Genome-wide DNA Methylation Signatures Research Articles

Metabolic and immune costs balance during natural acclimation of corals in fluctuating environments

Epigenetic modifications based on DNA methylation can rapidly improve the potential of corals to adapt to environmental pressures by increasing their phenotypic plasticity, a factor important for scleractinian corals to adapt to future global warming. However, the extent to which corals develop similar adaptive mechanisms and their specific adaptation processes remain unclear. Here, to reveal the regulatory mechanism by which DNA methylation improves thermal tolerance in Pocillopora damicornis under fluctuating environments, we analyzed genome-wide DNA methylation signatures in P. damicornis and compared the differences in the methylation and transcriptional responses of P. damicornis from fluctuating and stable environments using whole-genome bisulfite sequencing and nanopore-based RNA sequencingtranscriptome sequencing. We discovered low methylation levels in P. damicornis (average methylation 4.14%), with CpG accounting for 74.88%, CHH for 13.27%, and CHG for 11.85% of this methylation. However, methylation levels did not change between coral samples from the fluctuating and stable environments. The varied methylation levels in different regions of the gene revealed that the overall methylation level of the gene body was relatively high and showed a bimodal methylation pattern. Methylation occurs primarily in exons rather than introns within the gene body In P. damicornis, there was only a weak correlation between methylation and transcriptional changes at the individual gene level, and the methylation and gene expression levels generally exhibited a bell-shaped relationship, which we speculate may be due to the specificity of cnidarian species. Correlation analysis between methylation levels and the transcriptome revealed that the highest proportion of the top 20 enriched KEGG pathways was related to immunity. Additionally, P. damicornis collected from a high-temperature pool had a lower metabolic rate than those collected from a low-temperature pool. We hypothesize that the dynamic balance of energy–expenditure costs between immunity and metabolism is an important strategy for increasing P. damicornis tolerance. The fluctuating environment of high-temperature pools may increase the heat tolerance in corals by increasing their immunity and thus lowering their metabolism.

Peripheral Blood DNA Methylation Signatures and Response to Tofacitinib in Moderate-to-severe Ulcerative Colitis.

Predictive biomarkers for treatment efficacy of ulcerative colitis [UC] treatments are lacking. Here, we performed a longitudinal study investigating the association and potential predictive power of genome-wide peripheral blood [PB] DNA methylation signatures and response to tofacitinib treatment in UC. We recruited moderate-to-severe UC patients starting tofacitinib treatment, and measured PB DNA methylation profiles at baseline [T1], after 8 weeks [T2], and in a subset [n = 8] after a median of 20 weeks [T3] using the Illumina Infinium HumanMethylation EPIC BeadChip. After 8 weeks, we distinguished responders [R] from non-responders [NR] based on a centrally read endoscopic response [decrease in endoscopic Mayo score ≥1 or Ulcerative Colitis Endoscopic Index of Severity ≥2] combined with corticosteroid-free clinical and/or biochemical response. T1 PB samples were used for biomarker identification, and T2 and publicly available intraclass correlation [ICC] data were used for stability analyses. RNA-sequencing was performed to understand the downstream effects of the predictor CpG loci. In total, 16 R and 15 NR patients, with a median disease duration of 7 [4-12] years and overall comparable patient characteristics at baseline, were analysed. We identified a panel of 53 differentially methylated positions [DMPs] associated with response to tofacitinib [AUROC 0.74]. Most DMPs [77%] demonstrated both short- and long-term hyperstability [ICC ≥0.90], irrespective of inflammatory status. Gene expression analysis showed lower FGFR2 [pBH = 0.011] and LRPAP1 [pBH = 0.020], and higher OR2L13 [pBH = 0.016] expression at T1 in R compared with NR. Our observations demonstrate the utility of genome-wide PB DNA methylation signatures to predict response to tofacitinib.

DNA methylation analysis identifies key transcription factors involved in mesenchymal stem cell osteogenic differentiation

BackgroundKnowledge about regulating transcription factors (TFs) for osteoblastogenesis from mesenchymal stem cells (MSCs) is limited. Therefore, we investigated the relationship between genomic regions subject to DNA-methylation changes during osteoblastogenesis and the TFs known to directly interact with these regulatory regions.ResultsThe genome-wide DNA-methylation signature of MSCs differentiated to osteoblasts and adipocytes was determined using the Illumina HumanMethylation450 BeadChip array. During adipogenesis no CpGs passed our test for significant methylation changes. Oppositely, during osteoblastogenesis we identified 2462 differently significantly methylated CpGs (adj. p < 0.05). These resided outside of CpGs islands and were significantly enriched in enhancer regions. We confirmed the correlation between DNA-methylation and gene expression. Accordingly, we developed a bioinformatic tool to analyse differentially methylated regions and the TFs interacting with them. By overlaying our osteoblastogenesis differentially methylated regions with ENCODE TF ChIP-seq data we obtained a set of candidate TFs associated to DNA-methylation changes. Among them, ZEB1 TF was highly related with DNA-methylation. Using RNA interference, we confirmed that ZEB1, and ZEB2, played a key role in adipogenesis and osteoblastogenesis processes. For clinical relevance, ZEB1 mRNA expression in human bone samples was evaluated. This expression positively correlated with weight, body mass index, and PPARγ expression.ConclusionsIn this work we describe an osteoblastogenesis-associated DNA-methylation profile and, using these data, validate a novel computational tool to identify key TFs associated to age-related disease processes. By means of this tool we identified and confirmed ZEB TFs as mediators involved in the MSCs differentiation to osteoblasts and adipocytes, and obesity-related bone adiposity.

