Context: Philadelphia (Ph)-negative myeloproliferative neoplasms (MPNs), including essential thrombocythemia (ET), polycythemia vera (PV), and primary myelofibrosis (PMF) from a group of disorders characterized by dysregulated JAK-STAT functionality, abnormal hematopoiesis, as well as increased production of proliferative cytokines. In addition to JAK2^V617F mutation, additional gene alterations that are involved in epigenetic mechanisms, particularly de novo DNA methyltransferase 3A (DNMT3A), have been described in Ph-negative MPN biology. Aims: The aim of this study is to evaluate the H/C/S/P mutations in codon R882 of DNMT3A gene among patients with Ph-negative MPNs. Subjects and Methods: This study was conducted on 64 newly diagnosed patients with PV, ET, and PMF who referred to Shafa Hospital, Ahvaz, Iran. In the beginning, 5 mL whole blood was drawn from each patient, and the DNMT3A R882 codon mutations were investigated following the isolation of peripheral blood mononuclear cells by DNA amplification protocol using polymerase chain reaction and DNA sequencing techniques. Results: The R882H G > A mutation, which results in an amino acid substitution at position 882 of DNMT3A gene from arginine (R) to histidine (H), was observed in two patients (3.1%) with JAK2^V617F positive PV and JAK2^V617F negative PMFs. Conclusions: Based on the results, DNMT3A-R882 mutations occur at a low frequency in patients with Ph-negative MPNs. To the best of our knowledge, this is the first study to specifically estimate the prevalence of DNMT3A mutations among Ph-negative MPN patients living in the Middle East. It is recommended to investigate these mutants as a secondary defect along with common major complications in such patients.

Introduction

Myeloproliferative neoplasms (MPNs) are hematopoietic stem cell (HSC) malignancies that are associated with uncontrollable proliferation and development of myeloid lineages, causing progress toward bone marrow (BM) failure and myelofibrosis, ineffective hematopoiesis, or acute leukemia.[1] The identification of common genetic events, including mutations or rearrangements of genes encoding protein tyrosine kinases that are involved in a number of signaling pathways, has significantly changed the diagnostic approach of Philadelphia (Ph)-negative MPNs, especially polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF).[2] As the most important defect in this type of disorders, JAK2V617F mutation is found in over 95% of PV cases, as well as 50% of ET and PMF patients.[3] Nowadays, epigenetic studies have shown that the incidence of a somatic mutation is not sufficient by itself for the pathogenesis of Ph-negative MPN and is not the only critical factor for the progress of such diseases toward acute leukemia.[4] In addition, a significant number of patients with ET and PMF may have uncommon mutations of unknown clinical significance.

Epigenetic mechanisms, including DNA methylation, histone modifications, and noncoding RNAs act synergistically to regulate chromatin structure and gene expression. Meanwhile, DNA methylation is a fundamental factor to determine the fate of cells through controlling the balance between HSC self-renewal and differentiation, since the disruption of cellular balance leads to proliferative neoplasia.[5] The mammalian DNA methyltransferase (DNMT) family are responsible for this process, and in particular, de novo DNA methyltransferase 3A (DNMT3A) is reported to be highly expressed in CD34+ cells of BM, but its expression level is reduced following the differentiation of hematopoietic progenitor cells.[6]

Recognizing the effect of aberrant methylation causing the development of several malignancies, including acute myelogenous leukemia (AML),[7] acute lymphoblastic leukemia (ALL),[8] myelodysplastic syndromes (MDS),[9] and MPNs, the impaired expression of DNMT3A may account for the etiology of such disorders through induction of aberrant DNA methylation patterns. Several mutations have been detected in the gene encoding DNMT3A, in which the exon 23 missense variants, especially at position 882, are the most common type of mutations converting arginine to histidine (R882H), cysteine (R882C), serine (R882S), and phenylalanine (R882P).[10] Since there has been no detailed investigation monitoring the Ph-negative MPN patients in Iran, assessment of H/C/S/P mutations in codon R882 of DNMT3A gene may provide valuable information regarding early diagnosis, prognosis, and progression of the PV, ET, and PMF patients toward acute leukemia.

