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 Table of Contents  
ORIGINAL ARTICLE
Year : 2017  |  Volume : 6  |  Issue : 1  |  Page : 86-91

Sequencing myeloproliferative leukemia exon 10 mutations in iranian patients with breakpoint cluster region-Abelson murine leukemia viral oncogene homolog 1-negative myeloproliferative neoplasm


1 Thalassemia and Hemoglobinopathy Research Center, Research Institute of Health, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
2 Department of Medical Genetics, Faculty of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran

Date of Web Publication29-Jun-2017

Correspondence Address:
Najmaldin Saki
Research Center of Thalassemia and Hemoglobinopathy, Health research institute, Ahvaz Jundishapur University of Medical Sciences, Ahvaz
Iran
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ccij.ccij_11_17

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  Abstract 

Context: Breakpoint cluster region-Abelson murine leukemia viral oncogene homolog 1 (BCR-ABL1)-negative myeloproliferative neoplasms (MPNs), including essential thrombocythemia (ET), polycythemia vera (PV), and primary myelofibrosis (PMF), are distinguished by the dysregulated Janus kinase (JAK)-signal transducer and activator of transcription functionality, abnormal hematopoiesis, and spontaneous proliferation. Moreover, a mutation in JAK2V617F as well as myeloproliferative leukemia (MPL) mutations have been reported in these patients, which could be important in the pathogenesis of diseases. MPL plays a role in the development of megakaryocytes and platelets as well as self-renewal of hematopoietic stem cells. Aims: The aim of the present study was to investigate the frequency of MPL mutations in patients with BCR-ABL1-negative MPNs. Settings and Design: This study was a cross-sectional study conducted as an analytical, descriptive review. Subjects and Methods: This study was performed on 54 newly diagnosed patients with BCR-ABL1-negative MPN (PV, ET, and PMF) who referred to Shafa Hospital, Ahvaz, Iran. Five milliliter whole blood was drawn from these patients; JAK2V617Fmutation and mutations in exon 10 of MPL gene were investigated using polymerase chain reaction and DNA sequencing techniques following the isolation of mononuclear cells from the blood.Statistical Analysis: All the data were presented as mean ± standard deviation and were analyzed by SPSS. Results: JAK2V617Fmutation was present in 33 patients, among whom there were 6 ET (35.3%), 7 PMF (41.2%), and 20 PV cases (100%). MPLW515 L/Kmutation was found in only one case of PMF, which was negative for JAK2V617Fmutation. The prevalence of these mutations was 1.8%, and the patient had splenomegaly with lower white blood cell counts and hemoglobin concentration than normal. Conclusions: Based on the results, MPL mutations rarely occur in patients with MPN. These mutations could be co-expressed with JAK2 mutations and might be helpful for detecting MPN patients with no BCR-ABL1 translocation or JAK2V617Fmutation.

Keywords: Essential thrombocythemia, Janus kinase 2V617F, myeloproliferative leukemia mutation, myeloproliferative neoplasms, polycythemia vera, primary myelofibrosis


How to cite this article:
Kavianpour M, Far MA, Asl JM, Ahmadzadeh A, Vosughi T, Ketabchi N, Saki N. Sequencing myeloproliferative leukemia exon 10 mutations in iranian patients with breakpoint cluster region-Abelson murine leukemia viral oncogene homolog 1-negative myeloproliferative neoplasm. Clin Cancer Investig J 2017;6:86-91

How to cite this URL:
Kavianpour M, Far MA, Asl JM, Ahmadzadeh A, Vosughi T, Ketabchi N, Saki N. Sequencing myeloproliferative leukemia exon 10 mutations in iranian patients with breakpoint cluster region-Abelson murine leukemia viral oncogene homolog 1-negative myeloproliferative neoplasm. Clin Cancer Investig J [serial online] 2017 [cited 2019 Nov 19];6:86-91. Available from: http://www.ccij-online.org/text.asp?2017/6/1/86/209136


  Introduction Top


Myeloproliferative neoplasms (MPNs) include a group of clonal hematologic disorders caused by excessive proliferation of mutated multipotent hematopoietic stem cells (HSCs).[1] Among the subgroups of these disorders, polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF) show increased cellularity of bone marrow (BM), thrombosis, and bleeding, which are known as breakpoint cluster region-Abelson murine leukemia viral oncogene homolog 1 (BCR-ABL1)-negative MPNs due to the absence of Philadelphia chromosome.[2],[3] These disorders are caused by genetic changes that usually occur in Janus kinase 2 (JAK2), calreticulin, and myeloproliferative leukemia virus oncogene (MPL).[4],[5]