Distinct genome-wide DNA methylation and gene expression signatures in classical monocytes from African American patients with systemic sclerosis

BackgroundSystemic sclerosis (SSc) is a multisystem autoimmune disorder that has an unclear etiology and disproportionately affects women and African Americans. Despite this, African Americans are dramatically underrepresented in SSc research. Additionally, monocytes show heightened activation in SSc and in African Americans relative to European Americans. In this study, we sought to investigate DNA methylation and gene expression patterns in classical monocytes in a health disparity population.MethodsClassical monocytes (CD14+ + CD16−) were FACS-isolated from 34 self-reported African American women. Samples from 12 SSc patients and 12 healthy controls were hybridized on MethylationEPIC BeadChip array, while RNA-seq was performed on 16 SSc patients and 18 healthy controls. Analyses were computed to identify differentially methylated CpGs (DMCs), differentially expressed genes (DEGs), and CpGs associated with changes in gene expression (eQTM analysis).ResultsWe observed modest DNA methylation and gene expression differences between cases and controls. The genes harboring the top DMCs, the top DEGs, as well as the top eQTM loci were enriched for metabolic processes. Genes involved in immune processes and pathways showed a weak upregulation in the transcriptomic analysis. While many genes were newly identified, several other have been previously reported as differentially methylated or expressed in different blood cells from patients with SSc, supporting for their potential dysregulation in SSc.ConclusionsWhile contrasting with results found in other blood cell types in largely European-descent groups, the results of this study support that variation in DNA methylation and gene expression exists among different cell types and individuals of different genetic, clinical, social, and environmental backgrounds. This finding supports the importance of including diverse, well-characterized patients to understand the different roles of DNA methylation and gene expression variability in the dysregulation of classical monocytes in diverse populations, which might help explaining the health disparities.

Genome-wide DNA methylation signature predict clinical benefit of bevacizumab in non-small cell lung cancer

BackgroundThe efficacy of bevacizumab in non-small cell lung cancer (NSCLC) patients is unsatisfactory, and the selection of suitable patients is still challenging. Given the epigenetic modifications can contribute to an aberrant regulation of angiogenesis and microenvironment, we investigated DNA methylation profiles to determine clinical benefit of bevacizumab in NSCLC patients.MethodsGenome-wide DNA methylation profiling was performed in NSCLC patients treated with chemotherapy in combination with bevacizumab. Patients were divided into better prognosis group (A group) and inferior prognosis group (B group) based on their survival. The difference of methylation patterns and respective functional enrichment analysis were performed between two groups. Prognostic DNA methylation signature for bevacizumab was established with the least absolute shrinkage and selection operator regression analyses. TISIDB database was further used to infer immunological relationship for prognostic related DNA methylation.ResultsTwenty patients were included in this study, and significantly distinct methylation patterns were observed between patients with different prognosis. Related genes of different methylation regions were significantly enriched in the biological process of cell projection assembly, neutrophil mediated immunity, and pathway of VEGFA-VEGFR2 signaling pathway, neutrophil degranulation. A 10-gene DNA methylation signature for prognosis prediction was established with the C-index of 0.76. And host genes of signature were found to be related to the abundance of ActCD4, Th1, ActCD8, NKT and neutrophil cells.ConclusionThe 10-gene DNA methylation signature could serve as a novel biomarker to predict the clinical benefit of bevacizumab therapy and improve this anti-tumor approach for NSCLC patients.

Clinical Utility of a Unique Genome-Wide DNA Methylation Signature for KMT2A-Related Syndrome.

Wiedemann–Steiner syndrome (WDSTS) is a Mendelian syndromic intellectual disability (ID) condition associated with hypertrichosis cubiti, short stature, and characteristic facies caused by pathogenic variants in the KMT2A gene. Clinical features can be inconclusive in mild and unusual WDSTS presentations with variable ID (mild to severe), facies (typical or not) and other associated malformations (bone, cerebral, renal, cardiac and ophthalmological anomalies). Interpretation and classification of rare KMT2A variants can be challenging. A genome-wide DNA methylation episignature for KMT2A-related syndrome could allow functional classification of variants and provide insights into the pathophysiology of WDSTS. Therefore, we assessed genome-wide DNA methylation profiles in a cohort of 60 patients with clinical diagnosis for WDSTS or Kabuki and identified a unique highly sensitive and specific DNA methylation episignature as a molecular biomarker of WDSTS. WDSTS episignature enabled classification of variants of uncertain significance in the KMT2A gene as well as confirmation of diagnosis in patients with clinical presentation of WDSTS without known genetic variants. The changes in the methylation profile resulting from KMT2A mutations involve global reduction in methylation in various genes, including homeobox gene promoters. These findings provide novel insights into the molecular etiology of WDSTS and explain the broad phenotypic spectrum of the disease.

