 0.0258 and 0.0007, respectively, as compared with healthy controls, indicating the induction of higher expression of MDR1 gene by increasing the challenge of AML patients with chemotherapy regimen.


Introduction
Acute Myeloid Leukemia (AML) is a type of heterogeneous disorder with a diversity of genetic abnormalities and cytogenetic alterations that provide a significant disease prognosis and determining therapy responsiveness (Liu et al., 2007). AML resulted from a series of frequent hematopoietic stem cell genetic aberration accumulated with age (Ding et al., 2012). Childhood AML generally occurs de novo (Hamed et al., 2011).
Nucleophosmin 1 (NPM1) mutations are most frequently identified (25-30%) in AML patients, with female predominance. These mutations lead to the abnormal expression of NPM1, releasing the protein in the cytoplasm rather than the nucleus and stimulating myeloid proliferation and leukemia development (Falini et al., 2007). NPM1 gene consists of 12 exons and located at chromosome 5q35. The NP Mmolecule contains distinct domains responsible for its multiple biochemical functions (Hingorani et al., 2000). The traffic of NPM between the nucleus and cytoplasm is highly regulated and mostly the regulation comes through the Nuclear Localization Signal (NLS) and Nuclear Export Signal (NES) motifs. NLS directs NPM from the cytoplasm to the nucleoplasm, which then translocates to the nucleolus through its nucleolar binding domain, mainly tryptophan residues 288 and 290 (Yu et al., 2006). NPM remains in nucleoli, though it contains highly conserved hydrophobic leucine-rich NES was encoded by exon 2 and 3 motifs within residues 94-102 and 42-49, which drive it out of the nucleus (Wang et al., 2005;Yu et al., 2006).
Most studied NPM1 mutations were restricted to exon 12. All variants cause alterations at the Cterminus of the NPM leukemic mutants, within exon 12 region and this alteration is responsible for NPM dislocation into the cytoplasm, due to changes of tryptophan (s) 288 and 290 and creation of an additional NES. Thisprotein tends to be stable and accumulated in the cytoplasm (Suzuki et al., 2005). Limited studies were conducted in detecting mutations in NPM1 gene within exon 2 and/or exon 3 of AML patients. Both exons encoded for the N-terminal portion of NPM1 protein which plays a major role in protein oligomerization and chaperone activities (Herrera et al., 1996) also this region encode the Nuclear Export Signals (NES) that prove protein accumulation in the cytoplasm (Kazem et al., 2011).
Within Iraqi population, leukemia considered one of the most common types of cancer after breast cancer and lung cancer. The annual incidence of leukemia in Iraq is about 728 per 10,000 and it affects males 57% more than females 43%. As far as the prevalence is concerned, leukemia is the third most prevalent form of cancer after breast cancer in Iraqi population with annual significant increases each year (ICB, 2014).
On the other hand, treatment of leukemia considered as a challenge due to the development of resistance to chemotherapeutic agents. Assessing the drug resistance of leukemic cells is, therefore, an important aspect of treatment. One of the main mechanisms of resistance is rapid drug efflux mediated by various members of the ATP-binding cassette transporter super family, such as multidrug resistance gene 1 (MDR1), which encodes Pglycoprotein (Schaich et al., 2005). Therefore, the current study was aimed to detect possible mutation(s) within exon 2 and 3 in NPM1 gene and monitoring the expression of MDR1 gene by comparing the expression before and after the exposure to chemotherapy.

