DNA (cytosine-5-)-methyltransferase 3 Alpha Research Articles

Prognostic value of DNMT3A mutations in myelodysplastic syndromes: a meta-analysis

ABSTRACTObjectives: Although DNA (cytosine-5)-methyltransferase 3 alpha (DNMT3A) gene mutations have been widely reported in myelodysplastic syndromes (MDS), the prognostic significance of DNMT3A mutations is still controversial. In this study, we conducted a meta-analysis to determine the prognostic effect of DNMT3A mutations in patients with MDS.Methods: Eligible studies from PubMed, Embase, Web of Science, Clinical Trials and the Cochrane Library were searched. Hazard ratios (HRs) and their 95% confidence intervals (CIs) for overall survival (OS) and leukemia-free survival (LFS) were pooled to assess the effect of DNMT3A mutations on the prognosis in MDS patients.Results: A total of 12 studies with 2236 patients were included in this meta-analysis. The pooled HRs for OS and LFS revealed that MDS patients with DNMT3A mutations had a significantly poor prognosis as compared with those without mutations (OS: HR = 1.654, 95% CI = 1.387–1.973, p < 0.001; LFS: HR = 4.624, 95% CI = 3.121–6.851, p < 0.001).Discussion and Conclusion: This meta-analysis showed an adverse prognostic effect of DNMT3A mutations in patients with MDS, which will contribute to risk stratification and prognostic assessment in the disease.

DNA methylation mechanism of intracellular zinc deficiency-induced injury in primary hippocampal neurons in the rat brain

Objective: To explore Zn2+ deficiency-induced neuronal injury in relation to DNA methylation, providing valuable data and basic information for clarifying the mechanism of Zn2+ deficiency-induced neuronal injury.Methods: Cultured hippocampal neurons were exposed to the cell membrane-permeant Zn2+ chelator N,N,N',N'-Tetrakis (2-pyridylmethyl) ethylenediamine (TPEN) (2 μM), and to TPEN (2 μM) plus ZnSO4 (5 μM) for 24 hours. We analyzed intracellular Zn2+ levels, neuronal viability, and protein/mRNA levels for DNA (cytosine-5) methyltransferase 1 (DNMT1), DNA (cytosine-5-) methyltransferase 3 alpha (DNMT3a), methyl CpG binding protein 2 (MeCP2), Brain-derived neurotrophic factor (BDNF), and growth arrest and DNA-damage-inducible, beta (GADD45b) in the treated neurons.Results: We found that exposure of hippocampal neurons to TPEN (2 μM) for 24 hours significantly reduced intracellular Zn2+ concentration and neuronal viability. Furthermore, DNMT3a, DNMT1, BDNF, and GADD45b protein levels in TPEN-treated neurons were significantly downregulated, whereas MeCP2 levels were, as expected, upregulated. In addition, DNMT3a and DNMT1 mRNA levels in TPEN-treated neurons were downregulated, while MeCP2, GADD45b, and BDNF mRNA were largely upregulated. Addition of ZnSO4 (5 μM) almost completely reversed the TPEN-induced alterations.Conclusion: Our data suggest that free Zn2+ deficiency-induced hippocampal neuronal injury correlates with free Zn2+ deficiency-induced changes in methylation-related protein gene expression including DNMT3a/DNMT1/MeCP2 and GADD45b, as well as BDNF gene expression.

DNMT3A Mutations in AML Patients: Prognostic Impact and Comparative Analysis of Mutations Burden in Diagnostic Samples, after Standard Therapy, and after Allogeneic Stem Cell Transplantation