Genome-Wide DNA Methylation Signatures Predict the Early Asymptomatic Doxorubicin-Induced Cardiotoxicity in Breast Cancer.

Simple SummaryThis study examined whether the DNA methylation state of peripheral blood mononuclear cells (PBMCs) could predict cardiotoxicity caused by doxorubicin (DOX)-based chemotherapy in breast cancer patients. The results showed a significant difference in the pattern of DNA methylation of PBMCs associated with a risk of cardiotoxicity. These preliminary findings have the potential to further the goal of personalized medicine and tailor the treatment of breast cancer with DOX-based chemotherapy to reduce the toxicity to the heart.Chemotherapy with doxorubicin (DOX) may cause unpredictable cardiotoxicity. This study aimed to determine whether the methylation signature of peripheral blood mononuclear cells (PBMCs) prior to and after the first cycle of DOX-based chemotherapy could predict the risk of cardiotoxicity in breast cancer patients. Cardiotoxicity was defined as a decrease in left ventricular ejection fraction (LVEF) by >10%. DNA methylation of PBMCs from 9 patients with abnormal LVEF and 10 patients with normal LVEF were examined using Infinium HumanMethylation450 BeadChip. We have identified 14,883 differentially methylated CpGs at baseline and 18,718 CpGs after the first cycle of chemotherapy, which significantly correlated with LVEF status. Significant differentially methylated regions (DMRs) were found in the promoter and the gene body of SLFN12, IRF6 and RNF39 in patients with abnormal LVEF. The pathway analysis found enrichment for regulation of transcription, mRNA splicing, pathways in cancer and ErbB2/4 signaling. The preliminary results from this study showed that the DNA methylation profile of PBMCs may predict the risk of DOX-induced cardiotoxicity prior to chemotherapy. Further studies with larger cohorts of patients are needed to confirm these findings.

The palliative effects of folic acid on retinal microvessels in diabetic retinopathy via regulating the metabolism of DNA methylation and hydroxymethylation

ABSTRACT Diabetic retinopathy (DR) is one of the severe microvascular complications of diabetes. The protective effects of FA on retinal vascular endothelial cells against high glucose levels involve in multiple aspects in DR; however, the underlying mechanism is not fully elucidated. In present study, we investigated the transcriptome as well as genome-wide DNA methylation and hydroxymethylation signature in human retinal microvascular endothelial ACBRI 181 cells cultured within high glucose (HG) medium supplemented with or without FA by RNA-seq, MeDIP-seq, and hMeDIP-seq. Total 3308 differential expressed genes (DEGs) were involved in multiple biological processes and molecular functions containing angiogenesis, inflammation, S-adenosyl methionine metabolism, and hypoxia response. Moreover, the global DNA methylation and hydroxymethylation in ACBRI 181 cells with FA treatment were both compromised compared to HG. Combined with transcriptome data, four subclusters of DEGs with hyper- or hypomethylated promoters were further verified. Unexpectedly, promoters of these 487 genes all displayed a pattern of increased DNA hydroxymethylation. Furthermore, hyperglycemia rat model was established and administered with FA. The DNA methylation and hydroxymethylation changes of selected target genes COL1A1, ITGA7, MMP-14, and VEGFB confirmed by MeDIP-qPCR were consistent with the results in human ACBRI 181 cells. Finally, the presence of activated DNMT1 and TET2 induced by FA was determined in ACBRI 181 cells and hyperglycemia rat. Taken together, this research provided a resource of expression and epigenetic profiles in retinal microvascular endothelial cell, emphasizing a pharmacological mechanism of FA on DNA methylation and hydroxymethylation regulation in retinal microvessel cells of DR.

Alterations of DNA methylation were associated with the rapid growth of cortisol-producing adrenocortical adenoma during pregnancy

