Differentially Methylated CpGs Research Articles

Abstract A002: In-silico screening of race-specific endometrial cancer methylation markers using machine learning techniques

Abstract Targeted molecular therapy is a promising strategy for cancer treatment. Despite the benefits of these therapies, racial disparities in cancer therapy efficacy persist. To close this racial gap, there is a need to find race-specific biomarkers at the molecular level. Since abnormal DNA methylation has been linked with cancer and has a great potential to transform the diagnosis and treatment of cancer, the main objective of this study was to combine differential DNA methylation and machine learning techniques to identify race-specific methylation signature biomarkers in endometrial tumor samples from White and Black women. We performed genome wide Differentially Methylated CpG (DMCs) analysis in tumors from White patients (TWP) samples compared to tumors from Black patients (TBP) samples using Endometrial Carcinoma (EC) methylation and clinical data from The Cancer Genome Atlas (TCGA). We used supervised and unsupervised machine learning techniques to filter and select signature biomarkers. To evaluate the prognostic capacity of these genes, we performed univariate and multivariate survival analyses using Cox proportional hazards regression model from survival R package. By combining differential methylation CpGs and machine learning techniques, we reduced 704 differentially methylated CpGs to reliable race-specific epigenetic signature genes (ESGs) and Core- epigenetic signature genes (core-ESGs). An area under the curve (AUC) of 0.98 was achieved based on these signature genes. We further found that ESGs showed statistically significant overall survival differences between two racial groups. In addition, comparison of the mutational landscape showed differences in mutation frequencies in the ESGs between the two groups. By combining differential methylation analysis and machine learning techniques, we identified novel race-specific epigenetic signature genes in Black and White women with endometrial cancer. We further showed the potential of these gene in predicting survival differences, mutation, and transcriptomic differences in Black and White women. We propose that combined methylation and somatic mutation profiling of these novel race-specific signature genes can be used to develop potential therapeutic targets for diagnosis and treating endometrial cancer. Citation Format: Huma Asif, Grace Foley, Julie Kim. In-silico screening of race-specific endometrial cancer methylation markers using machine learning techniques [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr A002.

Aspirin Challenge-Induced Genome-Wide DNA Methylation Profile of Peripheral Blood Lymphocytes in Aspirin-Exacerbated Respiratory Disease.

Genetic variation and epigenetic factors are thought to contribute to the development of hypersensitivity to aspirin. DNA methylation fluctuates dynamically throughout the day. To discover new CpG methylation in lymphocytes associated with aspirin-exacerbated respiratory disease (AERD), we evaluated changes in global CpG methylation profiles from before to after an oral aspirin challenge in patients with AERD and aspirin-tolerant asthma (ATA). Whole-genome CpG methylation levels of peripheral blood mononuclear cells were quantified with an Illumina 860K Infinium Methylation EPIC BeadChip array and then adjusted for inferred lymphocyte fraction (ILF) with GLINT and Tensor Composition Analysis. Among the 866,091 CpGs in the array, differentially methylated CpGs (DMCs) were found in 6 CpGs in samples from all 12 patients with asthma included in the study (AERD, n = 6; ATA, n = 6). DMCs were found in 3 CpGs in the 6 ATA samples and in 615 CpGs in the 6 AERD samples. A total of 663 DMCs in 415 genes and 214 intergenic regions differed significantly in the AERD compared with the ATA. In promoters, 126 CpG loci were predicted to bind to 38 transcription factors (TFs), many of which were factors already known to be involved in the pathogenesis of asthma and immune responses. In conclusion, we identified 615 new CpGs methylated in peripheral blood lymphocytes by oral aspirin challenge in AERD but not in ATA. These findings indicate that oral aspirin challenge induces epigenetic changes in ILFs, specifically in AERD patients, possibly via changes in TF binding, which may have epigenetic effects on the development of AERD.

Tumor microenvironment deconvolution identifies cell-type-independent aberrant DNA methylation and gene expression in prostate cancer

