Specific Methylation Patterns Research Articles

MTHFR gene specific and global methylation patterns in normal pregnancy: A pilot study (India)

AimThe purpose of the present study is to evaluate and understand the association of global and MTHFR gene specific methylation in the progression of a normal pregnancy. MethodsThe subjects comprised of normal pregnant women and non pregnant women. Fasting blood sample (~5 ml) was drawn from all the participants followed by DNA extraction, global DNA methylation and MTHFR gene specific methylation. ResultsWomen with normal pregnancy were having relatively lower levels of global methylation in their first trimester as compared to non pregnant women. The trend of MTHFR gene specific methylation was exactly opposite of that of global methylation in the respective three trimesters and full term. Among normal pregnant women in the first trimester, a significant increase in MTHFR gene specific methylation is observed as compared to the non pregnant controls, followed with a relative decline in the second, third trimester and full term. ConclusionThe present study speculates that pregnancy is associated with decrease in global methylation levels and increase in MTHFR gene specific methylation as compared to non pregnant state. There is an inverse relationship between global and MTHFR gene specific methylation.

Whole Genome DNA Methylation Analysis of Commpass Identifies Biomarkers of Multiple Myeloma Survival

Multiple myeloma is a malignancy of terminally differentiated, antibody secreting B cells known as plasma cells. Normal B cell differentiation and cell fate are coupled to epigenetic and transcriptional reprogramming, including a proliferation-dependent global loss of DNA methylation (Barwick et al., 2016, 2018). However, relatively little is known about the epigenetic changes that underlie myelomagenesis and how these may contribute to pathogenesis. To this end, we are analyzing the DNA methylome of myeloma specimens from the MMRF CoMMpass trial (NCT01454297), which has already characterized the mutational, structural, and transcriptional landscape of nearly 1,000 myelomas from newly diagnosed patients.CoMMpass specimens were obtained from a centralized biobank with approval from the CoMMpass Tissue Use Committee and Emory IRB. DNA isolated from CD138+ myeloma specimens was subjected to reduced representation bisulfite sequencing (RRBS) or whole genome bisulfite sequencing (WGBS). In total, DNA methylation was derived for over 24 million CpGs with an average of 18x coverage. WGBS data from normal B cells and plasma cells was obtained with permission from the BluePrint project (Agirre et al., 2015) via the European Genome Archive. DNA methylation levels were associated with PFS and OS using a cox proportional regression.We have determined the DNA methylome for 36 primary myeloma specimens and an additional 84 specimens are currently being sequenced. Relative to normal B cells that had an average DNA methylation level of 89.1%, plasma cells and myelomas exhibited a progressive demethylation with mean levels of 71.3% and 43.7%, respectively. While this is consistent with previous observations (Agirre et al., 2015; Salhia et al., 2010), WGBS revealed that myeloma in particular was characterized by large hypomethylated domains. These large hypomethylated domains encompassed genes that were devoid of gene expression whereas DNA methylation remained unchanged in the bodies of genes that were highly expressed. Although the majority of these hypomethylated domains were common across myelomas, there existed many regions where methylation levels varied between myelomas and these differences commonly corresponded with local gene expression differences. To understand if these specific patterns of DNA methylation were indicative of disease pathogenesis, DNA methylation levels were compared to PFS and OS. This identified 2,594 regions where the level of DNA methylation was prognostic of outcome (P≤0.001). Reduced DNA methylation corresponded with poor outcome at 88.5% (N=2,298) of these regions, which included loci proximal to cell cycle genes such as MYC, E2F3, CCND1, and CCNE1. Only 11.5% (N=296) of regions associated with outcome had higher levels of DNA methylation associated with poor prognosis. These regions tended to be proximal to genes involved in B cell receptor signaling, such as PLCG2 and VAV2. Although the expression of several of these genes was also prognostic of survival, the majority were not, indicating that the epigenetic state contains a unique prognostic value.These data indicate that myeloma undergoes profound epigenetic remodeling that is co-ordinate with changes in gene expression. Perhaps the most striking feature were megabase domains of hypomethylation. That DNA methylation was preferentially retained in the bodies of expressed genes suggests that a molecular mechanism and/or cellular selection occurs to maintain methylation at genes whose expression is required for myeloma cell survival. Despite the small number (N=36) of myeloma specimens analyzed thus far, the large number of regions associated with survival indicates the potential prognostic value of DNA methylation in myeloma. Furthermore, DNA methylation indicative of outcome only partially overlapped with the prognostic value of gene expression, indicating DNA methylation has independent value as a biomarker of outcome in myeloma. This may be due, in part, to the fact that DNA methylation is a very stable modification that not only reflects the current gene expression program, but is also indicative of the cell history and potential. Integrative genetic, epigenetic, and transcriptional analysis from WGBS of 120 CoMMpass myeloma specimens will be presented, including matched baseline and relapsed specimens from 25 patients. DisclosuresLonial:Amgen: Research Funding. Boise:Abbvie: Consultancy; AstraZeneca: Honoraria.

