|Year : 2018 | Volume
| Issue : 4 | Page : 193-197
Clinical relevance of DNA methyltransferase 3a (dnmt3a) mutation in patients with acute myeloid leukemia
Amal Zidan1, Amina M Elnaggar1, Nahla I Zidan1, Fouad M Abo-Taleb2
1 Department of Clinical Pathology, Faculty of Medicine, Zagazig University, Zagazig, Egypt
2 Medical Oncology, Faculty of Medicine, Zagazig University, Zagazig, Egypt
|Date of Submission||17-Jan-2017|
|Date of Acceptance||02-Mar-2018|
|Date of Web Publication||10-Apr-2019|
Zagazig University Hospitals
Source of Support: None, Conflict of Interest: None
Background Acute myeloid leukemia (AML) is a complex and heterogeneous hematopoietic tissue neoplasm caused by gene mutations, chromosomal rearrangements, deregulation of gene expression, and epigenetic modifications. DNA methylation is altered in leukemia and can affect cells in three main ways: hypomethylation, hypermethylation, and loss of imprinting. Global hypomethylation has frequently been reported in the blast cells, and it is postulated that this promotes transcription of oncogenes and genes concerned with cell replication. Studies of DNA methylation in AML have revealed a series of subgroups with specific methylation signatures.
Patients and methods A total of 45 patients with newly diagnosed AML were included in the study. Moreover, 45 individuals matched for age and sex were selected as controls. Immunophenotyping, conventional cytogenetic analysis, and molecular detection of DNA methyltransferase 3A (DNMT3A) exon 23 mutations were performed. Patient follow-up was performed on day 28 after receiving induction therapy to evaluate the remission status.
Results DNMT3A exon 23 mutations were identified in 17.8% of patients with AML. All the mutations were missense and heterozygous. DNMT3A exon 23 mutations were significantly associated with AML with monocytic differentiation (75%) than the wild-type group (27%) (P=0.01). All DNMT3A mutations were observed in patients with intermediate-risk karyotype. There was a statistically significant decrease in the probability of achieving complete remission with shorter overall survival in the mutated group compared with the wild type, whereas no statistically significant difference between both groups in the probability of disease-free survival (P=0.14).
Conclusion DNMT3A mutations are associated with poor response to therapy conferring a poor outcome, and seem to add prognostic information in patients with AML harboring it with shorter overall survival. DNMT3A mutations can represent a valuable tool for making therapeutic decisions.
Keywords: acute myeloid leukemia, DNA methylation, DNA methyltransferase 3A mutations, prognosis
|How to cite this article:|
Zidan A, Elnaggar AM, Zidan NI, Abo-Taleb FM. Clinical relevance of DNA methyltransferase 3a (dnmt3a) mutation in patients with acute myeloid leukemia. Egypt J Haematol 2018;43:193-7
|How to cite this URL:|
Zidan A, Elnaggar AM, Zidan NI, Abo-Taleb FM. Clinical relevance of DNA methyltransferase 3a (dnmt3a) mutation in patients with acute myeloid leukemia. Egypt J Haematol [serial online] 2018 [cited 2019 Nov 13];43:193-7. Available from: http://www.ehj.eg.net/text.asp?2018/43/4/193/255875
| Introduction|| |
Acute myeloid leukemia (AML) is a complex hematopoietic cellular neoplasm characterized by clonal expansion of immature myeloid cells in the bone marrow, blood, or other organs. The affected cells undergo an uncontrolled proliferation and impaired differentiation program .
Genetic and epigenetic changes have been established in the pathogenesis of AML. DNA methylation is a major form of epigenetic modification . Methylation of mammalian genomic DNA is catalyzed by DNA methyltransferases (DNMTs). The mammalian DNMT family includes five active members: DNMT1, DNMT2, DNMT3A, DNMT3B, and DNMT3L. The DNMT1 is the major enzyme responsible for maintenance of the DNA methylation pattern during DNA replication . DNMT2 has the potential to methylate RNA instead of DNA . DNMT3A and DNMT3B appear to function as de novo methyltransferases , and finally, DNMT3L stimulates the enzymatic activity of DNMT3A both in vivo and in vitro .