BackgroundCortisol-producing adrenocortical adenoma (CPA) during pregnancy rarely occurs in clinic. Growing evidence suggests that DNA methylation plays a key role in adrenocortical adenomas. The present study aims to examine the genome-wide DNA methylation profiles and identify the differences in DNA methylation signatures of non-pregnant and pregnant patients with CPA.ResultsFour pregnant and twelve non-pregnant patients with CPA were enrolled. The pregnant patients with CPA had higher serum cortisol, Estradiol, Progesterone, and human chorionic gonadotropin concentration, while having lower serum FSH (follicle-stimulating hormone) and luteinizing hormone concentrations (P < 0.01). Compared with the non-pregnant patients, the duration is shorter, and the growth rate of the tumor is faster in pregnant patients with CPA (P < 0.05). Morphology and cell proliferation assay showed that the percentage of Ki-67 positive cells in CPA were higher in pregnant group than non-pregnant group (8.0% vs 5.5%, P < 0.05). The DNA methylation analysis showed that Genome-wide DNA methylation signature difference between pregnant and non-pregnant with CPA, that the pregnant group had more hypermethylated DMPs (67.94% vs 22.16%) and less hypomethylated DMPs (32.93% vs 77.84%). The proportion of hypermethylated DMPs was relatively high on chromosomes 1 (9.68% vs 8.67%) and X (4.99% vs 3.35%) but lower on chromosome 2(7.98% vs 12.92%). In pregnant patients with CPA, 576 hypomethylated DMPs and 1109 hypermethylated DMPs were identified in the DNA promoter region. Bioinformatics analysis indicated that the Wnt/β-Catenin pathway, Ras/MAPK Pathway and PI3K-AKT Pathway were associated with the development of CPA during pregnancy.ConclusionsGenome-wide DNA methylation profiling of CPA in non-pregnant and pregnant patients was identified in the present study. Alterations of DNA methylation were associated with the pathogenesis and exacerbation of CPA during pregnancy.

Epigenetic Phenocopying Expands Molecular Risk Assessment in Acute Myeloid Leukemia (Alliance)

Genetic profiling of leukemic cells forms the basis for risk stratification in acute myeloid leukemia (AML). Genetic markers in AML are used to make recommendations for distinct treatment approaches, such as remission consolidation with chemotherapy rather than stem cell transplant for patients with favorable risk genetics as defined by the current guidelines from the European LeukemiaNet (ELN). Yet, several limitations remain, such as overall rarity of many mutations, hierarchical complexity in cases with multiple mutations, conflicting reports of associations with outcomes for some genetic markers, and the absence of markers with prognostic significance in some patients. We have recently described genome-wide DNA methylation signatures that underlie biological features of AML cells and their utility to classify patients [Giacopelli et al. Genome Res. 2021;31:747]. The additional value of epigenetic information for risk assessment in AML in the context of current genetic and other clinical prognostic markers remains largely unexplored.In this study, we have first developed a targeted approach for assessment of DNA methylation-based signatures and employed it to classify 1,262 patients with de novo AML enrolled onto the Cancer and Leukemia Group B/Alliance for Clinical Trials in Oncology studies. We successfully classified 87.5% of patients into one of 13 DNA methylation subgroups, termed ‘epitypes’ (Figure 1A,B). We found that epitypes are composed of a majority of patients with a specific genetic alteration (or a unique combination of alterations) in 9 of 13 epitypes. However, we also identified subgroups of patients that lack these highly recurrent alterations, and, instead, represent an epigenetic phenocopy of the dominant genetic feature (epiphenocopy). Epiphenocopies within epitypes were often enriched in specific lower frequency mutations, suggesting convergence of biological function(s) for these rare mutations. Epiphenocopying was also exhibited by patients displaying a DNA methylation signature involving hypomethylation of STAT DNA sequence motifs (termed the STAT hypomethylation signature, SHS) that mimicked FLT3-ITD mutations. Epitype and SHS DNA methylation signatures affected clinical outcomes separately to ELN risk groups (P<0.0001; Figures 1C,D), and FLT3-ITD status (P<0.0001; Figure 1E), respectively.To broadly examine the prognostic power of DNA methylation signatures, we combined methylation-based classifications into a knowledge bank containing a compendium of other prognostic markers. Using a recently developed machine-learning approach [Gerstung et al. Nat Genet. 2017;49(3):332], we found that DNA methylation retained a high degree of importance for clinical outcomes, including overall survival (Figure 1F). Specifically, SHS and 6 epitypes were the most significant features negatively associated with overall survival along with age (P<0.0001; Figure 1G). SHS and epitype were among the most significantly associated features for all other endpoints, such as early death, remission and relapse (P<0.0001) and improved concordance between all predicted to actual outcomes. Finally, we used DNA methylation to reconstruct all 4 genetic features that define the ELN Favorable risk group. We found that patients with epiphenocopies of t(8;21)/inv(16) (CBF-AML), and CEBPA-dm had favorable outcome indistinguishable from that of patients with the respective genetic markers. NPM1-mutated, FLT3-ITD-negative patients displaying SHS-positivity had adverse risk despite lacking FLT3-ITD. Re-classifying patients with CBF-AML and CEBPA-dm epiphenocopies from more unfavorable risk groups into favorable group and excluding KMT2A/MLL-like and SHS-positive patients substantially improves the definition of favorable risk AML (P<0.0001; Figure 1H).Our study demonstrates that DNA methylation signatures advance our understanding of the biology of AML and improve risk stratification through the identification of patients with epiphenocopies that mimic genetic mutations and other biological features. Use of DNA methylation signatures may lead to more effective assignment of patients to existing and novel therapeutic approaches.Support: U10CA180821, U10CA180882, U24CA196171; https://acknowledgments.alliancefound.org; ClinicalTrials.gov Identifiers: NCT00048958 (8461), NCT00899223 (9665), and NCT00900224 (20202) [Display omitted] DisclosuresBlachly: KITE: Consultancy, Honoraria; INNATE: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; AstraZeneca: Consultancy, Honoraria. Blum: Abbvie: Honoraria; AmerisourceBergen: Honoraria; Celyad Oncology: Research Funding; Xencor: Research Funding; Nkarta: Research Funding; Forma Therapeutics: Research Funding; Leukemia and Lymphoma Society: Research Funding; Syndax: Honoraria. Stone: Agios Pharmaceuticals Inc, Novartis;: Research Funding; ACI Clinical, Syntrix Pharmaceuticals, Takeda Oncology: Other: Data Safety & Monitoring; AbbVie Inc, Actinium Pharmaceuticals Inc, Aprea Therapeutics, BerGenBio ASA, ElevateBio, Foghorn Therapeutics, GEMoaB, GlaxoSmithKline, Innate Pharma, Syndax Pharmaceuticals Inc, Syros Pharmaceuticals Inc, Takeda Oncology: Other: Advisory Committee. Eisfeld: Karyopharm (spouse): Current Employment. Byrd: Novartis, Trillium, Astellas, AstraZeneca, Pharmacyclics, Syndax: Consultancy, Honoraria; Vincerx Pharmaceuticals: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Newave: Membership on an entity's Board of Directors or advisory committees.