Cytoplasmic JAK2 tyrosine kinase is one of the most important factors in intracellular signal transduction of HSCs in response to erythropoietin, thrombopoietin (TPO), and other hematopoietic cytokines stimulating hematopoiesis.[6] A point mutation in this gene was observed to result in the increased activity and response to growth factors in BCR-ABL1-negative MPN patients due to the substitution of valine for alanine at position 617, which was reported in 95% of PV patients, 50% of ET patients, and 45% of PMF patients.[7],[8]MPL gene encodes TPO receptor and is the main regulator of megakaryopoiesis and platelet growth, which activates the JAK/signal transducer and activator of transcription (STAT) signaling pathway.[7],[9] Similar to JAK2 gene, acquired mutation in MPL causes cytokine-independent cell growth and increased sensitivity to TPO, which ultimately leads to sustained phosphorylation of JAK2, STAT3, STAT5, AKT, and ERK signaling proteins [10] [Figure 1].
Figure 1: Thrombopoietin receptor signaling pathway. After thrombopoietin binding to myeloproliferative leukemia, phosphorylation of Janus kinase 2 in the cytoplasmic domain of the receptor activates PI3K, signal transducer and activator of transcription, and RAS/MAPK pathways, and the transcription factors are recruited to the nucleus to enhance the expression of genes involved in survival, proliferation, and cell cycle. If myeloproliferative leukemiaW515 L/K mutations are present, spontaneous activation of this receptor leads to uncontrollable overactivity, which may result in myeloproliferative disorders

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The majority of mutations in this gene occur in juxtamembrane region of the receptor, including MPLW515 L(leucine to tryptophan) and MPLW515K(lysine to tryptophan) in position 515,[7],[11] which have been observed in 5%–10% of MPN patients,[12] involving 0%–10% of PMF, and nearly 0%–5% of ET cases.[13] In this study, we chose exon 10 mutations of MPL gene because mutations of other exons are uncommon in patients with BCR-ABL1-negative MPNs (PV, ET, and PMF). This mutation is analyzed along with molecular studies such as JAK2V617F mutation and clinical features in these patients. This was the first study to investigate MPL gene mutations in Khuzestan Province in southwest of Iran.


  Subjects and Methods Top


Sample collection

A total of 54 patients (17 ET, 17 PMF, and 20 PV patients) were referred to Shafa Hospital of Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran, between 2014 and 2015 and were diagnosed with MPN according to the World Health Organization criteria.[14] The initial diagnosis was based on peripheral blood (PB) cell morphology and BM aspiration as well as the information obtained by laboratory tests and clinical examinations. Five milliliter ethylenediaminetetraacetic acid-anticoagulated PB sample was drawn from all the patients before starting the treatment. This study was approved by Local Ethics Committee of Ahvaz Jundishapur University of Medical Sciences, and written informed consent forms were signed by all the patients (IR.AJUMS.REC.1394.344).

DNA extraction

Genomic DNA was isolated from mononuclear cells using a spin-column DNA isolation kit (Roche Diagnostics, Mannheim, Germany). To ensure the quality of extracted DNA, optical density of purified samples was confirmed at 260 and 280 nm wavelengths by a spectrophotometer (260/280 ratio ~ 1.8). The extracted samples were stored at −80°C until polymerase chain reaction (PCR) test was performed.

Polymerase chain reaction and sequencing

For JAK2V617F mutation, PCR was performed in 20 μL volume, containing 5.5 μL distilled water, 10 μL PCR buffer (×1), 1 μL MgCl2, 0.5 μL dNTPs, 0.5 μL of each forward and reverse primers, 1 μL Taq polymerase, and 1 μL DNA sample. The amplification process for JAK2V617F mutation included initial denaturation at 95°C for 5 min, 30 cycles of 95°C for 30 s, 62°C for 30 s, and 72°C for 1 min and a final extension at 72°C for 5 min. JAK2V617F primers included JAK2 common reverse (R), 5′-CTGAATAGTCCACAGTGTTTTCAGTTTCA-3′; JAK2 mutant 617 A-202 forward (F), 5'-AGCATTTGGTTTTAATATGGAGTATATT-3'; and JAK2 wild-type 363 bp (F), 5'-ATCTATAGTCATGC TGTTAGTAGGAGAAAG-3'.