DNA (cytosine-5)-methyltransferase 3 alpha (DNMT3A) mutations is one of the most frequent somatic mutations in acute myeloid leukemia (AML). Although DNMT3A mutations have been studied extensively, influence of mutations on AML prognosis and their role on the pathogenesis of AML remains not completely clear. Little is known about the significance of mutations burden in the time of diagnosis on the clinical and prognostic features of AML. Recently discovered persistence of DNMT3A mutations in complete remission after standard therapy indicates the presence of mutation in early pre-leukemic stem cells. In this aspect, it would be essential to analyze the existence of DNMT3A-positive cells after allogeneic stem cell transplantation (alloSCT).Using quantitative PCR, we analyzed mutations burden in diagnostic samples obtained from 65 AML patients with DNMT3A mutation. Then follow up samples were analyzed repeatedly, after induction, consolidation therapy, and after alloSCT with a mean follow-up of 33 months. Using well-established markers of minimal residual disease and donor engraftment, we monitored DNMT3A stability during complete molecular remission (CMR). In addition, we analyzed the combination of DNMT3A mutation with others mutations, such as FLT3, NPM1, IDH1, IDH,and others, at the time of diagnosis, as well as in relapse of AML. Finally, we analyzed the prognostic impact of DNMT3A mutations depending on the distinct combination of mutations with other genetic markers. For this, 150 matched-pairs AML patients without DNMT3A mutation were analyzed.We have found considerable variation of mutations burden (from 1% till 90%) in diagnostic samples. Although, the prognostic impact of mutations burden in diagnostic samples have not been verify, we have found a significant associations of higher mutations burden with M4/M5 FAB variant of AML (p=0,04) and presence of NPM1 mutation (p=0,023).Persistence of DNMT3A mutations in CMR after standard chemotherapy was found in all patients. The loss of correlation between DNMT3A and others could be explained by sub-clone architecture and clonal evolution of AML. After alloSCT, DNMT3A mutations were not discovered in patients with CMR and complete donor chimerism. This data suggests the removal of leukemic stem cells after alloSCT and indicate the importance of alloSCT for high risk AML patients. In relapse of leukemia, all samples showed an increasing of DNMT3A mutated alleles.In addition, matched-pairs analysis suggests that DNMT3A, particularly when accompanied by FLT3-ITD, is a significant prognostic factor for inferior outcome, even after alloSCT.We conclude that long-term quantitative monitoring of DNMT3A mutations could provide additional prognostic information and could explain the role of mutations in development and progression of AML. DisclosuresPezzutto:Cellgene, Novartis, Roche, Gilead: Membership on an entity's Board of Directors or advisory committees.

DNMT3A R882 Mutations Predict a Poor Prognosis in AML: A Meta-Analysis From 4474 Patients.

DNA (cytosine-5)-methyltransferase 3 alpha (DNMT3A) mutations were widely believed to be independently associated with inferior prognosis in acute myeloid leukemia (AML) patients. As dominant missense alterations in DNMT3A mutations, R882 mutations cause the focal hypomethylation phenotype. However, there remains debate on the influence of R882 mutations on AML prognosis. Thus, this meta-analysis aimed at further illustrating the prognostic power of DNMT3A R882 mutations in AML patients.Eligible studies were identified from 5 databases containing PubMed, Embase, Web of Science, Clinical Trials, and the Cochrane Library (up to October 25, 2015). Effects (hazard ratios [HRs] with 95% confidence interval [CI]) of relapse-free survival (RFS) and overall survival (OS) were pooled to estimate the prognostic power of mutant DNMT3A R882 in overall patients and subgroups of AML patients.Eight competent studies with 4474 AML patients including 694 with DNMT3A R882 mutations were included. AML patients with DNMT3A R882 mutations showed significant shorter RFS (HR = 1.40, 95% CI = 1.24-1.59, P < 0.001) and OS (HR = 1.47, 95% CI = 1.17-1.86, P = 0.001) in the overall population. DNMT3A R882 mutations predicted worse RFS and OS among the subgroups of patients under age 60 (RFS: HR = 1.44, 95% CI = 1.25-1.66, P < 0.001; OS: HR = 1.48, 95% CI = 1.15-1.90, P = 0.002), over age 60 (RFS: HR = 2.03, 95% CI = 1.40-2.93, P < 0.001; OS: HR = 1.85, 95% CI = 1.36-2.53, P < 0.001), cytogenetically normal (CN)-AML (RFS: HR = 1.52, 95% CI = 1.26-1.83, P < 0.001; OS: HR = 1.67, 95% CI = 1.16-2.41, P = 0.006), and non-CN-AML (RFS: HR = 1.96, 95% CI = 1.20-3.21, P = 0.006; OS: HR = 2.51, 95% CI = 1.52-4.15, P = 0.0038).DNMT3A R882 mutations possessed significant unfavorable prognostic influence on RFS and OS in AML patients.