FMRP Interacts with C/D Box snoRNA in the Nucleus and Regulates Ribosomal RNA Methylation

SummaryFMRP is an RNA-binding protein that is known to localize in the cytoplasm and in the nucleus. Here, we have identified an interaction of FMRP with a specific set of C/D box snoRNAs in the nucleus. C/D box snoRNAs guide 2’O methylations of ribosomal RNA (rRNA) on defined sites, and this modification regulates rRNA folding and assembly of ribosomes. 2’O methylation of rRNA is partial on several sites in human embryonic stem cells, which results in ribosomes with differential methylation patterns. FMRP-snoRNA interaction affects rRNA methylation on several of these sites, and in the absence of FMRP, differential methylation pattern of rRNA is significantly altered. We found that FMRP recognizes ribosomes carrying specific methylation patterns on rRNA and the recognition of methylation pattern by FMRP may potentially determine the translation status of its target mRNAs. Thus, FMRP integrates its function in the nucleus and in the cytoplasm.

Using Illumina Infinium HumanMethylation 450K BeadChip to explore genome‑wide DNA methylation profiles in a human hepatocellular carcinoma cell line.

Aberrant DNA methylation is the most common type of epigenetic alteration and is associated with many types of cancer. Although previous studies have provided a few novel DNA methylation markers in hepatocellular carcinoma (HCC), specific DNA methylation patterns and comparisons of the aberrant alterations in methylation between HCC and normal liver cell lines have not yet been reported. Therefore, in the present study the Illumina Infinium HumanMethylation 450K BeadChip was employed to identify the genome-wide aberrant DNA methylation profiles of Huh7 and L02 cells. Following Bonferroni adjustment, 102,254 differentially methylated CpG sites (covering 26,511 genes) were detected between Huh7 and L02 cells. Of those CpG sites, 62,702 (61.3%) sites were hypermethylated (covering 12,665 genes) and 39,552 (38.7%) sites were hypomethylated (covering 13,846 genes). The results of the present study indicated that 40.3% of the CpG sites were in CpG island regions, 20.7% were in CpG shores and 8.8% were in shelf regions. A total of 57.3% hypermethylated CpG sites and 39.4% of the hypomethylated CpG sites had a |β-Difference| ≥50%. Within the significant differentially methylated CpG sites, 490 genes were located within 598 differentially methylated regions. Gene Ontology enrichment analysis revealed that 2,107 differentially methylated genes were associated with ‘biological process’, 13,351 differentially methylated genes were associated with ‘molecular function’, and 18,041 differentially methylated genes were associated with ‘cellular component’. Kyoto Encyclopedia of Genes and Genomes pathway-based analysis revealed 43 signaling pathways that were associated with 5,195 differentially methylated genes. These results demonstrated that aberrant DNA methylation may be a key and common event underlying the tumorigenesis of Huh7 cells. The present study also identified many subsets of hypo- or hyper-methylated CpG sites, genes and signaling pathways, which have an importance in the occurrence and development of HCC.

DNA methylation profile at a satellite region is associated with aberrant placentation in cloned calves

IntroductionCloning via somatic cell nuclear transfer (SCNT) has been associated with a variety of pathologies, primarily in the placenta, and these alterations may be associated with aberrant epigenetic reprogramming of the donor cell genome. We tested the hypothesis that DNA methylation patterns are not appropriately established after nuclear transfer and that those altered patterns are associated with specific aberrant phenotypes. MethodsWe compared global and specific placental DNA methylation patterns between aberrant and healthy SCNT-produced calves. Foetal cotyledon samples of ten SCNT pregnancies were collected. Global DNA methylation and hydroxymethylation levels were measured using an ELISA-based assay and specific DNA methylation of satellite I, and α-satellite repeat elements were measured using bisulfite PCR. ResultsOur analysis revealed that the SCNT-produced calves, which showed aberrant phenotypes, exhibited a reduced methylation pattern of the satellite I region compared to that of healthy calves. In contrast, global methylation and hydroxymethylation analyses showed higher levels for both cytosine modifications in SCNT-produced female calves with aberrant phenotypes. The satellite I region showed most of the sequences to be hypermethylated in live cloned calves compared with those in deceased calves. DiscussionOur results suggest that this satellite I region could be used as an epigenetic biomarker for predicting offspring viability. Studies evaluating DNA methylation patterns of this satellite region in the donor cell genome or embryo biopsies could shed light on how to improve the efficiency of SCNT cloning.