For analysis of MPLW515K/L mutations, exon 10 of MPL gene was amplified by PCR, and PCR products were subsequently sequenced. Briefly, PCR was performed in 20 μL volume, containing 10 μL distilled water, 6 μL PCR buffer (×1), 0.5μL dNTPs, 1 μL MgCl2, 0.5 μL of each forward and reverse primers, 0.5 μL SmarTaq DNA polymerase, and 1 μL genomic DNA.

The exon 10 primers of MPL were as follows: F: 5'-ACCCAACTAGGGTGGAGACC-3' and R: 5'-AGAGGTGACGTGCAGGAAGT-3'. PCR reactions were carried out on an ABI 2720 Thermal Cycler (Applied Biosystems, USA). After denaturing at 95°C for 3 min, the amplification was conducted for 32 cycles at 95°C for 1 min, 60°C for 1 min, and 72°C for 1 min, followed by re-extension for 5 min at 72°C. PCR products were loaded onto a 1.5% agarose gel and were electrophoretically separated. After purification, PCR products were directly sequenced in both directions using ABI PRISM 3130xl Genetic Analyzer (Applied Biosystems, USA) to screen for the presence of mutations.

Statistical analysis

All data were presented as mean ± standard deviation; Statistical Package for the Social Sciences (IBM) was used in statistical analyses to evaluate the findings of this study.


  Results Top


In this study, 54 newly diagnosed patients (27 men and 27 women) with a mean age of 54 years (age range of 29–78 years) with BCR-ABL1-negative MPN whose disease was confirmed by clinical and laboratory studies were selected. There were 17 ET patients (31.4%), 17 PMF patients (31.4%), and 20 PV patients (37.2%).

The hematologic parameters examined in this study included white blood cells (WBC), red blood cells, and hemoglobin (Hb) concentration [Table 1]. The patients were evaluated for clinical symptoms, among whom 6 ET patients, 6 PMF patients, and 4 PV patients had splenomegaly, but hepatomegaly was observed only in one PMF patient.
Table 1: Laboratory parameters of the study participants

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The JAK2V617F mutation was positive in 6 ET patients, 7 PMF patients, and all the PV patients [Table 2]. After sequencing of PCR products in exon 10, only one patient with PMF showed the considered mutation of MPLW515Ltype, which is a function of single nucleotide substitution of TGG >TTG (Trp/Lue) in codon 515 [Figure 2]. This patient was negative for JAK2V617F mutation and showed splenomegaly as well as a normal liver function at the time of diagnosis [Table 3].
Table 2: Frequency of Janus kinase 2V617F mutation in all the participants in the study

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Figure 2: Sequencing of MPL exon 10 for screening W515L/K. The sequencing results of myeloproliferative leukemia gene at position 515, which is observed as wild-type TGG (a), indicates the substitution of TTG (b) in myeloproliferative leukemiaW515L mutation, leading to translation of leucine instead of tryptophan

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Table 3: Basic characteristics of myeloproliferative leukemia protein mutated patient

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  Discussion Top


PV, PMF, and ET are clonal disorders of HSCs. This group of heterogeneous disorders is defined by the increased proliferation and maturation of erythroid, myeloid, and megakaryocyte lineages.[15] In recent years, MPN has been classified according to molecular characteristics, which can be effective in the management of these disorders. Molecular analysis revealed the JAK2V617F mutation in BCR-ABL1-negative MPN patients and attracted the attention of researchers.[16]JAK2 and MPL genes play an important role in cell signaling and proliferation of myeloid cells.[17] Mutation in these genes results in the sustained activation of JAK/STAT pathway, as well as other signaling pathways, eventually leading to the proliferation and differentiation of several lineages.[15] Exon 10 of MPL gene at position 515 encodes tryptophan (W), which can be converted to five other amino acids such as leucine (L) or lysine (K) due to the mutation.[13],[18] There is a higher frequency of MPLW515K and MPLW515 L mutations, but the less prevalent MPLW515R and MPLW515A mutations have been reported in one and two patients, respectively.[19] Since the position 515 plays a key role in the formation and spontaneous inactivity of the receptor, mutations in this exon affect the severity of anemia, leading to a higher platelet count and proliferation of BM megakaryocytes.[20] In different studies, MPLW515L and MPLW515K mutations have been observed in almost 9% and 5% as well as 5% and 1% of PMF and ET cases, respectively.[13],[21] This mutation was observed in 1.8% of patients in our study, and the patient harboring MPLW515L mutation was afflicted with PMF. As shown in [Table 4], this type of MPLW515 L mutation has been mainly observed in PMF group in other studies.[28],[29]
Table 4: Myeloproliferative leukemia protein mutations among patients with breakpoint cluster region-Abelson murine leukemia viral oncogene homolog 1-negative myeloproliferative neoplasms