DNA methylation is a covalent chemical modification resulting in addition of a methyl group at the carbon position 5 of the cytosine ring in the CpG dinucleotides (cytosine with adjacent guanine) . Global methylation patterning (methylation of intron, exon, and transposon sequences) plays a role in gene expression control and maintenance of genomic stability .
DNA methylation is altered in leukemia and can affect cells in three main ways: hypomethylation, hypermethylation, and loss of imprinting. Global hypomethylation has frequently been reported in the blast cells, and it is postulated that this promotes transcription of oncogenes and genes concerned with cell replication. Local CpG promoter region hypermethylation, particularly in the promoter regions of tumor suppressor genes, also appears to play an important role in AML pathogenesis .
In vivo, DNMT3A mutations do not markedly alter global methylation levels. However, DNMT3A mutated AML demonstrates differential methylation patterns in certain regions of the genome ,. Among DNMT3A mutations, most frequently, codon R882 located in exon 23 is mutated, leading to a change from arginine to histidine (R882H), cysteine (R882C), or phenyl alanine (R882P) . DNMT3A mutations have been suggested to predict poor clinical outcome in AML.
The aim of this study was to detect the prevalence of mutation in exon 23 of DNMT3A gene in patients with AML and its value as a predictor for prognosis.
| Patients and methods|| |
Patients and treatment
This study was carried out in Clinical Pathology and Medical Oncology and Haematology Departments, Faculty of Medicine, Zagazig University Hospitals, during the period from June 2015 to June 2016. A total of 45 patients with newly diagnosed AML were included in the study after obtaining approval from the Institutional Review Board (IRB) of Zagazig University Hospital, approval of Ethical comittee in Faculty of Medicine and approval of Clinical pathology department committee. There were 19 males and 26 females. Their ages ranged from 17 to 75 years, with a mean±SD of 44.5±16.02 years. They were followed up for one year. Moreover, 45 individuals, age and sex matched, were selected as controls. There were 23 males and 22 females. Their ages ranged from 20 to 70 years, with a mean±SD of 44.2±15.8 years.
Peripheral blood and bone marrow samples were collected from all patients; samples were collected at the time of presentation, before therapy was initiated.
Patients were treated by an induction 3+7 regimen, consisting of continuous infusion of cytarabine (100 mg/m2) daily for 7 consecutive days combined with 3 days of doxorubicin (30 mg/m2). Patients 60 years of age or having poor performance status were treated by 2+5 (cytarabine 100 mg/m2 daily for 5 combined with 3 days of doxorubicin 25 mg/m2) regimen of low-dose cytarabine 10 mg/m2/12 h for 14 days. Consolidation comprised three to four courses of high-dose cytosine arabinoside (3 g/m2 every 12 h on days 1, 3 and 5; total, 18 g/m2) .
Bone marrow aspiration was performed on day 28 after receiving induction chemotherapy to evaluate morphological remission. Patients were followed once every 3 months with clinical examination and complete blood cell counts. Marrow examination was done if there was any doubt of a relapse on clinical examination or blood smear. The patients were followed up for one year to evaluate overall survival (OS) and disease-free survival (DFS).
Participants enrolled in the study were subjected to the following: full history taking; clinical examination; complete blood count; bone marrow aspiration and examination; immunophenotyping by flow cytometry using Becton Dickenson FacsCalibar device to detect the following markers: MPO, CD13, CD33, HLA-DR, TDT, CD14, CD64, CD34, CD3, CD20 and CD22; conventional cytogenetic analysis by G banding technique; and karyotyping according to International System for Human Chromosome, Nomenclature. A minimum of 20 metaphases were required to be examined for a patient to be classified and evaluated .
