The Prevalence of JAK2-V617F Mutation in Sudanese Patients with Chronic Myeloproliferative Neoplasms

Myeloproliferative neoplasms (MPNs) are clonal malignant diseases that represent a group of conditions including polycythemia vera (PV), essential thrombocythemia (ET), and myelofibrosis (MF). The JAK2-V617F mutation has been described as a frequent genetic event in majority of patients with MPNs. The aim of this study was to determine the frequency of JAK2-V617Fmutation in Sudanese patients with myeloproliferative disorders referred to Isotope Center, and to investigate the differences of laboratory parameters between patients with JAK2-V617F positive myeloproliferative neoplasms (MPNs) and JAK2 wild type MPNs. A total of 259 patients with MPNs; 159 with polycythemia vera (PV), 55 with essential thrombocytosis (ET) and 45 with primary myelofibrosis (PMF), and11 healthy adult individuals were enrolled in this study from March 2013 to November 2015. DNA was isolated from peripheral blood leukocytes by innuPREP kit, and JAK2-V617F mutation gene detected by allele specific PCR. The JAK2-V617F mutation was Original Research Article Abkar et al.; BJMMR, 14(5): 1-7, 2016; Article no.BJMMR.24741 2 detected in 71% (184/259) patients with MPNs. The prevalence of the mutation was 81.7% in PV, 56.4% inET, and 51.1% in PMF. Mutation was not detected in 11 healthy adult people. The presence of JAK2-V617F was not associated with Hb level, Hct, or the platelet count for PV and ET, whoever the mutation positively correlated with high Hb (P=.039), Hct (P=.04) in PMF patients, and with high erythrocytes count in PV. The JAK2-V617F mutation can be frequently detected in the Sudanese patients with MPNs.


INTRODUCTION
Myeloproliferative neoplasms (MPNs) are clonal disorders of the myeloid linage that clinically present as an excess of cells from one or more terminally differentiated myeloid compartments [1]. According to 2008 World Health Organization (WHO) classification of hematological neoplasms [2,3], MPNs are subclassified into eight separate entities; chronic myelogenous leukemia (CML), polycythemia vera (PV), essential thrombocythemia (ET), primary myelofibrosis (PMF), chronic neutrophilic leukemia, chronic eosinophilic leukemia-not otherwise specified, mastocytosis and MPN unclassifiable. Chronic myeloid leukemia (CML), PV, ET, and PMF are considered 'classic' MPNs [4]. As CML is invariably and specifically associated with BCR-ABL1, the other three (PV, ET and PMF) are operationally dubbed as 'BCR-ABL1-negative MPN' [5]. The most important discovery associated with classical BCR-ABL1-negative MPNs was the identification of JAK2-V617F mutation in 2005 [6,7], the mutation results from a somatic G to T mutation involving JAK2 exon 14, which leads to nucleotide change at position 1849 and the substitution of valine to phenylalanine at codon 617. The JAK2 is a cytoplasmic, non receptor, tyrosine kinase that via its association with cytokine receptors serves as a signaling mediator for hematopoietic cytokines such as erythropoietin (Epo), and thrombopoietin (Tpo) to regulate cell proliferation and growth [8]. V617F mutation affects the noncatalytic 'pseudo-kinase' domain (JH2) of JAK2 and disrupts its autoinhibitory function, resulting in constitutive activation of JAK2mediated signaling pathways, leading to growth factor independent autonomous proliferation of hematopoietic precursors [8,9]. JAK2-V617F was found in the majority of patients with PV and approximately half of the patients with ET andPMF [6,7,10]. Several studies revealed association of the JAK2-V617F mutation with clinical and hematological characteristics in MPNs. The aim of our study was to determine the frequency JAK2-V617F mutation, and to analyze the association of mutated JAK2 gene with laboratory characteristics among Sudanese MPNs patient. We used allele specific polymerase chain reaction (PCR) for the detection of JAK2-V617F mutation.

Study Population (Patients)
Over a period of about 2 years (2013-2015), a total of 259 patients with chronic MPNs; 159 with PV, 55 with ET and 45 with PMF, and 11 healthy adult individuals were randomly enrolled. Hematologic data obtained from patient records and hematological diagnoses and subtyping results were reconfirmed according to the 2008 WHO criteria.

Blood Sampling and DNA Extraction
5 ml of peripheral blood from each subject was collected in EDTA K3 containing vacutainer tube. Genomic DNA extraction from peripheral blood leukocytes was carried out following the protocol of innuPREP kit (Analytik Jena/Germany), then it was stored at -20℃ until the PCR was performed.