Subjects and Methods

Sample collection

This study was conducted on 65 newly diagnosed patients with Ph-negative MPNs who referred to Shafa Hospital of Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran during 2014–2015. Based on the WHO criteria, all the patients were assigned to three groups of PV, ET, and PMF.[11] The initial diagnosis was based on morphological examination of peripheral blood (PB) and BM aspiration in addition to the information obtained from laboratory assessments and clinical examination. Five ml ethylenediaminetetraacetic acid-anticoagulated PB sample was drawn from all the patients before the initiation of therapy. This study was approved by the Local Ethics Committee of the Ahvaz Jundishapur University of Medical Sciences, and written informed consent was obtained from all the studied patients.

DNA extraction

DNA was extracted from PB cells according to instructions of QIAamp DNA Blood Mini Kit (Qiagen, Germany). To ensure the quality of extracted DNA, the absorbance (optical density) of purified samples was confirmed at 260 and 280 nm by a spectrophotometer with purity in the range of ≤1.8. The extracted samples were stored at −80°C for polymerase chain reaction (PCR).

Polymerase chain reaction and sequencing

For DNMT3A mutation analysis, exon 23 of the target gene was amplified by PCR; then, the PCR products were sequenced. Briefly, PCR was performed in a 20 μL volume containing 1× PCR buffer, 0.6 mmol/L of deoxynucleotide triphosphates, 1 mmol/L of MgCl2, 0.5 mmol/L of forward and reverse primers, 0.5 U of Taq DNA polymerase, and 1 μL of genomic DNA. The primers of DNMT3A exon 23 were as follows: 5'-GAACTAAGCAGGCGTCAGAGG-3' (forward) and 5'-CTGGGTGCTGATACTTCTCTCC-3' (reverse). PCR reactions were carried out on an ABI 2720 Thermal Cycler (Applied Biosystems, USA). After denaturing at 95°C for 5 min, the amplification was conducted for 35 cycles at 95°C for 30 s, 60°C for 30 s, and 72°C for 30 s, followed by reextension for 5 min at 72°C. The PCR products were loaded onto 1.5% agarose gel containing ethidium bromide and were electrophoretically separated. After purification, the 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

Data were analyzed using SPSS (Statistical Package for Social Service Inc., Chicago, IL, USA) software version 19.0. In this study, descriptive statistics was used to show the results, including mean ± standard deviation and median.

Results

In this study, 64 newly diagnosed patients with Ph-negative MPN (30 men and 34 women) whose disease was confirmed by clinical and laboratory experiments were selected with a mean age of 52 years (age range of 21–78 years). There were 37 cases of PV (57.81%), 23 cases of ET (35.93%), and 4 cases of PMF (6.25%).

The clinical presentations of patients were evaluated, among whom only eight patients with PV, five patients with ET, and all the four patients with PMF had splenomegaly. Moreover, hepatomegaly was observed in three patients with PV, ET, and PMF. Hematologic parameters examined in this study included white blood cells, hemoglobin, and platelets [Table 1]. JAK2V617F mutation was positive in 35 PV patients, five ET patients, and three PMF patients.{Table 1}

Following DNA sequencing of exon 23 PCR products, DNMT3A-Arg882His (R882H) mutation was found in only two patients with PV and PMF, resulting in 882 R (CG C)→H (CA C) substitution [Figure 1]. These two mutated patients with PMF and PV were negative and positive for JAK2V617F mutation, respectively, and both had splenomegaly but showed no other evidence of hepatomegaly at the time of diagnosis [Table 2].{Figure 1}{Table 2}