Patients and Samples
The study consisted of 75 Iraqi AML patients (45 male and 30 female), all patients were above 18 years, with median age of 33. Peripheral Blood samples (3-5 mL) were collected from the patients during the period starting from December 2015 to March 2016, which were admitted to Baghdad Teaching Hospital (Baghdad-Iraq). All patients were diagnosed with AML based on updated French-American-British (FAB) classification (Vardiman et al., 2009).
Most patients were treated with chemotherapy. The induction chemotherapy regimens were, combined cytarabine plus adriamycin or combined vincristine plus doxorubicin or daunorubicin and All-Trans Retinoic Acid (ATRA) plus induction chemotherapy, depended on the subtype of AML. Accordingly, patients were categorized into four groups, newly diagnosed (No treatment started) 15 patients; 20 AML patients after first-course induction of chemotherapy; 20 AML patients after second induction and 20 AML patients in consolidation. In addition, another 25 blood samples were collected from healthy volunteers and considered as controls. The current study was approved by the Board of Baghdad Teaching Hospital and the College of Biotechnology at Al-Nahrain University.
Screening for NPM1 Gene Mutations DNA was extracted from both patients and health volunteers using SYNC™ DNA Extraction Kit (Genead, South Korea). Primers used for NPM1 gene amplification were: Gene mutations flanking the region start from 2380 to 4140 pb of NPM1 gene ( Fig. 1), which represent exons 2 and 3, were analyzed by amplifying the indicated region of the gene and analyzed on agarose gel (2%) in presence of 100 bp DNA ladder marker, followed by direct sequencing technique (Macrogen, South Korea). Sequencing products were compared with the information in gene bank of the National Center for Biotechnology Information (NCBI) for standard NPM1 gene, using Mega software, version 6.

MDR1 Expression in AML Patients
The expression of MDR1 transcripts was estimated by Real Time-qPCR. Primers were designed and supplied by BioNeer (South Korea) in a lyophilized form in the following sequence: MDR1F5'-GGAGGCCAACATACATGCCT-3', MDR1R5'-CAG GGCTTCTTGGACAACCT-3'. Lyophilized primers were dissolved in a free DNase/RNase water to give a final concentration of 100 pmol µL −1 ; it was stored at -20°C until use. In addition a working solution of 50 pmol µL −1 of oligo (dT) primer was prepared using deionized distilled water. The real-time PCR experiment was designed in singleplex assay, the chemistry used in real-time qPCR was DNA-binding dyes SYBR Green1.
RNA extraction was carried out by using Total RNA Mini Kit (Blood/Culture Cell) Protocol System (Geneaid, South Korea). The purity of RNA was measured at 260/280 nm absorbance using Nanodrop. While Reverse transcription of total RNA to cDNA was achieved using RT-PCR PreMix kit (Bioneer, South Korea). Real-Time qPCR was carried out according to the designed amplification program.

Data and Statistical Analysis
Relative expression of MDR1 gene in AML patient and healthy control was calculated using the equation: Ratio = 2-(∆∆ CT) (Livak, 2001). Statistical analysis was carried out using Graph Pad Prism (version 6). One way analysis of variance ANOVA (Duncan) and Chi-square were performed to test whether group variance were significant or not. Data were expressed as mean ± standard error. P-values were considered to be statistically significant if p≤0.01.

NPM1 Gene Amplification
The region of NPM1 was amplified from the extracted DNA of both AML patients and healthy controls. Results illustrated in Fig. 2 shows the amplified products appeared as clear bands after electrophoresis on agarose gel (2%). The product of NPMX1 primer ( Fig. 2A) was a single clear band with molecular size of 1150 bp, this product represents about 2380 to 3447 bp of amplified region and cover exon 2 from NPM1 gene, while the product of NPMX2 primer ( Fig. 2B) was also a single band with molecular size of 1337bp, which represent 2707 to 4140bp of amplified region that covered exon 3.

NPM1 Gene Sequencing and Mutations
The complete nucleotide sequence of PCR products (2380 to 4140bp) was analyzed to determine any genetic changes in this region. Results of sequencing that illustrated in Table 1 shows the genetic variations and frequencies of mutations associated with the studied AML cases.
Three different Single Nucleotide Polymorphism (SNP) G/A792, G/A794 and G/A797 were detected in the intron region. G/A792 was found in 68 of 75 AML patients (90.6%), G/A794 was found in 60 of 75 AML patients (80%), while G/A797 was found in all AML patients. All of the three were detected in 17 of 25 healthy controls (68%). These SNPs were not before correlated with mutant NPM1 Iraqi AML patients and are first described. All statistical analysis that compares between AML cases and healthy controls were summarized in Table 2.
Single nucleotide variant was identified in exon 3 that encode NES, it was found in 53 of 75 AML patients (70.6%). This variant was not detected in healthy subjects. Based on statistical analysis, this NPM1 mutation was significantly associated with AML (p = 0.0022). In addition, single nucleotide variant was observed in two sites G/A635 and G/A660 within intron region that were identified in 80% of AML patients. These SNPs were significantly correlated with mutant NPM1 (p = 0.0153). No genetic variation was observed in exon 2 during sequences analysis of amplified NPM1 gene.
Amino acids sequence encoded by exon 3 of NPM1 gene in AML patients were examined and compared with the reference sequence recorded in NCBI. Results (Table 3)  The position of amino acid (threonine, T) related to the expected rs753788683 SNP occurred in exon 3 at position 75 within the reference sequence. The results showed no changes in the sequence of amino acids (100% similarity) between AML sequence and the reference sequence. The codon that encodes the threonine at position 75 was ACA. The SNP was a base substitution mutation that changed A to G 1275 (rs753788683) causes transversion in the codon from ACA to ACG, however, this altered codon encoded the same amino acid threonine, so the resulted mutation was silent and not affected the amino acid sequence.