Genome-wide DNA methylation pattern in systemic sclerosis microvascular endothelial cells: Identification of epigenetically affected key genes and pathways.

The etiology of systemic sclerosis is not clear, but there is evidence suggesting a critical role for epigenetic alterations in disease pathogenesis and clinical expression. We sought, in this study, to characterize the genome-wide DNA methylation signature in systemic sclerosis microvascular endothelial cells. We performed a genome-wide DNA methylation study in microvascular endothelial cells derived from seven diffuse cutaneous systemic sclerosis patients compared to seven age-, sex-, and ethnicity-matched healthy controls. We paired matched samples on Illumina HumanMethylation450 (three diffuse cutaneous systemic sclerosis microvascular endothelial cells and three controls), and reproduced the results in an independent set of matched patient and controls using Illumina Infinium MethylationEPIC (four diffuse cutaneous systemic sclerosis patients and four controls) to identify differentially methylated genes. We identified 71,353 differentially methylated CpG sites in systemic sclerosis microvascular endothelial cells using Infinium MethylationEPIC microarray in the first group (0.081% of representative probes) and 33,170 CpG sites in the second group using HumanMethylation450 microarray (0.073% of representative probes) in diffuse cutaneous systemic sclerosis microvascular endothelial cells. Among the two groups of subjects, we identified differential methylation of 2455 CpG sites, representing 1301 genes. Most of the differentially methylated CpG sites were hypermethylated (1625 CpG), corresponding to 910 genes. Common hypermethylated genes in systemic sclerosis microvascular endothelial cells include NOS1, DNMT3A, DNMT3B, HDAC4, and ANGPT2. We also identified hypomethylation of IL17RA, CTNNA3, ICAM2, and SDK1 in systemic sclerosis microvascular endothelial cells. Furthermore, we demonstrate significant inverse correlation between DNA methylation status and gene expression in the majority of genes evaluated. Gene ontology analysis of hypermethylated genes demonstrated enrichment of genes involved in angiogenesis (p = 0.0006). Pathway analysis of hypomethylated genes includes genes involved in vascular smooth muscle contraction (p = 0.014) and adherens junctions (p = 0.013). Our data suggest the presence of significant genome-wide DNA methylation aberrancies in systemic sclerosis microvascular endothelial cells, and identify novel affected genes and pathways in systemic sclerosis microvascular endothelial cells.

Stable DNMT3L overexpression in SH-SY5Y neurons recreates a facet of the genome-wide Down syndrome DNA methylation signature

BackgroundDown syndrome (DS) is characterized by a genome-wide profile of differential DNA methylation that is skewed towards hypermethylation in most tissues, including brain, and includes pan-tissue differential methylation. The molecular mechanisms involve the overexpression of genes related to DNA methylation on chromosome 21. Here, we stably overexpressed the chromosome 21 gene DNA methyltransferase 3L (DNMT3L) in the human SH-SY5Y neuroblastoma cell line and assayed DNA methylation at over 26 million CpGs by whole genome bisulfite sequencing (WGBS) at three different developmental phases (undifferentiated, differentiating, and differentiated).ResultsDNMT3L overexpression resulted in global CpG and CpG island hypermethylation as well as thousands of differentially methylated regions (DMRs). The DNMT3L DMRs were skewed towards hypermethylation and mapped to genes involved in neurodevelopment, cellular signaling, and gene regulation. Consensus DNMT3L DMRs showed that cell lines clustered by genotype and then differentiation phase, demonstrating sets of common genes affected across neuronal differentiation. The hypermethylated DNMT3L DMRs from all pairwise comparisons were enriched for regions of bivalent chromatin marked by H3K4me3 as well as differentially methylated sites from previous DS studies of diverse tissues. In contrast, the hypomethylated DNMT3L DMRs from all pairwise comparisons displayed a tissue-specific profile enriched for regions of heterochromatin marked by H3K9me3 during embryonic development.ConclusionsTaken together, these results support a mechanism whereby regions of bivalent chromatin that lose H3K4me3 during neuronal differentiation are targeted by excess DNMT3L and become hypermethylated. Overall, these findings demonstrate that DNMT3L overexpression during neurodevelopment recreates a facet of the genome-wide DS DNA methylation signature by targeting known genes and gene clusters that display pan-tissue differential methylation in DS.