Smartamine M and MetaSmart supplementation during the peripartal period alter hepatic expression of gene networks in 1-carbon metabolism, inflammation, oxidative stress, and the growth hormone–insulin-like growth factor 1 axis pathways

Peripartal cows likely require greater amounts of Met not only at the tissue and cell level for methylation reactions but also for milk protein synthesis after calving. Thirty-nine Holstein cows were fed throughout the peripartal period (−21 d to 30 d in milk) a basal control (CON) diet (n=14) with no Met supplementation, CON plus MetaSmart (MS; Adisseo Inc., Antony, France; n=12), or CON plus Smartamine M (SM; Adisseo Inc.; n=13). The Met supplements were adjusted daily and top-dressed over the total mixed ration at a rate of 0.19 or 0.07% (dry matter) of feed for MS or SM. Liver tissue was collected on −10, 7, and 21 d for transcriptome profiling of genes associated with Met and glutathione metabolism as well as components of the inflammation, oxidative stress, growth hormone/insulin-like growth factor-1 axis, and DNA methylation pathways. Data were analyzed using PROC MIXED of SAS (SAS Institute Inc., Cary, NC) with the preplanned contrasts CON versus SM + MS and SM versus MS. The S-adenosylhomocysteine hydrolase (SAHH) gene was the most abundant among all genes evaluated, with overall greater expression in Met-supplemented cows than CON, and in SM than MS. Expression of Met adenosyltransferase 1A (MAT1A) was greater in Met-supplemented cows than CON by 21 d postpartum. A greater overall expression of 5-methyltetrahydrofolate-homocysteine methyltransferase (MTR) occurred in Met-supplemented cows than CON. In contrast, the expression of glutathione synthase (GSS); glutamate-cysteine ligase, catalytic subunit (GCLC); and superoxide dismutase 1, cytosolic (SOD1) was lower in Met-supplemented cows than CON. A greater overall expression of nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 (NFKB1) and greater upregulation of haptoglobin (HP) on d 7 occurred in Met-supplemented cows than CON. Expression of DNA cytosine-5-methyltransferase 3 alpha (DNMT3A) was greater but expression of DNMT1 was lower in Met-supplemented cows than CON. The response observed in SAHH reflects its importance to Met supplementation during the peripartum period. Despite greater HP expression after calving, the lower expression of glutathione (GSS and GCLC) metabolism genes and SOD1 due to Met reflect a lower oxidative stress and mild inflammatory status. The extent to which changes in expression of DNMT3A and DNMT1 result in epigenetic effects partly responsible for the previously observed enhanced performance in Met-supplemented cows remains to be examined. Increasing the supply of Met as SM or MS can affect expression of genes in the Met cycle to various extents and, hence, the supply of methyl donors such as S-adenosylmethionine and antioxidants such as glutathione. These compounds likely are in high demand during the peripartum period.

The evolving molecular genetic landscape in acute myeloid leukaemia

Acute myeloid leukaemia (AML) is a heterogeneous disease with a variable response to therapy. The heterogeneity of AML is evident from variations in morphology, immunophenotype, cytogenetics and molecular abnormalities. The introduction of genome-wide technologies has enabled an even more detailed molecular analysis of AML. As a result, the molecular landscape of AML is rapidly evolving. The purpose of this review is to discuss the recent advances made in this field, with a special focus on risk stratification of AML. Clinical AML has been analysed in great molecular detail by gene expression profiling and more recently through epigenetic profiling and next generation sequencing. This has resulted in the identification of novel biomarkers, some of which appear to have a consistent clinical impact in AML, that is mutations in the genes encoding DNA (cytosine-5)-methyltransferase 3 alpha (DNMT3A), additional sex combs-like 1 (ASXL1), tet methylcytosine dioxygenase 2 (TET2) and Runt-related transcription factor 1 (RUNX1). In addition, massively parallel sequencing has revealed a great mutational heterogeneity as well as temporal clonal evolution in AML. The list of acquired mutations with clinical value in AML is growing. Clinical implementation of this multitude of markers will require integrated approaches and selection of markers to facilitate AML risk stratification in the future. The revealed molecular heterogeneity and evolution in AML will have implications for developing targeted therapies.

