MIR29B mediates epigenetic mechanisms of HBG gene activation

Sickle cell disease (SCD) affects over 2 million people worldwide with high morbidity and mortality in underdeveloped countries. Therapeutic interventions aimed at reactivating fetal haemoglobin (HbF) is an effective approach for improving survival and ameliorating the clinical severity of SCD. A class of agents that inhibit DNA methyltransferase (DNMT) activity show promise as HbF inducers because off-target effects are not observed at low concentrations. However, these compounds are rapidly degraded by cytidine deaminase when taken by oral administration, creating a critical barrier to clinical development for SCD. We previously demonstrated that microRNA29B (MIR29B) inhibits de novo DNMT synthesis, therefore, the goal of our study was to determine if MIR29 mediates HbF induction. Overexpression of MIR29B in human KU812 cells and primary erythroid progenitors significantly increased the percentage of HbF positive cells, while decreasing the expression of DNMT3A and the HBG repressor MYB. Furthermore, HBG promoter methylation levels decreased significantly following MIR29B overexpression in human erythroid progenitors. We subsequently, observed higher MIR29B expression in SCD patients with higher HbF levels compared to those with low HbF. Our findings provide evidence for the ability of MIR29B to induce HbF and supports further investigation to expand treatment options for SCD.     

Persistence of a component of DNA methylation in gastric mucosae after Helicobacter pylori eradication

Background  

Helicobacter pylori (HP) infection potently induces aberrant DNA methylation in gastric mucosae, and its accumulation is associated with gastric cancer risk. Cross-sectional analysis of methylation levels (fraction of methylated DNA molecules) and temporal analysis of methylation incidence suggested that methylation levels decrease after HP infection discontinues. We aimed to demonstrate the decrease in methylation levels.     

DNMT3B Promoter Polymorphism and Risk of Gastric Cancer

To investigate the association of single-nucleotide polymorphism (SNP) in DNA methyltransferase 3B (DNMT3B) gene and the risk of gastric cancer (GC), we detected -149C>T and -579G>T in the promoter region of the DNMT3B gene by polymerase chain reaction restriction fragment length polymorphism (PCR–RFLP) and DNA sequencing analysis. The DNMT3B genotype was determined in 259 gastric cancer patients and 262 healthy controls that were frequency matched for age and gender. Results showed that individuals with at least one -579G allele were also at significantly decreased risk of gastric cancer [odds ratio (OR), 0.43; 95% confidence interval (CI) 0.26–0.72] compared with those having a -579TT genotype. The -149C>T genotype distribution was irrelevant to the risk of gastric cancer (OR, 1.49; 95% CI, 0.17–17.94) in the studied Chinese population. In addition, data suggested that DNMT3B genetic polymorphism varied among different races, ethnic groups, and geographic areas.     

Insight into the molecular pathophysiology of myelodysplastic syndromes: targets for novel therapy

Myelodysplastic syndromes (MDS) are clonal hematopoietic stem cell disorders characterized by abnormal cellular differentiation and maturation with variable progression to acute leukemia. Over the last decade, scientific discoveries have unraveled specific pathways involved in the complex pathophysiology of MDS. Prominent examples include aberrations in cytokines and their signaling pathways (such as tumor necrosis factor‐alpha, interferon‐gamma, SMAD proteins), mutations in genes encoding the RNA splicing machinery (SF3B1, SRSF2, ZRSR2, and U2AF1 genes), mutations in genes disrupting the epigenetic machinery (TET2, DNMT3A, DNMT3B, EZH2, ASXL1). In addition, abnormalities in regulatory T‐cell dynamics and atypical interactions between the bone marrow microenvironment, stroma and progenitor cells, and abnormal maintenance of telomeres are also notable contributors to the complex pathogenesis of MDS. These pathways represent potential targets for novel therapies. Specific therapies include drugs targeting aberrant DNA methylation and chromatin remodeling, modulating/activating the immune system to enhance tumor‐specific cellular immune responses and reduce anomalous cytokine signaling, and blocking abnormal interaction between hematopoietic progenitors and stromal cells.     

Role of Ucp1 enhancer methylation and chromatin remodelling in the control of Ucp1 expression in murine adipose tissue

Aims/hypothesis  

Increasing the expression of the brown adipose tissue-specific gene uncoupling protein-1 (Ucp1) is a potential target for treating obesity. We investigated the role of DNA methylation and histone modification in Ucp1 expression in adipose cell lines and ex vivo murine adipose tissues.     

Identification of Disease-Associated DNA Methylation in B Cells from Crohn’s Disease and Ulcerative Colitis Patients

Background

Changes in the methylation status of inflammatory bowel disease (IBD)-associated genes could significantly alter levels of gene expression, thereby contributing to disease onset and progression. We previously identified seven disease-associated DNA methylation loci from intestinal tissues of IBD patients using the Illumina GoldenGate BeadArray assay.

Aims

In this study, we extended this approach to identify IBD-associated changes in DNA methylation in B cells from 18 IBD patients [9 Crohn??s disease (CD) and 9 ulcerative colitis (UC)]. B cell DNA methylation markers are particularly favorable for diagnosis due to the convenient access to peripheral blood.