BackgroundAmong men, prostate cancer (PCa) is the second most common cancer and the second leading cause of cancer death. Etiologic factors associated with both prostate carcinogenesis and somatic alterations in tumors are incompletely understood. While genetic variants associated with PCa have been identified, epigenetic alterations in PCa are relatively understudied. To date, DNA methylation (DNAm) and gene expression (GE) in PCa have been investigated; however, these studies did not correct for cell-type proportions of the tumor microenvironment (TME), which could confound results.MethodsThe data (GSE183040) consisted of DNAm and GE data from both tumor and adjacent non-tumor prostate tissue of 56 patients who underwent radical prostatectomies prior to any treatment. This study builds upon previous studies that examined methylation patterns and GE in PCa patients by using a novel tumor deconvolution approach to identify and correct for cell-type proportions of the TME in its epigenome-wide association study (EWAS) and differential expression analysis (DEA).ResultsThe inclusion of cell-type proportions in EWASs and DEAs reduced the scope of significant alterations associated with PCa. We identified 2,093 significantly differentially methylated CpGs (DMC), and 51 genes associated with PCa, including PCA3, SPINK1, and AMACR.ConclusionsThis work illustrates the importance of correcting for cell types of the TME when performing EWASs and DEAs on PCa samples, and establishes a more confounding-adverse methodology. We identified a more tumor-cell-specific set of altered genes and epigenetic marks that can be further investigated as potential biomarkers of disease or potential therapeutic targets.

Methylation analysis by targeted bisulfite sequencing in large for gestational age (LGA) newborns: the LARGAN cohort

BackgroundIn 1990, David Barker proposed that prenatal nutrition is directly linked to adult cardiovascular disease. Since then, the relationship between adult cardiovascular risk, metabolic syndrome and birth weight has been widely documented. Here, we used the TruSeq Methyl Capture EPIC platform to compare the methylation patterns in cord blood from large for gestational age (LGA) vs adequate for gestational age (AGA) newborns from the LARGAN cohort.ResultsWe found 1672 differentially methylated CpGs (DMCs) with a nominal p < 0.05 and 48 differentially methylated regions (DMRs) with a corrected p < 0.05 between the LGA and AGA groups. A systems biology approach identified several biological processes significantly enriched with genes in association with DMCs with FDR < 0.05, including regulation of transcription, regulation of epinephrine secretion, norepinephrine biosynthesis, receptor transactivation, forebrain regionalization and several terms related to kidney and cardiovascular development. Gene ontology analysis of the genes in association with the 48 DMRs identified several significantly enriched biological processes related to kidney development, including mesonephric duct development and nephron tubule development. Furthermore, our dataset identified several DNA methylation markers enriched in gene networks involved in biological pathways and rare diseases of the cardiovascular system, kidneys, and metabolism.ConclusionsOur study identified several DMCs/DMRs in association with fetal overgrowth. The use of cord blood as a material for the identification of DNA methylation biomarkers gives us the possibility to perform follow-up studies on the same patients as they grow. These studies will not only help us understand how the methylome responds to continuum postnatal growth but also link early alterations of the DNA methylome with later clinical markers of growth and metabolic fitness.

Epigenome-wide association analysis of infant bronchiolitis severity: a multicenter prospective cohort study

Bronchiolitis is the most common lower respiratory infection in infants, yet its pathobiology remains unclear. Here we present blood DNA methylation data from 625 infants hospitalized with bronchiolitis in a 17-center prospective study, and associate them with disease severity. We investigate differentially methylated CpGs (DMCs) for disease severity. We characterize the DMCs based on their association with cell and tissues types, biological pathways, and gene expression. Lastly, we also examine the relationships of severity-related DMCs with respiratory and immune traits in independent cohorts. We identify 33 DMCs associated with severity. These DMCs are differentially methylated in blood immune cells. These DMCs are also significantly enriched in multiple tissues (e.g., lung) and cells (e.g., small airway epithelial cells), and biological pathways (e.g., interleukin-1-mediated signaling). Additionally, these DMCs are associated with respiratory and immune traits (e.g., asthma, lung function, IgE levels). Our study suggests the role of DNA methylation in bronchiolitis severity.

Epigenetic responses to rhinovirus exposure in airway epithelial cells are correlated with key transcriptional pathways in chronic rhinosinusitis.

Viruses may drive immune mechanisms responsible for chronic rhinosinusitis with nasal polyposis (CRSwNP), but little is known about the underlying molecular mechanisms. To identify epigenetic and transcriptional responses to a common upper respiratory pathogen, rhinovirus (RV), that are specific to patients with CRSwNP using a primary sinonasal epithelial cell culture model. Airway epithelial cells were collected at surgery from patients with CRSwNP (cases) and from controls without sinus disease, cultured, and then exposed to RV or vehicle for 48 h. Differential gene expression and DNA methylation (DNAm) between cases and controls in response to RV were determined using linear mixed models. Weighted gene co-expression analysis (WGCNA) was used to identify (a) co-regulated gene expression and DNAm signatures, and (b) genes, pathways, and regulatory mechanisms specific to CRSwNP. We identified 5585 differential transcriptional and 261 DNAm responses (FDR <0.10) to RV between CRSwNP cases and controls. These differential responses formed three co-expression/co-methylation modules that were related to CRSwNP and three that were related to RV (Bonferroni corrected p < .01). Most (95%) of the differentially methylated CpGs (DMCs) were in modules related to CRSwNP, whereas the differentially expressed genes (DEGs) were more equally distributed between the CRSwNP- and RV-related modules. Genes in the CRSwNP-related modules were enriched in known CRS and/or viral response immune pathways. RV activates specific epigenetic programs and correlated transcriptional networks in the sinonasal epithelium of individuals with CRSwNP. These novel observations suggest epigenetic signatures specific to patients with CRSwNP modulate response to viral pathogens at the mucosal environmental interface. Determining how viral response pathways are involved in epithelial inflammation in CRSwNP could lead to therapeutic targets for this burdensome airway disorder.