Screening for mutation R882 in the DNMT3A gene in Chinese patients with hematological disease

The DNA (cytosine-5-)-methyltransferase 3 alpha (DNMT3A) gene is actively involved in epigenetic regulation. Mutations in this gene affect disease progression and response to therapy, particularly in hematological disease. Recent studies have demonstrated that DNMT3A gene mutations affecting codon R882 are actively involved in acute myelogenous leukemia (AML), and cause abnormal alteration of DNA methylation and poor survival. In this study, we screened DNMT3A mutations in a total of 389 Chinese patients with hematological disease by sequencing the coding region of exon 23 covering residue R882. Three heterozygous mutations (p.R882S, p.R882C and p.R882H) were identified in three of 61 AML patients, whereas none of patients with other hematological disorders harbored any mutation. Our results support the notion that DNMT3A R882 was a frequent mutation in AML patients but rare in other types of hematological disorder.

Genome Wide DNA Methylation Analysis Reveals Significant Differences Between Blasts of Patients with Acute Promyelocytic Leukemia and CD34+-Hematopoietic Stem Cells of Healthy Individuals

Abstract 1452 Background:Acute promyelocytic leukemia (APL) accounts for approximately 5 % of all acute myeloid leukemias. Recent experiments have shown that aberrant DNA-promotor-methylation of tumor suppressor genes may be an important mechanism contributing to leukemogenesis and oncogenic transformation. Figueroa et al. recently identified characteristic methylation profiles in a large cohort of acute myeloid leukemia patients using HpaII tiny fragment enrichment by ligation-mediated PCR and array-based methylation analysis. However, as APL is a rare entity, little is still known about the role of deregulated DNA methylation in APL. Methods:Bone marrow blast cells of 45 APL patients at initial diagnosis were analyzed (median age 47 years, gender: 28 female, 17 male). These samples have previously been characterized by high-density-SNP-array analysis (Nowak D et al., Abstract# 38779, ASH 2011), which allowed a segregation into defined molecular and clinical risk subgroups (samples with deletion of chromosome 7q: n = 6; samples with > 5 copy-number-alterations as detected by SNP array: n = 8; samples with white blood cell count at initial diagnosis > 10000/μl: n = 5; samples of patients who suffered relapse: n = 7; samples of patients with initial white blood cell count < 10000/μl: n=19). Furthermore, CD34+-cells from the bone marrow of 10 healthy donors (median age 32 years, gender: 5 female, 5 male) were analyzed as a healthy control group. DNA was extracted using the QIAGEN Allprep Kit® (Qiagen, Hilden, Germany). Genome wide DNA methylation analysis was performed using the HumanMethylation450 BeadChip (Illumina, San Diego, USA). Differential methylation of CpGs between subgroups was defined by a minimum mean methylation difference of 25% as expressed by the beta-value of the array data and statistical significance set at p ≤ 0.05 in a two-sided t-test. Analysis of array data was performed using Genome-Studio Software® (Illumina, San Diego, USA), SAS 9.2./JMP-Genomics 4.2® (SAS Software, Cary, USA) and Microsoft Excel 10.1® (Microsoft Software, Redmond, USA). Results:Using a detection p-value of p ≤ 0.01 for the beta-value of the array and excluding X-chromosomal CpGs, 472.261 CpGs were evaluable for analysis. Highly significant differential methylation profiles between APL-blasts and CD34+-cells of healthy donors could be detected for 18163 CpGs (3.8 %). CpGs differentially hypermethylated in APL versus healthy individual affected the coding regions of interesting candidate genes such as transcriptional regulators microRNA 125b1 (MIR125B1), wilms tumor gene 1 (WT1), WT1 antisense RNA (WIT1), v-ets erythroblastosis virus E26 oncogene homolog 1 (ETS1), chromodomain helicase DNA binding protein 5 (CHD5), deleted in colon cancer (DCC) and homeobox genes like iroquois homeobox 1 (IRX5) and NK6 homeobox 2 (NKX6–2). CpGs in the vicinity of genes involved in regulation of the epigenetic landscape such as jumonji, AT rich interactive domain 2 (JARID2) and DNA (cytosine-5-)-methyltransferase 3 alpha (DNMT3A) were significantly hypomethylated in APL as compared to the control group. The analysis of molecular and clinical subgroups revealed that methylation profiles frequently clustered according to the respective molecular lesions in the analyzed samples. Conclusions:We present a comprehensive genome wide methylation analysis of different APL patient subgroups defined by molecular aberrations and clinical risk profiles. Thereby we have detected sets of significantly differentially methylated CpGs in all subgroups. Integration of this data with results from high density SNP array analysis and detailed clinical outcome data may allow to elucidate the pathomechanistic contribution of aberrant methylation in APL and possibly establish novel prognostic markers based on differential methylation. Disclosures:No relevant conflicts of interest to declare.