Methods

We examined DNA methylation profiles of B cell lines using the Illumina GoldenGate BeadArray assay. Disease-associated CpGs/genes with changes in DNA methylation were identified by comparison of methylation profiles between B cell lines from IBD patients and their siblings without IBD. BeadArray data were validated using a bisulfite polymerase chain reaction (PCR)-based restriction fragment length polymorphism (RFLP) method. To verify that observed changes in DNA methylation were not due to virus transformation, we compared specific CpG DNA methylation levels of GADD45A and POMC between B cell lines and matching peripheral blood B lymphocytes from five individuals.

Results

Using this approach with strict statistical analysis, we identified 11 IBD-associated CpG sites, 14 CD-specific CpG sites, and 24 UC-specific CpG sites with methylation changes in B cells.

Conclusions

IBD- and subtype-specific changes in DNA methylation were identified in B cells from IBD patients. Many of these genes have important immune and inflammatory response functions including several loci within the interleukin (IL)-12/IL-23 pathway.     

Evaluation of <Emphasis Type="Italic">DNMT1</Emphasis> gene expression profile and methylation of its promoter region in patients with ankylosing spondylitis

Ankylosing spondylitis (AS) is an autoimmune disease with a chronic inflammatory arthritis. The critical role of methylation in the biology of immunocytes has increasingly been surveyed to discover disease etiology. DNA methyltransferase 1 (DNMT1) is an enzyme, which establishes and regulates patterns of methylated cytosine residues. The aim of the current investigation was to unveil if methylation circumstances of CpG sites in DNMT1 promoter could affect the mRNA expression level of this gene in peripheral blood mononuclear cells (PBMCs) from AS patients. PBMCs were isolated from whole blood of 40 AS patients and 40 healthy individuals. Total RNA and DNA contents of leukocytes were extracted. Afterward, quantitative analysis was carried out by real-time PCR using the SYBR Green PCR Master Mix. Finally, to determine the methylation level, PCR products of bisulfite-treated DNA from patients and controls were sequenced. Compared with healthy controls, expression level of DNMT1 in AS patients was significantly downregulated. Methylation of DNMT1 promoter was significantly higher in AS patients in comparison to controls. While a negative correlation between methylation and expression level of DNMT1 was observed in AS patients, both methylation and expression level of DNMT1 did not correlate with clinical manifestations. Considering the observation that decreased expression level of DNMT1 was associated with hypermethylation of DNMT1 promoter in PBMCs from AS patients, this survey suggests that dysregulation of DNMT1 expression through altered methylation level of other target genes would probably contribute to AS development.     

Polymorphisms in Interleukin-1B (IL-1B) and Interleukin 1 Receptor Antagonist (IL-1RN) Genes Associate with Gastric Cancer Risk in the Chinese Population

Background  

Gastric cancer is one of the most common malignancies afflicting the Chinese population. Polymorphisms in interleukin-1B (IL-1B) and interleukin-1 receptor antagonist (IL-1RN) genes have been associated with increased gastric cancer risk.     

Persistence of DNMT3A mutations at long‐term remission in adult patients with AML

Mutations in DNMT3A, the gene encoding DNA methyltransferase 3 alpha, have been identified as molecular drivers in acute myeloid leukaemia (AML) with possible implications for minimal residual disease monitoring and prognosis. To further explore the utility of DNMT3A mutations as biomarkers for AML, we developed assays for sensitive detection of recurrent mutations affecting residue R882. Analysis of DNA from 298 diagnostic AML samples revealed DNMT3A mutations in 45 cases (15%), which coincided with mutations in NPM1, FLT3 and IDH1. DNMT3A mutations were stable in 12 of 13 patients presenting with relapse or secondary myelodysplastic syndrome, but were also present in remission samples from 14 patients (at allele frequencies of <1–50%) up to 8 years after initial AML diagnosis, despite the loss of all other molecular AML markers. The mutant DNMT3A allele burden was not related to the clinical course of disease. Cell sorting demonstrated the presence of DNMT3A mutations in leukaemic blasts, but also at lower allele frequencies in T and B‐cells from the same patients. Our data are consistent with the recent finding of preleukaemic stem cells in AML, which are resistant to chemotherapy. The persistence of DNMT3A mutations during remission may have important implications for the management of AML.     