Transgenerational Inheritance of DNA Hypomethylation in Daphnia magna in Response to Salinity Stress.

Epigenetic mechanisms have been found to play important roles in environmental stress response and regulation. These can, theoretically, be transmitted to future unexposed generations, yet few studies have shown persisting stress-induced transgenerational effects, particularly in invertebrates. Here, we focus on the aquatic microcrustacean Daphnia, a parthenogenetic model species, and its response to salinity stress. Salinity is a serious threat to freshwater ecosystems and a relevant form of environmental perturbation affecting freshwater ecosystems. We exposed one generation of D. magna to high levels of salinity (F0) and found that the exposure provoked specific methylation patterns that were transferred to the three consequent nonexposed generations (F1, F2, and F3). This was the case for the hypomethylation of six protein-coding genes with important roles in the organisms' response to environmental change: DNA damage repair, cytoskeleton organization, and protein synthesis. This suggests that epigenetic changes in Daphnia are particularly targeted to genes involved in coping with general cellular stress responses. Our results highlight that epigenetic marks are affected by environmental stressors and can be transferred to subsequent unexposed generations. Epigenetic marks could therefore prove to be useful indicators of past or historic pollution in this parthenogenetic model system. Furthermore, no life history costs seem to be associated with the maintenance of hypomethylation across unexposed generations in Daphnia following a single stress exposure.

DNA Methylation Profiles in Whole Blood of Huntington's Disease Patients.

Epigenetic mechanisms, especially DNA methylation, are suggested to play a role in the age-of-onset in Huntington's disease (HD) based on studies on patient brains, and cellular and animal models. Methylation is tissue-specific and it is not clear how HD specific methylation in the brain correlates with the blood compartment, which represents a much more clinically accessible sample. Therefore, we explored the presence of HD specific DNA methylation patterns in whole blood on a cohort of HDM and healthy controls from Slovenia. We compared CpG site-specific DNA methylation in whole blood of 11 symptomatic and 9 pre-symptomatic HDM (HDM), and 15 healthy controls, by using bisulfite converted DNA on the Infinium® Human Methylation27 BeadChip microarray (Illumina) covering 27,578 CpG sites and 14,495 genes. Of the examined 14,495 genes, 437 were differentially methylated (p < 0.01) in pre-symptomatic HDM compared to controls, with three genes (CLDN16, DDC, NXT2) retaining statistical significance after the correction for multiple testing (false discovery rate, FDR < 0.05). Comparisons between symptomatic HDM and controls, and the comparison of symptomatic and pre-symptomatic HDM further identified 260 and 198 differentially methylated genes (p < 0.01), respectively, whereas the comparison of all HDM (symptomatic and pre-symptomatic) and healthy controls identified 326 differentially methylated genes (p < 0.01), however, none of these changes retained significance (FDR < 0.05) after the correction for multiple testing. The results of our study suggest that methylation signatures in the blood compartment are not robust enough to prove as valuable biomarkers for predicting HD progression, but recognizable changes in methylation deserve further research.