Temporal genome-wide DNA methylation signature of post-smolt Pacific salmon challenged with Piscirickettsia salmonis

ABSTRACT Piscirickettsiosis is the most important bacterial disease in the Chilean salmon industry, which has sorted several efforts to its control, generating enormous economic losses. Epigenetic alterations, such as DNA methylation, can play a relevant role in the modulation of the metazoans response to pathogens. Bacterial disease may activate global and local immune responses generating intricate responses with significant biological impact in the host. However, it is scarcely understood how bacterial infections influence fish epigenetic alterations. In the present study, we utilized Pacific salmon and Piscirickettsiosis as model, to gain understanding into the dynamics of DNA methylation among fish-bacterial infection interactions. A genome-wide analysis of DNA methylation patterns in female spleen tissue of Pacific salmon was achieved by reduced representation bisulphite sequencing from a time course design. We determined 2,251, 1,918, and 2,516 differentially methylated regions DMRs among infected and control Pacific salmon in 1 dpi, 5 dpi, and 15 dpi, respectively. The mean methylation difference per DMR among control and infected groups was of ~35%, with an oscillatory pattern of hypo, hyper, and hypomethylation across the disease. DMCs, among the control and infected group, showed that they were statistically enriched in intergenic regions and depleted in exons. Functional annotation of the DMR genes demonstrated three KEGG principal categories, associated directly with the host response to pathogens infections. Our results provide the first evidence of epigenetic variation in fish provoked by bacterial infection and demonstrate that this variation can be modulated across the disease.

DNA Methylation Profiling and Genomic Analysis in 20 Children with Short Stature Who Were Born Small for Gestational Age.

In a significant proportion of children born small for gestational age (SGA) with failure of catch-up growth, the etiology of short stature remains unclear after routine diagnostic workup. We wanted to investigate if extensive analysis of the (epi)genome can unravel the cause of growth failure in a significant portion of these children. Twenty SGA children treated with GH because of short stature were selected from the BELGROW database of the Belgian Society for Pediatric Endocrinology and Diabetology for exome sequencing, single-nucleotide polymorphism (SNP) array and genome-wide methylation analysis to identify the (epi)genetic cause. First-year response to GH was compared with the response of SGA patients in the KIGS database. We identified (likely) pathogenic variants in 4 children (from 3 families) using exome sequencing and found pathogenic copy number variants in 2 probands using SNP array. In a child harboring a NSD1-containing microduplication, we identified a DNA methylation signature that is opposite to the genome-wide DNA methylation signature of Sotos syndrome. Moreover, we observed multilocus imprinting disturbances in 2 children in whom no other genomic alteration could be identified. Five of 6 children with a genetic diagnosis had an "above average" response to GH. The study indicates that a more advanced approach with deep genotyping can unravel unexpected (epi)genomic alterations in SGA children with persistent growth failure. Most SGA children with a genetic diagnosis had a good response to GH treatment.

Genome-Wide DNA Methylation Signatures of Sea Cucumber Apostichopus japonicus during Environmental Induced Aestivation.

Organisms respond to severe environmental changes by entering into hypometabolic states, minimizing their metabolic rates, suspending development and reproduction, and surviving critical ecological changes. They come back to an active lifestyle once the environmental conditions are conducive. Marine invertebrates live in the aquatic environment and adapt to environmental changes in their whole life. Sea cucumbers and sponges are only two recently known types of marine organisms that aestivate in response to temperature change. Sea cucumber has become an excellent model organism for studies of environmentally-induced aestivation by marine invertebrates. DNA methylation, the most widely considered epigenetic marks, has been reported to contribute to phenotypic plasticity in response to environmental stress in aquatic organisms. Most of methylation-related enzymes, including DNA methyltransferases, Methyl-CpG binding domain proteins, and DNA demethylases, were up-regulated during aestivation. We conducted high-resolution whole-genome bisulfite sequencing of the intestine from sea cucumber at non-aestivation and deep-aestivation stages. Further DNA methylation profile analysis was also conducted across the distinct genomic features and entire transcriptional units. A different elevation in methylation level at internal exons was observed with clear demarcation of intron/exon boundaries during transcriptional unit scanning. The lowest methylation level occurs in the first exons, followed by the last exons and the internal exons. A significant increase in non-CpG methylation (CHG and CHH) was observed within the intron and mRNA regions in aestivation groups. A total of 1393 genes were annotated within hypermethylated DMRs (differentially methylated regions), and 749 genes were annotated within hypomethylated DMRs. Differentially methylated genes were enriched in the mRNA surveillance pathway, metabolic pathway, and RNA transport. Then, 24 hypermethylated genes and 15 hypomethylated genes were Retrovirus-related Pol polyprotein from transposon (RPPT) genes. This study provides further understanding of epigenetic control on environmental induced hypometabolism in aquatic organisms.