In vitro and in vivo induction of fetal hemoglobin with a reversible and selective DNMT1 inhibitor

Pharmacological induction of fetal hemoglobin (HbF) expression is an effective therapeutic strategy for the management of β-hemoglobinopathies such as sickle cell disease. DNA methyltransferase (DNMT) inhibitors 5-azacytidine (5-aza) and 5-aza-2′-deoxycytidine (decitabine) have been shown to induce HbF expression in both preclinical models and clinical studies, but are not currently approved for the management of hemoglobinopathies. We report here the discovery of a novel class of orally bioavailable DNMT1-selective inhibitors as exemplified by GSK3482364. This molecule potently inhibits the methyltransferase activity of DNMT1, but not DNMT family members DNMT3A or DNMT3B. In contrast with cytidine analog DNMT inhibitors, the DNMT1 inhibitory mechanism of GSK3482364 does not require DNA incorporation and is reversible. In cultured human erythroid progenitor cells, GSK3482364 decreased overall DNA methylation resulting in derepression of the -globin genes HBG1 and HBG2 and increased HbF expression. In a transgenic mouse model of sickle cell disease, orally administered GSK3482364 caused significant increases in both HbF levels and in the percentage HbF-expressing erythrocytes, with good overall tolerability. We conclude that in these preclinical models, selective, reversible inhibition of DNMT1 is sufficient for the induction of HbF, and is well-tolerated. We anticipate that GSK3482364 will be a useful tool molecule for the further study of selective DNMT1 inhibition both in vitro and in vivo.     

Abnormal DNA methylation in CD4+ T cells from people with latent autoimmune diabetes in adults

Aberrant DNA methylation in T cells has been linked to pathogenesis of autoimmune diseases. To investigate genomic and gene-specific DNA methylation levels in CD4⁺ T cells from patients with latent autoimmune diabetes in adults (LADA), and to investigate changes in the expression of genes that regulate methylation as well as the autoimmune-related gene FOXP3 in these patients. Global CD4⁺ T cell DNA methylation was measured in 15 LADA patients and 11 healthy controls using a methylation quantification kit. mRNA levels of DNA methytransferases (DNMTs), methyl-DNA binding domain proteins (MBDs) and FOXP3 were measured by real time PCR. Methylation of a FOXP3 regulatory element region was determined by bisulphite genomic sequencing. Genomic DNA methylation in CD4⁺ T cells from LADA patients was significantly increased compared to controls. DNMT3b mRNA levels were higher in CD4⁺ T cells from LADA patients than in controls. DNMT3b expression positively correlated with global DNA methylation in LADA CD4⁺ T cells. FOXP3 expression was decreased, and the FOXP3 promoter region was hypermethylated in CD4⁺ T cells from LADA patients compared with controls. DNA methylation levels are altered in CD4⁺ T cells from LADA patients, which may contribute to disease onset and progression by affecting the expression of autoimmune-related genes.     

No effect by the common gene variant rs10830963 of the melatonin receptor 1B on the association between sleep disturbances and type 2 diabetes: results from the Nord-Trøndelag Health Study

Aims/hypothesis  

Genetic variation in the melatonin receptor 1B (MTNR1B) is associated with type 2 diabetes. Melatonin contributes to the regulation of sleep, and sleep problems are a documented risk factor for type 2 diabetes. The aim of this study was to investigate whether the MTNR1B gene variant rs10830963 is associated with sleep problems and whether this variant contributes to the association between sleep disturbances and type 2 diabetes.     

Hydralazine may induce autoimmunity by inhibiting extracellular signal–regulated kinase pathway signaling

Objective

To determine whether hydralazine might decrease DNA methyltransferase (DNMT) expression and induce autoimmunity by inhibiting extracellular signal–regulated kinase (ERK) pathway signaling.

Methods

The effect of hydralazine on DNMT was tested in vitro using enzyme inhibition studies, and in vivo by measuring messenger RNA (mRNA) levels and enzyme activity. Effects on ERK, c‐Jun N‐terminal kinase, and p38 pathway signaling were tested using immunoblotting. Murine T cells treated with hydralazine or an ERK pathway inhibitor were injected into mice and anti‐DNA antibodies were measured by enzyme‐linked immunosorbent assay.

Results

In vitro, hydralazine did not inhibit DNMT activity. Instead, hydralazine inhibited ERK pathway signaling, thereby decreasing DNMT1 and DNMT3a mRNA expression and DNMT enzyme activity similar to mitogen‐activated protein kinase kinase (MEK) inhibitors. Inhibiting T cell ERK pathway signaling with an MEK inhibitor was sufficient to induce anti–double‐stranded DNA antibodies in a murine model of drug‐induced lupus, similar to the effect of hydralazine.

Conclusion

Hydralazine reproduces the lupus ERK pathway signaling abnormality and its effects on DNMT expression, and inhibiting this pathway induces autoimmunity. Hydralazine‐induced lupus could be caused in part by inducing the same ERK pathway signaling defect that occurs in idiopathic lupus.

     

Association of polymorphisms MTHFR C677T and A1298C with risk of colorectal cancer, genetic and epigenetic characteristic of tumors, and response to chemotherapy

Background and aims  

The enzyme MTHFR plays an important role in folate metabolism, and folate is implicated in carcinogenesis due to its role in DNA methylation, repair, and synthesis. We analyze the relationship of MTHFR C677T and A1298C polymorphisms with biological, clinicopathological, genetic and epigenetic features of tumors, and the patient outcome after treatment with 5-FU-based chemotherapy to determine the contribution of MTHFR genotypes in the risk of colorectal cancer (CRC) and in the response to therapy.