Analysis of the JAK2-V617F Mutation
Mutation analysis of JAK2-V617F was performed using allele-specific PCR. Amplifications were done in a total volume of 25 µL PCR mix containing 12.5 µL of TaqMan Universal Master Mix, 5 µL of nuclease-free PCR grade water, 2.5 µL of primers and probes mix and 50 ng/µL of DNA template. PCR primers were; forward wildtype 5'-TCC TCA GAA CGT TGA TGG CAG-3' and reverse wild-type 5'-ATT GCT TTC CTT TTT CAC AAG AT-3', forward mutant 5'-GCA TTT GGT TTT AAA TTA TGG AGT ATA TG-3' and a reverse mutant 5'-GTT TTA CTT ACT CTC GTC TCC ACA AAA-3'. The PCR conditions used were denaturation at 95℃ for 1 min, annealing at 58℃ for 40 s, and extension at 72℃ for 45 s. Products were electrophoresed on agarose gels and visualized using ethidium bromide staining.

Statistical Analysis
Statistical analyses were performed using the SPSS version 11.2.

RESULTS
The JAK2-V617F mutation was detected in 71% (184/259) patients with MPNs. The prevalence of the mutation in subtypes were 81.7% in PV, 56.4% inET, and 51.1% in PMF. JAK2-V617F was not detected in 11 healthy adult people. At diagnosis, the presence of JAK2-V617F was found to be significantly associated with high Hb (P=.039) and Hct (P=.04) in PMF patients. JAK2-V617F positive ET patients were older than ET patients without the mutation (P=.033). But no significant correlations were detected between JAK2-V617F mutation and white blood cell count among the three groups. No significant differences in Hb, Hct, or platelet count were observed between JAK2-V617F positive and JAK2-V617F negative PV and ET patient. These results are summarized in (Tables 1 and 2).

DISCUSSION
A somatic single point mutation (V617F) in the JAK2gene is the most commonly described mutation associated with Philadelphia-negative MPNs; it was detected in >70% of MPNs [6,11,12,13]. The frequency of the JAK2-V617F mutation varied between the MPNs subtypes, it can be found in approximately 95% cases of PV and in approximately 50-60% cases of ET or PMF [6,10]. In numerous published studies, the JAK2-V617F mutation was detected in 80% -97.0% in PV [13,14,15,16,17]. In agreement with these reports, we have detected the JAK2-V617F mutation in 71% (184/259) of patients with MPNs. The prevalence of the mutation was 81.7% in PV, 56.4% in ET, and 51.1% in PMF. Our results were similar to the results obtained by Baxter et al. [6]; they were detected the JAK2 mutation in 29 (57%) of 51 with ET, eight (50%) of 16 with idiopathic myelofibrosis, and 71 (97%) of 73 patients with PV. Similarly, Ayad et al. [16] detected the JAK2-V617F mutation in 81.4% of PV, 50% of ET, 46.1% of PMF. The study by Karkucak et al. [18], was in line with our study. They found that 80% of the PV group and 42% of the ET group were positive for the JAK2-V617F mutation. Also our study was consistent with the study by Zhu et al. [13]; who detected the mutation in 83.9%, 60% in PV and ET, respectively. In numerous studies, the JAK2-V617F mutation has been variably associated with higher Hb, WBC, older age and unchanged or decreased platelet count [19,20,21,22]. Another study by Campbell et al. [23] showed that patients with the mutation present with higher hemoglobin levels, higher white cell counts in PV. In the study of Singh et al. [24], it has been showed that JAK2-V617F negative patients with PMF were significantly associated with more severe anemia, younger age and thrombocytopenia. Several studies [25,26] revealed positive correlation with the JAK2-V617Fmutation and higher Hb, WBC, older age and lowest platelet countin patients with ET. In contrast other studies [27,28] showed no significant correlations between V617F mutation and patient age, white blood cell count, Hb, Hct, or the platelet count for ET or PMF patients. In our study, we have observed significant association between older patients with JAK2-V617F positive ET, with high Hb (P=.039) and Hct (P=.04) in PMF patients, and high erythrocyte count in PV patients. In contrast, no significant differences in Hb, Hct, or the platelet count between V617F positive and V617F negative patients for PV and ET were detected. Overall our study of the JAK2-V617F status of Sudanese MPN patients is consistent with the above studies of MPN patients in Europe, the USA and Asia.

CONCLUSION
The JAK2-V617F mutation can be frequently detected in the Sudanese patients with MPNs, and our study is consistent with previous published studies of MPN patients in Europe, the USA and Asia.

CONSENT
All authors declare that written informed consent was obtained from the patient for publication of this paper.

ETHICAL APPROVAL
It is not applicable.

JAK2-V617Fnegative), 279bp e) and 229bp negative)
V617F mutation can be frequently detected in the Sudanese patients with MPNs, our study is consistent with previous published studies of MPN