MDR1 Gene Expression Assessment in AML Patients
The expression of MDR1 gene in AML patients was achieved by RT-qPCR. The patients were categorized depending on their chemotherapy induction, as newly diagnosed (No treatment started), first-course induction of chemotherapy, second induction and AML patients in consolidation. The healthy control (untreated) used as calibrator.
The result of amplification reaction was summarized in the Fig. 3. The amplification reaction increase dependent on mRNA copy of MDR1 gene in each group that treated with chemotherapy. Also, real-time PCR products showed only a single melting temperature peak was observed for each reaction Fig. 4, thus suggesting nonspecific amplification occurred, like primer dimmer, DNA contamination and other non-specific bindings.  The expression MDR1 gene was compared between the different groups ( Fig. 5 and 6). The Y-axis represents the mean CT value of expression while X-axis represents the five group of analysis. Result (Fig. 5) showed that the expression of the standard GADPH gene had no significant differences between healthy control and the four groups of AML patients. No significant differences in MDR1 expression between the newly diagnosed AML group and first course AML patients as compared with controls (Fig. 6). Nevertheless significant decreases in CT value were recorded for both the second induction AML group and AML consolidation group with p value of 0.0258 and 0.0007, respectively, as compared with healthy controls, indicating the induction of higher expression of MDR1 gene by increasing the challenge of AML patients with chemotherapy regimen.
Further assessment of different MDR1 gene expression in AML patients was done by calculating the relative ratio for testing patient groups as compared with healthy controls (Fig. 7). Results showed no significant coloration between healthy and newly diagnosed since the relative expression ratio was -0.34 fold (p = 0.1591), therefore both groups have similar MDR1 gene expression. This suggested no induction in gene expression. Also no significant differences between healthy calibrator and single chemotherapy AML induction group with relative expression ratio of 2.22 fold, p value 0.2222. Upregulation of MDR1 gene was clearly observed in AML patients with second induction by 5.6 fold increase with significant differences as compared with calibrator (p<0.0001), in addition MDR1 high gene expression, was also observed in AML consolidation group with a relative ratio of 7.58 fold increase and significant differences comparing with control group (p<0.0001).