Abstract 1289: Crosstalk between methylation and alternative splicing in cancer

Abstract Background: DNA methylation in promoter regions leads to transcriptional silencing, such as of tumor suppressor genes in cancer. However, the functions of DNA methylation in the gene body (intragenic, i.e., exons and introns) have not yet been completely elucidated. Recently, hyper-methylation has been implicated in enhancing exon recognition by recruiting methyl-CpG-binding protein (MECP2) to hyper-methylated sites. In this study, we examined intragenic methylation in splicing regulatory elements (SREs: exonic splicing enhancer (ESE), exon splicing silencer (ESS), intronic splicing enhancer (ISE)) as a mechanism to understand how epigenetic factors contribute to alternative splicing events in cancer. Hypothesis: Methylation patterns of SREs are involved in directing alternative splicing events in cancer. Methods: We developed a splicing decision model to identify actionable methylation loci potentially affecting splicing events (i.e., exon skipping) among CpG sites in putative intragenic SRE regions. We used DNA methylation and RNA-Seq data for breast and lung cancer samples and normal cases from The Cancer Genome Atlas (TCGA). We additionally used hyper- and hypo-methylation data published in Selamat et al. (2013) as an independent lung cancer dataset. To identify regions hyper- and hypo-methylated in cancer, we performed an unpaired t-test for normally distributed data and a Mann-Whitney-Wilcox test for non-normally distributed data for differential methylation status (beta value) between cancer and normal cases. Multiple testing was accounted for by controlling the false discovery rate (p&amp;lt; 0.05 with FDR&amp;lt; 0.1). Results: We investigated whether specific patterns of methylation were enriched in SRE regions affecting exon skipping. We found that for breast cancer samples, differential methylation status was enriched in SRE regions but not in non-SRE regions. Particularly, hypo-methylation in cancer showed a greater enrichment in ESE regions with putative exon skipping events. Similar results were observed in the both the TCGA lung cancer cases and an independent lung cancer dataset. We calculated GC contents and the methylation frequencies at each position of the hexameric sequence of SREs and further investigated whether certain SREs are prevalent to be with methylation or not. These data suggest that hypo-methylation in SRE regions may cause failure to recruit MECP2, therefore leading to exon skipping. Conclusion: Our study suggests that intragenic methylation status is important for splicing regulation and may improve our understanding of how intragenic epigenetic markers play a role in gene regulation by affecting alternative splicing. Citation Format: Dongwook Kim, Seonggyun Han, Younghee Lee. Crosstalk between methylation and alternative splicing in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1289.

Abstract 5305: DNA methylation and gene expression in melanoma: A large-scale integrated analysis

Abstract Dysregulation of DNA methyltransferase activity, through global hypomethylation and localized hypermethylation phenomena, has been associated with cancer development. Even though the role of promoter methylation in gene regulation has been widely described, the regulatory function of methylation status of intergenic and intragenic regions has been not yet clarified. High-throughput technology and bioinformatics analysis represent useful tools to investigate the functional role of global methylation patterns in gene regulation. Therefore, a specific bionformatic tool was generated to identify methylation hotspots as well as extended genomic methylation regions in order to analyze specific genetic methylation patterns associated with cancer gene deregulation. In the present study, melanoma was selected as tumor model for its aggressive phenotype. Melanoma samples were obtained from the Cancer Genome Atlas (TCGA) to analyze DNA methylation and gene expression profiling data. We developed an algorithm, using R language, that performs cyclic correlation analysis between gene expression and methylation levels of all genes. Our results show that in melanoma there are different methylation patterns that positively and negatively correlate with gene expression. In particular, the hypomethylation of the promoter (TSS1500, TSS200, 5'UTR) leads to the over-expression of selected gene (p&amp;lt;0.01) while the hypermethylation of gene body and 3'UTR regions were associated with gene over-expression. Furthermore, the frequency of genes showing a global hypomethylation status was higher compared to those that were hypermethylated (70% vs 30%). We have also identified the main individual CG probesets and methylated regions that are differentially correlated with gene expression. A further pathway analysis shows that the identified genes are implicated in central cellular processes associated with melanoma. Our algorithm allows the identification of methylation hotspots involved in the regulation of key genes for melanoma development. This may contribute to the development of new therapeutic strategies targeting methylome alterations in melanoma cells. Moreover, the identification of methylation hotspots associated to cancer initiation and progression can represent useful biomarkers for patients' risk stratification. Citation Format: Saverio Candido, Giuseppe A. Parasiliti Palumbo, Giulia Russo, Luca Falzone, Giulia C. Leonardi, Francesco Pappalardo, Massimo Libra. DNA methylation and gene expression in melanoma: A large-scale integrated analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5305.

Abstract 3658: DNA methylation of circulating tumor educated leukocytes predicts IDH1/2 mutation status in adult patients with diffuse gliomas