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The results of Ghotaslou et al.[22] investigation in Iranian people indicated that from a total of 60 patients, 34 (56.6%) and 1 (1.7%) patients had JAK2V617F and MPL mutation, respectively. Patients with JAK2V617F mutation had higher WBC counts and Hb concentrations than those without the mutation (P = 0.005, P = 0.003). In addition, the mutation was negative in all the healthy participants of the control group. Their study revealed that unlike the JAK2V617F mutations, MPL mutations rarely occur in Iranian patients with Ph-negative MPNs, and this low mutation rate should be considered in the design of screening strategies for MPN patients.

Furthermore, Karimzadeh et al.[23] showed that 26 out of 30 PV patients (86%), 8 out of 13 PMF patients (61%), 8 out of 15 ET patients (53%), and none of 31 chronic myeloid leukemia (CML) patients were positive for JAK2V617F mutation. The PV patient harboring this mutation displayed higher WBC counts (P = 0.03). Sixteen out of 26 JAK2V617F- positive patients were female, which demonstrates the correlation between the presences of a mutant allele with gender. The differences in other groups were not significant, and the results of their study showed that a single acquired point mutation in JAK2 is present in virtually all the patients with PV and about 50% of those with ET or PMF. However, in another study, JAK2V617Fmutation has been detected in the vast majority of patients with PV (65%–95%), which was less frequent in patients with ET (23%–57%), PMF (23%–57%), and CML (19%, 3 out of 16 Ph-negative CML patients).

In 2011, Asghari et al.[24] reported that the prevalence of JAK2V617F mutation in patients was 58.2%, and the highest prevalence was observed among PV patients. There were significant differences in age, WBC, and PLT in PV patients regarding the prevalence of JAK2V617Fmutation. Their study indicated a high level of association between JAK2V617Fmutation in patients with PV, ET, and PMF in Iranian patients. Therefore, screening for JAK2V617Fmutation can be incorporated into the initial evaluation of patients suspected to chronic MPNs. This test can be used to determine the association between JAK2V617Fmutation with prognosis and treatment of patients with abnormal blood indices.

MPL mutation is rarely observed in PV patients or other myeloid disorders,[13] and there was no case of MPL mutation in PV patients in our study. The evaluation of JAK2V617Fmutation is important for the detection of simultaneous mutations in this gene and MPL. In the study of Dos Santos and Rumi, JAK2V617F and MPLW515 L mutations have been simultaneously detected in ET patients [25],[26] [Table 4]. In our study, the JAK2V617F mutation was observed in 100% of PV patients, 35.3% of ET patients, and 41.2% of PMF patients, but the patient harboring MPL mutation was negative for JAK2V617F mutation, and we were not able to identify the simultaneous occurrence of these two mutations with our low sample size. Mutation in JAK2 gene leads to erythropoiesis, but a mutation in MPL is associated with thrombosis, extramedullary hematopoiesis, and myelofibrosis.[27]


  Conclusions Top


It can be concluded from the current study that JAK2V617F and MPLW515 L/K mutations are rarely seen in patients with MPN but might be helpful for detecting MPN patients with no BCR-ABL1 translocation or JAK2V617F mutation.

Acknowledgments

This work was financially supported by grant TH94/7 from the Vice Chancellor for Research Affairs of Ahvaz Jundishapur University of Medical Sciences. This paper is issued from the thesis of Maria Kavianpour.

Financial support and sponsorship

Nil

Conflicts of interest

There are no conflicts of interest.

 
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