Discussion
The exon 3 and exon 2 encode the N-terminal region of NPM1 protein. In this study a mutation in exon 3 (rs753788683) was recorded, even though this mutation was not affected on amino acid sequences. Mutations in intron region not affecting on amino acids sequences but may affect gene expression. The N-terminal portion of NPM1 protein was important for oligomerization and chaperon activities (Herrera et al., 1996) and any mutation in this region cause defect in portion function. NPM1 mutations are restricted to exon 12 of AML patients (Kazem et al., 2011) and all mutations tend to defect NLS signal and modify it into new NES signal in exon 12, the NES signal was important in mutant NPM1 protein to accumulated the protein in the cytoplasm (Chou et al., 2006). More than 40 molecular variants of NPM1 mutations have been described in AML patients (Tan et al., 2008). The most common mutation (socalled mutation A12) duplicates a TCTG tetra nucleotide (Szankasi et al., 2008). Mutations B and D are observed in about 10 and 5% of cases, respectively; other mutations are very rare (Falini et al., 2007). In addition the N-terminal region contains high conserved hydrophobic leucine-rich NES motifs (Wang et al., 2005;Falini et al., 2009), therefore it is very difficult that mutations to occur in this region. The region that encodes for NES was important for protein function and any genetic variation in NES signal may attribute in increasing or decreasing protein function.
The goal of chemotherapy treatment is to achieve a complete remission of AML and then to prevent relapse in post-remission therapy. In the last few years, there have developing changes in the diagnosis and treatment of AML based on molecular genetic assessment of MDR1 expression for designing novel curative regimens that reversing regulation of drug-resistant phenotype of AML cells (Cianfriglia, 2013). Response to treatments is affected by many factors according to risk categories associated with morphological features, genetic criteria and age (Almeida and Ramos, 2016;Webber et al., 2008). Longley and Johnston (2005), stated that such findings correlate with the concept of AML being an intrinsically resistant disease and that such up-regulation could be acquired during induction treatment. This support our study that higher expression of MDR1 significantly observed in group two 5.6 fold and group three 7.58 fold and in another hand the non-difference in MDR1 gene expression between the calibrator and newly diagnosed ratio -0.34 fold. Up-regulation of MDR1 after induction has been reported by others. showed in vitro study, the MDR1 and MRP1 (drug resistance proteins) mediated in multidrug resistance of human leukemic cells through the mechanism of resistance to doxorubicin-induce cell death in human HL60 AML cells, they indicated the continuous exposure of leukemic cells to stepwise increasing concentration of doxorubicin resulted in the selection of HL60/DOX cells, which expressed about 10.7 fold resistance as compared to parental sensitive (Baran et al., 2007). The existence of Cancer Stem Cells (CSCs) has been proposed as a potential mechanism ofdrug resistance and a potential therapeutic target (Gupta et al., 2009). CSCs are a putative population of malignant cells within a tumor that possess the characteristics associated with normal stem cells. In this theory's most elaborate conception, CSCs can self-renew and divide by asymmetric division, reconstituting the heterogeneity of the original tumor. CSCs are also considered to be highly resistant to chemotherapeutic agents (Honoki, 2010). In this model, chemotherapy kills most of the cancer cells that are some what more differentiated and do not have long-term self-renewal capacity. When therapy is discontinued, the CSCs repopulate cancer, there by mediating disease relapse. In contrast, molecular remission after consolidation has important prognostic value. This implies that despite CR, in these patients a number of cancer cells survive treatment and can grow out to cause a relapse. The increased MDR1 expression after induction may cause early relapse and progression disease. These studies agreed with our finding that revealed in one case showed complete remission after induction then relapsed occurred after consolidation.
In addition to the involvement of the ATP-Binding Cassette (ABC) family in the efflux and reduction of intracellular drug concentration in malignant conditions, it has recently been shown that these proteins have a role in transportation of prostaglandins, leukotrienes, Sphingosine-1-Phosphate (S1P) and Platelet-Activating Factor (PAF) out of the cells. These lipids cause activation of different signaling pathways, which results in cell proliferation, migration, survival and activation of angiogenesis, metastases and the escape of leukemic cells from immune surveillance (Fletcher et al., 2010). Various ABC transporters are specific to lipids. MDR1 is involved in the efflux of PAF; a PAF molecule activates its G-protein-coupled receptor, PAER and induces up regulation of the anti-apoptotic proteins Bcl-2 and Bcl-XL. These molecules prevent programmed cell death and cause expansion of resistant cells (Raggers et al., 2001). Therefore MDR1 protein has another function that important in AML proliferation. When the AML patients take chemotherapy the expression of MDR1 gene will increase, so the function of MDR1 gene will increase cell proliferation of leukemia cells. This suggested that when patient taken chemotherapy then relapsed occurred after consolidation, the chemotherapy will be ineffective in our patient and also increase the severity of AML disease.

Conclusion
In conclusion, reported mutations detected within NPM1 of exon 3 showed significant correlation with AML as compared with healthy controls, however such mutations did not affect NES amino acid sequence. No genetic variations were detected in exon 2 of AML patient. In addition, MDR1 gene expression was significantly correlated with chemotherapy induction in AML patients.