Abstract INTRODUCTION: Identification of IDH1/2 mutations and association with better outcome revolutionized diagnosis and management of patients with gliomas. Liquid biopsy can detect driver mutation from blood, however, detection of circulating tumor DNA or circulating tumor cells from patients with brain tumors is difficult due to the low amount of circulating tumor material in the peripheral blood. We hypothesized that brain tumors alter the epigenetic profiles of circulating leukocytes and sought to identify specific methylation patterns in peripheral blood that would identify molecular subtypes of gliomas. METHODS: We analyzed brain tumors and matched whole peripheral blood collected in EDTA tubes from 63 patients with primary gliomas. Tumor IDH1/2 mutation status was analyzed using next-generation sequencing and DNA methylation was analyzed using Illumina Human EPIC array. A complete blood count with differential (CBC) was analyzed to control for variations in CBC profiles. Peripheral blood methylation profile was compared to blood of 40 individuals with no known history of brain tumors. DNA methylation of Acute Myeloid Leukemia (AML) samples (source: TCGA) was used to identify overlapping DNA methylation changes induced by IDH1/2 mutations. Methylation data were analyzed with the R Bioconductor package minfi, including quality control, data normalization and differentially methylated CpG site analysis. Subsequent filtering was performed using a p-value cutoff = 0.01 and a minimal mean difference of the Beta-value of 0.1. RESULTS: Peripheral blood methylation profiles of patients with brain tumors are distinctly different from normal controls. DNA methylation of IDH1/2 mutated brain tumors and AML showed significant overlap (enrichment = 3, p-value 2.2x10-16) and DNA methylation of IDH1/2 mutated AML cells showed significant overlap with circulating leukocytes from patients with IDH1/2 mutated brain tumors (enrichment = 6.5, p-value 2.2x10-8 ). Using this classifier supported by AML data, DNA methylation of circulating leukocytes was able to predict IDH1/2 mutation status of diffuse gliomas with 98% accuracy, nearly perfectly separating IDH status by clustering. CONCLUSIONS: Diffuse gliomas induce epigenetic changes in the methylome of the leukocytes, which resembles methylation changes of IDH1/2 mutated AML. IDH wildtype and mutant gliomas can be distinguished with high accuracy by profiling the epigenome of leukocytes. Our study demonstrates the potential of peripheral blood DNA methylation assessment for non-invasive diagnosis of brain tumors. Citation Format: Andreas Kloetgen, Jonathan Serrano, Seema Patel, Christopher Bowman, Guomiao Shen, David Zagzag, Matthias A. Karajannis, John G. Golfinos, Dimitris Placantonakis, Aristotelis Tsirigos, Andrew S. Chi, Matija Snuderl. DNA methylation of circulating tumor educated leukocytes predicts IDH1/2 mutation status in adult patients with diffuse gliomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3658.

Abstract 557: Aberrant methylation of tumor-suppressor genes in pediatric myelodysplastic syndrome/acute myeloid leukemia

Abstract Introduction: Not only somatic mutations, but also epigenetic modifications in acute myeloid leukemia (AML) are estimated to have a pivotal role in leukemogenesis and it could be a therapeutic target. Especially, aberrant methylation of the promotor region of tumor suppressor genes (TSGs) were the causative events in leukemogenesis, however, there was only limited information in pediatric myelodysplastic syndrome (MDS) and AML. Materials and Methods: We analyzed the aberrant methylation of promotor region of 25 TSGs in pediatric 7 low grade MDS, 4 advanced MDS and 22 AML patients by FAB classification by Methylation-Specific Multiplex Ligation-dependent Probe Amplification (MS-MLPA) method (10 Male, 12 female, 0-14 years old, median 6 years ols). Simultaneously, detection of SNVs, In/del, and copy number variants were also analyzed by target sequence of more than 150 leukemia related genes. Especially, specific chimeras were analyzed in each patient. Results: Total 12/22(54.5%) patients had more than one methylated TSGs (number of methylated TSGs was 0-5 in each patient, median 1). More than 20% of patients had methylated TSGs including CDKN2B, CADM1, CDH13 and ESR1. Especially, there was a specific methylation pattern of TSGs, for example, all (4/4) t(8;21) patients had CDH13 methylation, 3/4 (75%) patients with inv(16) had ESR1 mutation and normal karyotype did not. Normal control samples, complete remission samples, and low grade MDS samples were not completely methylated. All advanced MDS patients had 1 or 2 methylated TSGs. Simultaneously, several leukemogenic genes were mutated such as GATA1, IKZF1, KRAS, KIT, FLT3, SH2B3, PIK3CD, PU.1, SMARC1, ALK, etc. Several genes were deleted such as CTCF, RB1, SRP72, ATM, NF1, U2AF1 etc. Discussion: In adult MDS, the increased number of methylated TSGs correlated to the development of AML and the poor prognosis. In this study, a few TSGs were methylated as the adult study (Hess CJ., Leuk Lymphoma 2008), furthermore, many genes related to epigenetic modification such as ASXL1, TET2 and EZH2 were not mutated in this study. Recently, anti-methylation drug were used in adult MDS/AML, larger study will be needed for adjusted use of epigenetic modifier in pediatric MDS/AML. Citation Format: Akira Shimada, Hirotaka Kanzaki, Hisashi Ishida, Takehiro Matsubara, Michinori Aoe. Aberrant methylation of tumor-suppressor genes in pediatric myelodysplastic syndrome/acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 557.