Analysis of DNMT3A mutations
Genomic DNA was extracted from diagnostic bone marrow or peripheral blood mononuclear cells using standard procedures. To screen the DNMT3A mutations, polymerase chain reaction for amplification of DNMT3A exon 23 was carried out by using primers DNMT3A-F (5′-TCCTGCTGTGTGGTTAGACG-3′) and DNMT3A-R (5′-TTTTTCTTCTGGGTGGTGA-3′). PCR amplification was performed in a 25-µl reaction mixture, containing 150 ng of genomic DNA, 1 µl of 10 pmol of each forward and reverse primers, and 12.5 µl of the ready master mix containing dNTPs, MgCL2, hot start Taq DNA polymerase, and Taq buffer. Then, purified distilled water was added to reach the total volume of 25 µl. PCR was performed using thermal cycler (AB Applied Biosystem, USA) with cycling condition of initial denaturation step at 95°C for 10 min followed by 35 cycles of denaturation at 95°C for 30 s. Annealing was done at 50°C for 30 s, and this permits the annealing of the oligopeptides to the DNA template but only at their specific complementary sequences. Extension was done at 72°C for 1 min and final extension at 72°C for 10 min. The Taq polymerase efficiently synthesizes DNA. Purified PCR products were subsequently subjected to cycle sequencing with the forward primer using Big dye terminator ready reaction cycle sequencing kit V3.1 (Applied Biosystem, USA). Then DNA sequencing using ABI PRISM 350 Genetic Analyzer (Applied Biosystem, USA) was done, and finally, the sequencing results were analyzed by using Basic Local Alignment Search Tool (BLAST) web site, which was provided by the National Center for Biotechnology Information (NCBI), to find regions of local similarity between sequences.
Analysis of data was performed using SPSS computer program (version 20; SPSS Inc. Chicago, Illinois, USA). χ2-test, t-test, and Mann–Whitney test were used for statistical analysis. DFS and OS were estimated by the Kaplan–Meier method and compared using the log-rank test. A P value less than 0.05 was considered statistically significant.
| Results|| |
DNMT3A exon 23 mutations were identified in 17.8% of patients with AML. All the identified mutations in exon 23 were clustered in the codon R882 (100%) and ranked as follows: R882H (n=4; 50%), followed by R882C (n=3; 37.5%) and R882P (n=1; 12.5%). No mutations were found in the control group ([Table 1]).
|Table 1 Description of the identified DNA methyltransferase 3A mutations in the patient group|
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The wild-type and mutated groups have statistically significant difference in terms of age, total leukocyte count, hemoglobin level, and platelet count but did not differ regarding sex and percentage of bone marrow blasts. DNMT3A mutations were significantly enriched in AML with monocytic lineage involvement as 75% of DNMT3A mutated cases fall into the FAB subtypes M4 or M5 versus 27% of DNMT3A wild-type cases (P=0.01). Moreover, DNMT3A mutated cases most frequently associated with normal cytogenetics (87.5%) versus DNMT3A wild-type cases (56.8%) (P=0.04) ([Table 2]).
|Table 2 Comparison between the wild-type and mutated groups regarding laboratory data, FAB, and cytogenetic|
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Therapeutic outcome and prognostic effect of DNMT3A mutations on patients with acute myeloid leukemia
After induction therapy, complete remission was statistically significantly different between DNMT3A mutated and wild-type patients (37.5 vs. 78.4%; P=0.02). Regarding the prognostic effect of DNMT3A mutations on patients with AML, compared with DNMT3A wild-type patients, DNMT3A mutated patients had a statistically significant shorter OS (1 year OS: 37.5 vs. 75.7%; P=0.03) but nonstatistically significant DFS (1 year DFS: 66.7 vs. 93.2%; P=0.13) ([Table 3] and [Figure 1]).
|Table 3 Comparison between the wild-type and mutated groups regarding response to induction therapy and survival rates|
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|Figure 1 Kaplan–Meier curve shows probability of (a) overall survival and (b) disease-free survival for the wild-type and mutated groups.|
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| Discussion|| |
In this comprehensive analysis of 45 patients with de novo AML, the incidence of DNMT3A exon 23 mutations was 17.8% in patients with AML, which corresponds with the reports of Thol et al.  (17.8%) and Ostronoff et al.  (19%). It was higher than the ones found in South Brazil (8%)  and China (6.6%) . However, it was lower than that found in France (29%) .