Abstract LB-174: Genome-wide DNA methylation signatures predict the early asymptomatic doxorubicin-induced cardiotoxicity in breast cancer

Abstract Background: Doxorubicin (DOX)-induced cardiotoxicity is cumulative-dose-dependent that begins with the first dose, but epigenetic changes after the first dose have not been examined. This study had two aims: Aim 1: to examine whether DNA methylation profile of peripheral blood cells (PBCs) induced by the first cycle of DOX-based chemotherapy can predict the risk of cardiotoxicity; and Aim 2 to determine if there are pretreatment methylation signatures at baseline to predict the risk of cardiotoxicity. Methods: Whole-genome DNA methylation of PBCs from 19 breast cancer patients was examined prior to the start of DOX-based chemotherapy and after the first cycle using Infinium HumanMethylation 450 BeadChip. ChAMP R package was used for the data normalization by the BMIQ algorithm and the COMBAT algorithm to adjust for batch effects. Chemotherapy-induced cardiotoxicity was determined by the assessment of left ventricle ejection fraction (LVEF) with multigated acquisition (MUGA) scan before chemotherapy and at its completion, and any LVEF decline by &amp;gt;10% was considered abnormal. Results: Pre- and post- DOX treatment were compared between patients with abnormal LVEF decline and those who maintained normal LVEF to assess the differences in the cytosine methylation states. The multiple correction testing resulted in a total of 515 differentially methylated CpGs between the patients with abnormal versus normal LVEF post-DOX treatment. There were 379 differentially methylated CpGs at baseline and 136 CpGs after the first cycle which significantly correlated with LVEF status after applying the Holm-Bonferroni method for multiple testing correction. The intersection of the probes resulted in 38 differentially methylated genes (DMG) that when used in hierarchical clustering, correctly clustered baseline samples into those that would result in normal and abnormal LVEF after treatment. DMG genes included SEPT5, IRF6, KCNQ1, NELL1, SIX6, HOXC9 and BCAN. IRF6, which encodes interferon regulatory factor 6, was identified as being significant in the subsequent analysis at the probe and region level. Comparisons of the samples before and after DOX treatment did not result in any probes associated with the treatment that were significant after multiple testing correction. Conclusions: The results from this study provide evidence that DNA methylation profile of peripheral blood cells has the potential to predict the risk of DOX-induced cardiotoxicity. The important finding was that the extent of methylation at baseline correlated with the post-DOX LVEF reduction, indicating that it may have the potential to predict the subsequent development of cardiotoxicity. Further studies with larger cohort of patients are needed to confirm these findings as well as narrowing down candidate methylation markers that can be implemented in a blood test. These finding may help guide treatment or identify patients that need to be followed closely to mitigate heart damage after DOX treatment. Citation Format: Michael A. Bauer, Issam Makhoul, Ping-Ching Hsu, Jeanne Wei, L. Joseph Su, Annjanette Stone, Weleetka Carter, Valentina K. Todorova. Genome-wide DNA methylation signatures predict the early asymptomatic doxorubicin-induced cardiotoxicity in breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-174.

Genome-wide DNA Methylation Signatures Are Determined by DNMT3A/B Sequence Preferences.

Cytosine methylation is an important epigenetic mark, but how the distinctive patterns of DNA methylation arise remains elusive. For the first time, we systematically investigated how these patterns can be imparted by the inherent enzymatic preferences of mammalian de novo DNA methyltransferases in vitro and the extent to which this applies in cells. In a biochemical experiment, we subjected a wide variety of DNA sequences to methylation by DNMT3A or DNMT3B and then applied deep bisulfite sequencing to quantitatively determine the sequence preferences for methylation. The data show that DNMT3A prefers CpG and non-CpG sites followed by a 3′-pyrimidine, whereas DNMT3B favors a 3′-purine. Overall, we show that DNMT3A has a sequence preference for a TNC[G/A]CC context, while DNMT3B prefers TAC[G/A]GC. We extended our finding using publicly available data from mouse Dnmt1/3a/3b triple-knockout cells in which reintroduction of either DNMT3A or DNMT3B expression results in the acquisition of the same enzyme specific signature sequences observed in vitro. Furthermore, loss of DNMT3A or DNMT3B in human embryonic stem cells leads to a loss of methylation at the corresponding enzyme specific signatures. Therefore, the global DNA methylation landscape of the mammalian genome can be fundamentally determined by the inherent sequence preference of de novo methyltransferases.