PO-372 Investigating the epigenetic changes underlying combination treatment of acute promyelocytic leukaemia with all-trans retinoic acid and arsenic trioxide

IntroductionAcute promyelocytic leukaemia (APL) results in the interrupted differentiation of granulocytes. Combination induction therapy using all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) is the most effective treatment of APL. DNA methylation and histone modifications are epigenetic mechanisms of gene regulation aberrant in APL. While ATRA treatment of APL NB4 cells restores granulocyte differentiation and induces significant genome-wide changes in histone modifications, it does not affect the aberrant DNA methylation. Similarly, ATO has comparable effects. Therefore, we hypothesise that the increased effectiveness of ATRA and ATO when combined is due to epigenetic changes at both the histone and DNA methylation level, resulting in profound changes in gene expression.Material and methodsWe identified the concentration of ATRA and ATO in combination that induced enhanced differentiation and cell death of NB4 cells in comparison to single agent treatment through fluorescence activated cells sorting (FACS) of CD11b and 7-AAD labelled cells at 72 hours and 96 hours post treatment termination. Quantitative PCR (qPCR) was performed to determine the expression levels of a panel of genes after 72 hours of treatment and 96 hours post treatment termination.Results and discussionsATRA and ATO in combination induced significant differentiation and cell death of NB4 cells after 72 hours. Interestingly, only the combination-treated cells maintained differentiation when treatment was terminated for 96 hours. Additionally,combination treatment significantly increased the expression of several genes important in APL compared to single agent treatment after 72 hours as well as 96 hours post treatment termination. Chromatin immunoprecipitation (ChIP) is currently being utilised to investigate the enrichment of chromatin with acetylated and methylated histones, and qPCR was performed to assess the enrichment of several genes of interest. Finally, bisulfite pyrosequencing is being performed to characterise the specific methylation pattern of these genes of interest.ConclusionATRA and ATO in combination synergistically induces the differentiation of APL NB4 cells. Together, the ChIP and bisulfite pyrosequencing experiments will enable us to characterise the effects of combination treatment at the epigenetic level. Ultimately, understanding the epigenetic changes induced by ATO and ATRA synergy can help improve treatment in the clinic.

Evaluating NISCH and CDH1 Promoter Hypermethylation in Nonsmokers, Cancer Free Smokers and Lung Cancer Patients: A Case Control Study.

Lung cancer has very high mortality due to late stage diagnosis not amenable to curative resection. Cancer specific methylation patterns of tumor suppressor genes may precede precursor lesions of lung cancer. Our aim was to evaluate the promoter hypermethylation of tumor suppressor gene NISCH and CDH1 in cfDNA from plasma of lung cancer patients and its possible correlation with smoking status and various clinicopathological parameters. Forty histopathologically confirmed lung cancer cases, thirty smoker and thirty nonsmoker controls were enrolled. Plasma cfDNA was extracted and subjected to bisulfite treatment followed by MS-PCR. Serum nischarin levels were estimated by ELISA. The frequency of promoter hypermethylation of NISCH and CDH1 was significantly higher in lung cancer patients as compared to lifelong non-smoker controls (p < 0.05). It did not vary with smoking status among cancer cases. No significant association was found with staging or histological grading. NISCH methylation was found to be significantly higher among smoker controls. Pack years and packs per day were significantly higher in the methylated group. Serum nischarin levels showed no significant association with NISCH methylation or clinicopathological variables. NISCH is highly methylated in both high risk smoker controls as well as cancerousnon-smokers and may mark the convergence of varied etiologies of lung cancer. Hence NISCH and CDH1 are highly methylated in plasma cfDNA of lung cancer patients.