Discussion

Ph-negative MPNs, including PV, ET, and PMF, are caused by a clonal proliferation of malignant HSCs. Despite numerous attempts to identify the etiology and molecular mechanisms of these disorders, there has been no comprehensive understanding with respect to the pathogenesis of this type of hematologic malignancy to be able to eliminate the leukemic stem cells (LSCs). In addition to molecular defects in cytosolic signaling molecules and transcription factors controlling hematopoiesis, there is evidence on the important role of mutations in epigenetic regulators involved in the development of hematological malignancies.[12] DNA methylation is an important epigenetic mechanism, controlling the expression of a wide range of genes in the human body. Aberrant expression or structural mutations of DNMT genes can lead to a number of malignancies associated with altered DNA methylation patterns in the form of hypomethylation or hypermethylation.[13] Several studies have indicated a high prevalence of DNMT3A mutation among AML-M4/M5 patients, especially in a hotspot fragment of the R882codon, which is associated with an older age and hyperleukocytosis in such patients.[14] However, this type of mutation has rarely been reported in other hematologic malignancies such as ALL, MPN, and MDS. Grossmann et al. indicated a strong correlation between DNMT3A mutation with decreased survival and poor prognosis of T-ALL patients.[8] Moreover, Wang et al. showed that this type of mutation has a poorer prognosis relative to JAK2V617F among MPN and MDS/MPN patients.[15] Obviously, the occurrence of mutations with a poor prognosis should be a diagnostic priority in such patients; however, the low prevalence of these genetic complications and the absence of adequate studies complicates this matter.

DNMT3A mutation in patients with Ph-negative MPNs was first studied by Stegelmann et al.[16] They were only able to identify two PV and three PMF cases out of 115 MPN patients harboring mutations in DNMT3A exon 2–23 together with JAK2V617F somatic mutation [Table 3].{Table 3}

Preliminary results highlighted that these mutations might play a role in the development of malignancy, but other effective mutations are needed for the persistence of malignant conditions. In this study, DNMT3A mutation was assessed in 64 patients with PV, ET, and PMF, among whom only two patients with PV and PMF were detected to harbor the mutation. R882H was the observed nucleotide sequence alteration in both JAK2V617F positive PV and JAK2V617F negative PMF patients. Considering the fact that DNMT3 mutation occurs with or without JAK2V617F mutation, it cannot be regarded as an independent risk factor for the development of these diseases Nangalia J et al.[17] On the other hand, unlike AML patients, the role of DNMT3A mutations in the prediction of disease severity and progression toward acute phase remains to be unknown in Ph-negative MPN population. In this regard, DNMT3A mutation does not seem to be a common finding in early stages of MPNs in comparison to patients progressing to acute leukemia. Rao et al. have reported two M880V and R882C mutations following their exon 23 sequence analysis of 75 patients with PV.[20] They also assessed the frequency of an R882C allele in CD14+, CD3+, and CD19+ cells, which was revealed to be limited to the myeloid lineage. Lin et al. have studied the lineage specificity of DNMT3A mutation in only one mutated ET patient out of 130 Ph-negative MPN cases and found that the R882H allele was limited to myeloid cells as well.[22] In fact, by providing favorable conditions, DNMT3A mutation is likely to be conducive to the growth and proliferation of the myeloid lineage. By genotyping the burst forming unit-erythroid colonies of a PV patient, Rao et al. observed the R882C mutation (but not the JAK2 mutation) in all the investigated colonies.[20] These findings address the hypothesis that whether the development of these epigenetic changes predisposes to other defects related to the pathogenesis of MPNs.

Identification of the exact role of DNMT3A in controlling the genes involved in hematopoiesis is an important issue in this background since the decreased or increased activity of this enzyme causes irreversible complications in myeloid precursors, as well as the incidence of malignancy.[23] Experimental results on DNMT3A gene represent mutation in R882 codon of both PV and PMF patients, which would be associated with poor prognosis in such cases. Studies investigating this mutation in different hematological disorders have suggested the aberrant methylation of tumor suppressor genes involved in such malignancies.[24],[25] It seems that further studies on the evaluation of quantitative DNMT3A gene promoter methylation among MPNs, as well as the comparison of their methylation patterns with expression profiles of other genes involved in disease recurrence, could be helpful for the prediction of clinical course of the patients.

Conclusions

In addition to aberrant DNA methyltransferase activity, the probable functions of DNMT3A mutations, including the involvement in histone modifications or other regulatory epigenetic mechanisms, demand further investigations. Hence, an important question arises: is the DNMT3A enzyme responsible for the control of gene expression within LSCs or malignant precursor cells could affect the activity of genes involved in the cell cycle in ways other than epigenetic DNA methylation modification?

Acknowledgments

This paper is issued from the thesis of Neda Ketabchi, MSc student of hematology and blood banking. This work was financially supported by grant IR. AJUMS. REC. TH94/9 from vice chancellor for Research Affairs of Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.