Expression Patterns and miRNA Regulation of DNA Methyltransferases in Chicken Primordial Germ Cells

DNA methylation is widespread in most species, from bacteria to mammals, and is crucial for genomic imprinting, gene expression, and embryogenesis. DNA methylation occurs via two major classes of enzymatic reactions: maintenance-type methylation catalyzed by DNA (cytosine-5-)-methyltransferase (DNMT) 1, and de novo methylation catalyzed by DNMT 3 alpha (DNMT3A) and -beta (DNMT3B). The expression pattern and regulation of DNMT genes in primordial germ cells (PGCs) and germ line cells has not been sufficiently established in birds. Therefore, we employed bioinformatics, RT-PCR, real-time PCR, and in situ hybridization analyses to examine the structural conservation and conserved expression patterns of chicken DNMT family genes. We further examined the regulation of a candidate de novo DNA methyltransferase gene, cDNMT3B by cotransfection of cDNMT3B 3′UTR- and cDNMT3B 3′UTR-specific miRNAs through a dual fluorescence reporter assay. All cDNMT family members were differentially detected during early embryonic development. Of interest, cDNMT3B expression was highly detected in early embryos and in PGCs. During germ line development and sexual maturation, cDNMT3B expression was reestablished in a female germ cell-specific manner. In the dual fluorescence reporter assay, cDNMT3B expression was significantly downregulated by four miRNAs: gga-miR-15c (25.82%), gga-miR-29b (30.01%), gga-miR-383 (30.0%), and gga-miR-222 (31.28%). Our data highlight the structural conservation and conserved expression patterns of chicken DNMTs. The miRNAs investigated in this study may induce downregulation of gene expression in chicken PGCs and germ cells.

Benzo[a]pyrene diol epoxide suppresses retinoic acid receptor-β2 expression by recruiting DNA (cytosine-5-)-methyltransferase 3A

Tobacco smoke is an important risk factor for various human cancers, including esophageal cancer. How benzo [a]pyrene diol epoxide (BPDE), a carcinogen present in tobacco smoke as well as in environmental pollution, induces esophageal carcinogenesis has yet to be defined. In this study, we investigated the molecular mechanism responsible for BPDE-suppressed expression of retinoic acid receptor-beta2 (RAR-β2) in esophageal cancer cells. We treated esophageal cancer cells with BPDE before performing methylation-specific polymerase chain reaction (MSP) to find that BPDE induced methylation of the RAR-β2 gene promoter. We then performed chromatin immunoprecipitation (ChIP) assays to find that BPDE recruited genes of the methylation machinery into the RAR-β2 gene promoter. We found that BPDE recruited DNA (cytosine-5-)-methyltransferase 3 alpha (DNMT3A), but not beta (DNMT3B), in a time-dependent manner to methylate the RAR-β2 gene promoter, which we confirmed by reverse transcription-polymerase chain reaction (RT-PCR) analysis of the reduced RAR-β2 expression in these BPDE-treated esophageal cancer cell lines. However, BPDE did not significantly change DNMT3A expression, but it slightly reduced DNMT3B expression. DNA methylase inhibitor 5-aza-2'-deoxycytidine (5-Aza) and DNMT3A small hairpin RNA (shRNA) vector antagonized the effects of BPDE on RAR-β2 expressions. Transient transfection of the DNMT3A shRNA vector also antagonized BPDE's effects on expression of RAR-β2, c-Jun, phosphorylated extracellular signal-regulated protein kinases 1/2 (ERK1/2), and cyclooxygenase-2 (COX-2), suggesting a possible therapeutic effect. The results of this study form the link between the esophageal cancer risk factor BPDE and the reduced RAR-β2 expression.