In an Egyptian study, Ghannam et al.  reported a higher incidence of DNMT3A mutations in patients with AML (27%) than ours; this could be attributed to that they sequenced only the DNA fragment spanning R882 codon.
This great discrepancy between different studies could be attributed to the fact that the addressed problem is a genetic disease with a mutation that ultimately may have a different prevalence in different ethnic populations. Other possible explanations for this difference are different distribution of FAB subtypes of patients with AML, mutation detection methods, a limited number of patients, and variability in the sequenced fragment of DNMT3A gene, as some studies sequenced not only exon 23 but the entire DNMT3A genome.
In this study, all the identified mutations in exon 23 were clustered in the codon R882 (100%) and ranked as follows: R882H (n=4; 50%), followed by R882C (n=3; 37.5%) and R882P (n=1; 12.5%). Of note, the highest rate of R882 mutation was R882H (50%). This finding was previously reported by Ghannam et al.  who detected R882H mutation in 60% of patients, followed by R882C (34%) and R882P (6%).
DNMT3A mutations were more prevalent at older age in accordance with other findings ,,. The mutations were also associated with a statistically significant increase in total leukocyte count and platelet count at diagnosis compared with patients without this mutation, which is in agreement with those reported in numerous studies ,,.
DNMT3A mutations were significantly associated with AML with monocytic differentiation (75%) than the wild group (27%) (P=0.01). Our results were in agreement with Renneville et al. , Ghannam et al. , and Amal et al. (2015)  who reported that DNMT3A mutations were more frequently belonged to FAB groups M4 and M5 when compared with DNMT3A wild type.
There was a statistically significant difference between both groups regarding risk stratification. DNMT3A mutations were found in patients with intermediate-risk cytogenetic (100%), which was dominated by patients with normal karyotype (7/8 mutations; 87.5%), which was reported by Thol et al. , Lin et al. , and Amal et al. .
The virtual exclusion of DNMT3A mutations in patients with a favorable-risk profile is not random and may reflect the leukemogenic properties of the fusion proteins created by the favorable chromosomal rearrangements. The PML-RARA fusion protein, which is created by t (15; 17), physically interacts with DNMT3A; this fusion protein alters the methylation of specific promoters . Both PML and DNMT3A regulate telomere function, and all-transretinoic acid, which is part of the therapy for patients with t (15; 17), downregulates DNMT3A expression . Together, these data suggest that DNMT3A mutations and the favorable-risk fusion oncogenes (e.g. PML-RARA and AML-ETO) may not be found in the same AML genomes because they both act to alter the function of DNA methyltransferases and are therefore redundant. However, the outcomes for patients with DNMT3A mutations and those with a favorable-risk cytogenetic profile are dramatically different, for reasons that are currently unclear .After induction therapy, it was noted that CR rate was significantly lower in the mutated group than that in the wild group, indicating that this mutation confers a poor outcome. Same finding was reported by Shen et al.  who mentioned that this mutation may not only associated with cell proliferation but also with reduced apoptosis of the leukemic cells; either of these mechanisms could be considered as inducing chemoresistance.
DNMT3A mutations seem to add prognostic information in patients with AML harboring it. However, its predictive value is still unclear. In the current study, OS was shortened in patients with DNMT3A mutations compared with ones with no mutation; on the contrary, no statistically significant difference was observed between the mutated and wild-type groups regarding DFS, as reported in other studies ,. A possible explanation for its adverse effect on OS is the functional relationship between the absence of hypermethylation phenotype and mutation in DNMT3A with hypomethylation of promoter regions of tumor suppressor genes leading to their inactivation with a negative prognostic effect owing to the loss of their role as genomic keepers .
| Conclusion|| |
DNMT3A mutations could serve as a valuable prognostic marker and may allow stratification for risk-directed therapy in patients with AML. Therefore, early detection of DNMT3A mutations should become a standard procedure for patients with AML because they are molecular targets for specific therapies.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Pezzi A, Moraes L, Valim V, Amorin B, Olivera F, Silva M et al.