Dissecting the Methylomes of EGFR-Amplified Glioblastoma Reveals Altered DNA Replication and Packaging, and Chromatin and Gene Silencing Pathways.

Glioblastoma IDH wildtype is the most frequent brain tumor in adults. It shows a highly malignant behavior and devastating outcomes. To date, there is still no targeted therapy available; thus, patients' median survival is limited to 12-15 months. Epithelial growth factor receptor (EGFR) is an interesting targetable candidate in advanced precision medicine for brain tumor patients. In this study, we performed integrated epigenome-wide DNA-methylation profiling of 866,895 methylation specific sites in 50 glioblastoma IDH wildtype samples, comparing EGFR amplified and non-amplified glioblastomas. We found 9849 significantly differentially methylated CpGs (DMCGs) with Δβ ≥ 0.1 and p-value < 0.05 in EGFR amplified, compared to EGFR non-amplified glioblastomas. Of these DMCGs, 2380 were annotated with tiling (2090), promoter (117), gene (69) and CpG islands (104); 7460 are located at other loci. Interestingly, the list of differentially methylated genes allocated eleven functionally relevant RNAs: five miRNAs (miR1180, miR1255B1, miR126, miR128-2, miR3125), two long non-coding RNAs (LINC00474, LINC01091), and four antisense RNAs (EPN2-AS1, MNX1-AS2, NKX2-2-AS1, WWTR1-AS1). Gene ontology (GO) analysis showed enrichment of "DNA replication-dependent nucleosome assembly", "chromatin silencing at rDNA", "regulation of gene silencing by miRNA", "DNA packaging", "posttranscriptional gene silencing", "gene silencing by RNA", "negative regulation of gene expression, epigenetic", "regulation of gene silencing", "protein-DNA complex subunit organization", and "DNA replication-independent nucleosome organization" pathways being hypomethylated in EGFR amplified glioblastomas. In summary, dissecting the methylomes of EGFR amplified and non-amplified glioblastomas revealed altered DNA replication, DNA packaging, chromatin silencing and gene silencing pathways, opening potential novel targets for future precision medicine.

DNA methylation analysis reveals the effect of arsenic on gestational diabetes mellitus

BackgroundArsenic (As) exposure is one of the risk factors for gestational diabetes mellitus (GDM). This study aimed to explore the effect of As-exposure on DNA methylation in GDM and to establish a risk assessment model of GDM in As exposed pregnant women. MethodWe collected elbow vein blood of pregnant women before delivery to measure As concentration and DNA methylation data. Then compared the DNA methylation data and established a nomogram. ResultWe identified a total of 10 key differentially methylated CpGs (DMCs) and found 6 corresponding genes. Functions were enriched in Hippo signaling pathway, cell tight junction, prophetic acid metabolism, ketone body metabolic process, and antigen processing and presentation. A nomogram was established that can predict GDM risks (c-index = 0.595, s:p = 0.973). ConclusionWe found 6 genes associated with GDM with high As exposure. The prediction of the nomograms has been proven to be effective.

DNA-Methylation Signatures of Tobacco Smoking in a High Cardiovascular Risk Population: Modulation by the Mediterranean Diet.