A genome-wide DNA methylation signature for SETD1B-related syndrome

SETD1B is a component of a histone methyltransferase complex that specifically methylates Lys-4 of histone H3 (H3K4) and is responsible for the epigenetic control of chromatin structure and gene expression. De novo microdeletions encompassing this gene as well as de novo missense mutations were previously linked to syndromic intellectual disability (ID). Here, we identify a specific hypermethylation signature associated with loss of function mutations in the SETD1B gene which may be used as an epigenetic marker supporting the diagnosis of syndromic SETD1B-related diseases. We demonstrate the clinical utility of this unique epi-signature by reclassifying previously identified SETD1B VUS (variant of uncertain significance) in two patients.

OTHR-09. IDENTIFYING EPIGENETIC SIGNATURES IN LUNG ADENOCARCINOMAS THAT PREDICT DEVELOPMENT OF BRAIN METASTASIS

INTRODUCTION: Metastases are the most common adult brain tumor with half spreading from lung cancers and they reduce median overall survival from 26 to 12 months. There are no robust patient-specific predictors of brain metastasis. Epigenetic signatures predict disease recurrence in other cancers and identifying brain metastasis methylation-based signatures may allow for treatment approaches to high-risk patients that prevent their development. METHODS: In 207 lung adenocarcinomas, multivariate cox time to brain metastasis analyses including clinically-relevant variables (lung tumor size and TNM nodal score) along with significant covariates on univariate analyses were performed. DNA was extracted from 142 of these tumors and profiled on the Illumina Infinium EPIC array. A generalized boosted regression classification model used differentially methylated CpG sites significantly predicting time to brain metastasis in a 70% training cohort cox analysis (p< 0.05). Resulting methylation-based risk scores were compared to size and nodal status in a multivariate analysis of the independent 30% testing cohort. RESULTS: Of 207 patients with 72 brain metastatic events, tumor size (HR=1.5, 95%CI 1.1–2.0, p=0.01), N status (N3 vs. N0, HR=9.9, 95%CI 3.1–31, p=0.0001), EGFR status (HR=0.4, 95%CI 0.2–0.8, p=0.014), and age (HR=0.7, 95%CI 0.5–1.0, p=0.039) independently predicted their development. Methylation-based risk scores significantly predicted time to brain metastasis in a univariate analysis of the testing cohort (p=0.03). A multivariate analysis of testing cohort patients identified methylation score as the only independent predictor of brain metastasis (HR=4.3, 95%CI 1.1–17, p=0.038) accounting for tumor size and N score. CONCLUSIONS: Genome-wide DNA methylation signatures predict brain metastasis development in lung adenocarcinomas independent of tumor size and nodal disease. The design of a nomogram combining methylation profile other clinical factors may be used to determine patient specific brain metastasis risk values to guide patient counselling, extent of treatment, and screening.

Abstract 815: Genome-wide DNA methylation signatures to predict pathologic complete response from combined neoadjuvant chemotherapy with bevacizumab in breast cancer

Abstract Neoadjuvant chemotherapy is given before surgery to patients with primarily unresectable, advanced-stage cancers to render the tumor resectable, and to facilitate breast conservation. Previously, we have reported a prospective phase II trial for women with stage IIA-B/IIIA-B-C breast cancer with improved pathologic complete response (pCR) when using bevacizumab in the neoadjuvant setting. Chemotherapy agents are given orally or intravenously during multiple cycles of systemic treatments. However, the effect of the treatment can only be evaluated upon the completion of therapy. Here, we aim to identify epigenetic signatures in blood for patients at baseline as non-invasive markers to predict their pCR from the treatment, and to determine if treatment-related changes in epigenetic profiles reflect responsiveness to therapy. We performed genome-wide DNA methylation profiling using blood-derived DNA, and found that pre-treatment methylation status of BRD9 was predictive of responsiveness to therapy. Post-treatment global methylation differences were also observed between responders and non-responders. Most differentially methylated CpGs were located in promoter CpG island regions for responders, but in the open sea region for non-responders. DNMT3B was hypomethylated while most of the other genes were hypermethylated after 4 cycles of treatment. Hypomethylation of DNMT3B could potentially lead to the increased methylation of oncogenes and genes responsible for cell growth and proliferation, facilitating responsiveness to the therapy. These results support the development of BRD9 as a biomarker for treatment selection before neoadjuvant therapy with bevacizumab, and indicate DNMT3B as a potential target to improve clinical response. Citation Format: Ping-Ching Hsu, Susan Kadlubar, Eric Siegel, Lora J. Rogers, Valentina K. Todorova, Joseph Su, Issam Makhoul. Genome-wide DNA methylation signatures to predict pathologic complete response from combined neoadjuvant chemotherapy with bevacizumab in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 815.