DNMT3A mutations in patients with acute myeloid leukemia in South Brazil. Adv Hematol
Hajkova H, Markova J, Haskovec C, Sarova I, Haskove C, Sarova L et al.
Decreased DNA methylation in acute myeloid leukemia patients with DNMT3A mutations and prognostic implications of DNA methylation. Leuk Res
Miremadi A, Oestergaard Z, Pharoah D, Caldas C. Cancer genetics of epigenetic genes. Hum Mol Genet
Goll M, Kirpekar F, Maggert K, Yoder J, Liu C. Methylation of tRNAAsp by the DNA methyltransferase homolog Dnmt2. Science
Okano M, Bell D, Haber D, Li E. DNA methyltransferases DNMT3A and DNMT3B are essential for de novo methylation and mammalian development. Cell
Hata K, Okano M, Lei H, Li E. DNMT3L cooperates with the DNMT3 family of de novo DNA methyltransferases to establish maternal imprints in mice. Development
Shih A, Abdel-Wahab O, Patel J, Levine R. The role of mutations in epigenetic regulators in myeloid malignancies, Nat Rev Cancer
Saied M, Marzec J, Khalid S, Smith P, Down T, Rakyan V et al.
Genome wide analysis of acute myeloid leukemia reveal leukemia specific methylome and subtype specific hypomethylation of repeats. PLoS One
Ley T, Ding L, Walter M, McLellan M, Larson D, Randoth C et al.
DNMT3A mutations in acute myeloid leukemia. N Engl J Med
Renneville A, Boissel N, Nibourel O, Berthon C, Heleraut N, Gardin C et al.
Prognostic significance of DNA methyltransferase 3A mutations in cytogenetically normal acute myeloid leukemia: a study by the Acute Leukemia French Association. Leukemia
Tafferi A, Latendre L. Going beyond 7+3 regimens in the treatment of adult acute myeloid leukemia. J Clin Oncol
Swansbury J. Cancer cytogenetics: methods and protocols; cytogenetic techniques for myeloid disorders. Mol Biol
Thol F, Damm F, Lüdeking A, Winschel C, Wagner K, Morgan M et al.
Incidence and prognostic influence of DNMT3A mutations in acute myeloid leukemia. J Clin Oncol
Ostronoff F, Othus M, Ho P, Kutny M, Geraghty D, Petersdorf S et al.
Mutations in DNMT3A exon 23 independently predict poor outcome in older patients with acute myeloid leukemia: a SWOG report. Leukemia
Lin J, Yao D, Chen Q, Li Y, Yang J, Wang C, Chai H et al.
Recurrent DNMT3A R882 mutations in chienese patients with acute myeloid leukemia and myelodysplastic syndrom. PLoS One
Ghannam D, Taalab M, Ghazy H, Eneen A. DNMT3A R882 mutations in patients with cytogenetically normal acute myeloid leukemia and myelodysplastic syndrome. Blood Cells Mol Dis
Hou H, Kuo Y, Liu C, Chou W, Lee M, Chen C et al.
DNMT3A mutations in acute myeloid leukemia stability during disease evolution and the clinical implications. Blood
Shen Y, Zhu Y, Fan X, Shi J, Wang Q, Yan X et al.
Gene mutation patterns and their prognostic impact in a cohort of1185 patients with acute myeloid leukemia. Blood
Gaidzik V, Schlenk R, Paschka P, Stölzle A, Spath D, Brugger W et al.
Clinical impact of DNMT3A mutations in younger adult patients with acute myeloid leukemia: results of the AML Study Group (AMLSG). Blood
Amal M, Ines S, Hind B, Ichraf R, Samia M et al.
DNMT3A mutations in Tunisian patients with acut myeloid leukemia. J Blood Disord
Fazi F, Zardo G, Gelmetti V, Travaglini L, Croce L, Rosa A et al.
Heterochromatic gene repression of the retinoic acid in acute myeloid leukemia. Blood
Gonzalo S, Jaco I, Fraga M, Chen T, Blasco M. DNA methyltransferases control telomere length and telomere recombination in mammalian cells. Nat Cell Biol
[Table 1], [Table 2], [Table 3]