HGG-20. DNA METHYLATION ANALYSIS OF HIGH-GRADE GLIOMA IN PATIENTS WITH MISMATCH REPAIR DEFICIENCIES

Abstract Patients with constitutional mismatch repair deficiency (CMMRD) are prone to developing high-grade glioma (HGG). These tumours acquire DNA polymerase mutations and become ultra-hypermutant harbouring hundreds of mutations per megabase. The impact of these mutations on methylation profile and the ability of the tool to differentiate MMRD tumours from others is unknown. In order to answer these questions, we performed either 450k/850K methylation analysis on a cohort of 52 CMMRD-HGG and compared them to 148 non-CMMRD HGG and normal brain controls. CMMRD HGG harbouring classic mutations in histone 3 or IDH genes had a methylation profile which clustered closely with non-MMRD tumours harbouring these mutations. Tumours without these alterations exhibited a tendency to hypomethylation with some tumours being extremely hypomethylated in comparison to other HGG. Hypomethylation was unrelated to mutational burden and type of DNA polymerase mutation present. Gene set analysis of methylation patterns revealed enrichment of hypomethylation for cellular pathways involved in cellular metabolism, organelle maintenance, mitotic cell cycle and gene expression. This pattern persisted in subgroup analysis of IDH mutant tumours in patients with and without MMRD. Importantly, this pattern was present in MMRD HGG with mutational burdens <10 mutations/MB and shared between primary and recurrent tumours suggesting that hypomethylation is an early event. CMMR-HGG have unique pattern of hypomethylation which can distinguish them from other paediatric HGG. Several plausible explanations include that hypomethylation in specific pathways confer a survival advantage on the cells which acquire it, or that hypermutations in specific CpG affect methylation patterns in these genes.

Specific breast cancer prognosis-subtype distinctions based on DNA methylation patterns.

Tumour heterogeneity is an obstacle to effective breast cancer diagnosis and therapy. DNA methylation is an important regulator of gene expression, thus characterizing tumour heterogeneity by epigenetic features can be clinically informative. In this study, we explored specific prognosis‐subtypes based on DNA methylation status using 669 breast cancers from the TCGA database. Nine subgroups were distinguished by consensus clustering using 3869 CpGs that significantly influenced survival. The specific DNA methylation patterns were reflected by different races, ages, tumour stages, receptor status, histological types, metastasis status and prognosis. Compared with the PAM50 subtypes, which use gene expression clustering, DNA methylation subtypes were more elaborate and classified the Basal‐like subtype into two different prognosis‐subgroups. Additionally, 1252 CpGs (corresponding to 888 genes) were identified as specific hyper/hypomethylation sites for each specific subgroup. Finally, a prognosis model based on Bayesian network classification was constructed and used to classify the test set into DNA methylation subgroups, which corresponded to the classification results of the train set. These specific classifications by DNA methylation can explain the heterogeneity of previous molecular subgroups in breast cancer and will help in the development of personalized treatments for the new specific subtypes.

Locus-specific control of the de novo DNA methylation pathway in Arabidopsis by the CLASSY family.

DNA methylation is essential for gene regulation, transposon silencing, and imprinting. Although the generation of specific DNA methylation patterns is critical for these processes, how methylation is regulated at individual loci remains unclear. Here we show that a family of four putative chromatin remodeling factors, CLASSY (CLSY) 1–4, are required for both locus-specific and global regulation of DNA methylation in Arabidopsis. Mechanistically, these factors act in connection with RNA polymerase-IV (Pol-IV) to control the production of 24-nucleotide small interfering RNAs (24nt-siRNAs), which guide DNA methylation. Individually, the CLSYs regulate Pol-IV-chromatin association and 24nt-siRNA production at thousands of distinct loci, and together, they regulate essentially all 24nt-siRNAs. Depending on the CLSYs involved, this regulation relies on different repressive chromatin modifications to facilitate locus-specific control of DNA methylation. Given the conservation between methylation systems in plants and mammals, analogous pathways likely operate in a broad range of organisms.

Enrichment of methylated molecules using enhanced-ice-co-amplification at lower denaturation temperature-PCR (E-ice-COLD-PCR)for the sensitive detection of disease-related hypermethylation.

The detection of specific DNA methylation patterns bears great promise as biomarker for personalized management of cancer patients. Co-amplification at lower denaturation temperature-PCR (COLD-PCR) assays are sensitive methods, but have previously only been able to analyze loss of DNA methylation. Enhanced (E)-ice-COLD-PCR reactions starting from 2ng of bisulfite-converted DNA were developed to analyze methylation patterns in two promoters with locked nucleic acid (LNA) probes blocking amplification of unmethylated CpGs. The enrichment of methylated molecules was compared to quantitative (q)PCR and quantified using serial dilutions. E-ice-COLD-PCR allowed the multiplexed enrichment and quantification of methylated DNA. Assays were validated in primary breast cancer specimens and circulating cell-free DNA from cancer patients. E-ice-COLD-PCR could prove a useful tool in the context of DNA methylation analysis for personalized medicine.

ASB1 differential methylation in ischaemic cardiomyopathy: relationship with left ventricular performance in end-stage heart failure patients.

AimsIschaemic cardiomyopathy (ICM) leads to impaired contraction and ventricular dysfunction, causing high rates of morbidity and mortality. Epigenomics allows the identification of epigenetic signatures in human diseases. We analyse the differential epigenetic patterns of the ASB gene family in ICM patients and relate these alterations to their haemodynamic and functional status.Methods and resultsEpigenomic analysis was carried out using 16 left ventricular (LV) tissue samples, eight from ICM patients undergoing heart transplantation and eight from control (CNT) subjects without cardiac disease. We increased the sample size up to 13 ICM and 10 CNT for RNA sequencing and to 14 ICM for pyrosequencing analyses. We found a hypermethylated profile (cg11189868) in the ASB1 gene that showed a differential methylation of 0.26Δβ (P = 0.016). This result was validated by a pyrosequencing technique (0.23Δβ, P = 0.048). Notably, the methylation pattern was strongly related to LV ejection fraction (r = −0.849, P = 0.008), stroke volume (r = −0.929, P = 0.001), and end‐systolic and diastolic LV diameters (r = −0.743, P = 0.035 for both). ASB1 showed a down‐regulation in messenger RNA levels (−1.2‐fold, P = 0.039).ConclusionsOur findings link a specific ASB1 methylation pattern to LV structure and performance in end‐stage ICM, opening new therapeutic opportunities and providing new insights regarding which is the functionally relevant genome in the ischaemic failing myocardium.

Base-Resolution Analysis of DNA Methylation Patterns Downstream of Dnmt3a in Mouse Naïve B Cells.

The DNA methyltransferase, Dnmt3a, is dynamically regulated throughout mammalian B cell development and upon activation by antigenic stimulation. Dnmt3a inactivation in hematopoietic stem cells has been shown to drive B cell-related malignancies, including chronic lymphocytic leukemia, and associates with specific DNA methylation patterns in transformed cells. However, while it is clear that inactivation of Dnmt3a in hematopoietic stem cells has profound functional effects, the consequences of Dnmt3a inactivation in cells of the B lineage are unclear. To assess whether loss of Dnmt3a at the earliest stages of B cell development lead to DNA methylation defects that might impair function, we selectively inactivated Dnmt3a early in mouse B cell development and then utilized whole genome bisulfite sequencing to generate base-resolution profiles of Dnmt3a+/+ and Dnmt3a−/− naïve splenic B cells. Overall, we find that global methylation patterns are largely consistent between Dnmt3a+/+ and Dnmt3a−/− naïve B cells, indicating a minimal functional effect of DNMT3A in mature B cells. However, loss of Dnmt3a induced 449 focal DNA methylation changes, dominated by loss-of-methylation events. Regions found to be hypomethylated in Dnmt3a−/− naïve splenic B cells were enriched in gene bodies of transcripts expressed in B cells, a fraction of which are implicated in B cell-related disease. Overall, the results from this study suggest that factors other than Dnmt3a are the major drivers for methylome maintenance in B cell development.

Epigenetic and Posttranscriptional Regulation of CD16 Expression during Human NK Cell Development.

The surface receptor FcγRIIIA (CD16a) is encoded by the FCGR3A gene and is acquired by human NK cells during maturation. NK cells bind the Fc portion of IgG via CD16a and execute Ab-dependent cell-mediated cytotoxicity, which is critical for the effectiveness of several antitumor mAb therapies. The role of epigenetic regulatory mechanisms controlling transcriptional and posttranscriptional CD16 expression in NK cells is unknown. In this study, we compared specific patterns of DNA methylation and expression of FCGR3A with FCGR3B, which differ in cell type-specific expression despite displaying nearly identical genomic sequences. We identified a sequence within the FCGR3A promoter that selectively exhibits reduced methylation in CD16a+ NK cells versus CD16a- NK cells and neutrophils. This region contained the transcriptional start site of the most highly expressed CD16a isoform in NK cells. Luciferase assays revealed remarkable cell-type specificity and methylation-dependent activity of FCGR3A- versus FCGR3B-derived sequences. Genomic differences between FCGR3A and FCGR3B are enriched at CpG dinucleotides, and mutation of variant CpGs reversed cell-type specificity. We further identified miR-218 as a posttranscriptional negative regulator of CD16a in NK cells. Forced overexpression of miR-218 in NK cells knocked down CD16a mRNA and protein expression. Moreover, miR-218 was highly expressed in CD16a- NK cells compared with CD16a+ NK cells. Taken together, we propose a system of FCGR3A regulation in human NK cells in which CpG dinucleotide sequences and concurrent DNA methylation confer developmental and cell type-specific transcriptional regulation, whereas miR-218 provides an additional layer of posttranscriptional regulation during the maturation process.