Biomarkers based on DNA methylation are relevant in the field of environmental health for precision health. Although tobacco smoking is one of the factors with a strong and consistent impact on DNA methylation, there are very few studies analyzing its methylation signature in southern European populations and none examining its modulation by the Mediterranean diet at the epigenome-wide level. We examined blood methylation smoking signatures on the EPIC 850 K array in this population (n = 414 high cardiovascular risk subjects). Epigenome-wide methylation studies (EWASs) were performed analyzing differential methylation CpG sites by smoking status (never, former, and current smokers) and the modulation by adherence to a Mediterranean diet score was explored. Gene-set enrichment analysis was performed for biological and functional interpretation. The predictive value of the top differentially methylated CpGs was analyzed using receiver operative curves. We characterized the DNA methylation signature of smoking in this Mediterranean population by identifying 46 differentially methylated CpGs at the EWAS level in the whole population. The strongest association was observed at the cg21566642 (p = 2.2 × 10-32) in the 2q37.1 region. We also detected other CpGs that have been consistently reported in prior research and discovered some novel differentially methylated CpG sites in subgroup analyses. In addition, we found distinct methylation profiles based on the adherence to the Mediterranean diet. Particularly, we obtained a significant interaction between smoking and diet modulating the cg5575921 methylation in the AHRR gene. In conclusion, we have characterized biomarkers of the methylation signature of tobacco smoking in this population, and suggest that the Mediterranean diet can increase methylation of certain hypomethylated sites.

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.

Perinatal Lead Exposure Promotes Sex-Specific Epigenetic Programming of Disease-Relevant Pathways in Mouse Heart.

Environmental contaminants such as the metal lead (Pb) are associated with cardiovascular disease, but the underlying molecular mechanisms are poorly understood. In particular, little is known about how exposure to Pb during early development impacts the cardiac epigenome at any point across the life course and potential differences between sexes. In a mouse model of human-relevant perinatal exposures, we utilized RNA-seq and Enhanced Reduced Representation Bisulfite Sequencing (ERRBS) to investigate the effects of Pb exposure during gestation and lactation on gene expression and DNA methylation, respectively, in the hearts of male and female mice at weaning. For ERRBS, we identified differentially methylated CpGs (DMCs) or differentially methylated 1000 bp regions (DMRs) based on a minimum absolute change in methylation of 10% and an FDR < 0.05. For gene expression data, an FDR < 0.05 was considered significant. No individual genes met the FDR cutoff for gene expression; however, we found that Pb exposure leads to significant changes in the expression of gene pathways relevant to cardiovascular development and disease. We further found that Pb promotes sex-specific changes in DNA methylation at hundreds of gene loci (280 DMCs and 99 DMRs in males, 189 DMCs and 121 DMRs in females), and pathway analysis revealed that these CpGs and regions collectively function in embryonic development. In males, differential methylation also occurred at genes related to immune function and metabolism. We then investigated whether genes exhibiting differential methylation at weaning were also differentially methylated in hearts from a cohort of Pb-exposed mice at adulthood. We found that a single gene, Galnt2, showed differential methylation in both sexes and time points. In a human cohort investigating the influence of prenatal Pb exposure on the epigenome, we also observed an inverse association between first trimester Pb concentrations and adolescent blood leukocyte DNA methylation at a locus in GALNT2, suggesting that this gene may represent a biomarker of Pb exposure across species. Together, these data, across two time points in mice and in a human birth cohort study, collectively demonstrate that Pb exposure promotes sex-specific programming of the cardiac epigenome, and provide potential mechanistic insight into how Pb causes cardiovascular disease.

Genome-wide methylation profiling identify hypermethylated HOXL subclass genes as potential markers for esophageal squamous cell carcinoma detection

BackgroundNumerous studies have revealed aberrant DNA methylation in esophageal squamous cell carcinoma (ESCC). However, they often focused on the partial genome, which resulted in an inadequate understanding of the shaped methylation features and the lack of available methylation markers for this disease.MethodsThe current study investigated the methylation profiles between ESCC and paired normal samples using whole-genome bisulfite sequencing (WGBS) data and obtained a group of differentially methylated CpGs (DMC), differentially methylated regions (DMR), and differentially methylated genes (DMG). The DMGs were then verified in independent datasets and Sanger sequencing in our custom samples. Finally, we attempted to evaluate the performance of these genes as methylation markers for the classification of ESCC.ResultsWe obtained 438,558 DMCs, 15,462 DMRs, and 1568 DMGs. The four significantly enriched gene families of DMGs were CD molecules, NKL subclass, HOXL subclass, and Zinc finger C2H2-type. The HOXL subclass homeobox genes were observed extensively hypermethylated in ESCC. The HOXL-score estimated by HOXC10 and HOXD1 methylation, whose methylation status were then confirmed by sanger sequencing in our custom ESCC samples, showed good ability in discriminating ESCC from normal samples.ConclusionsWe observed widespread hypomethylation events in ESCC, and the hypermethylated HOXL subclass homeobox genes presented promising applications for the early detection of esophageal squamous cell carcinoma.

Dementia with Lewy bodies post-mortem brains reveal differentially methylated CpG sites with biomarker potential

Dementia with Lewy bodies (DLB) is a common form of dementia with known genetic and environmental interactions. However, the underlying epigenetic mechanisms which reflect these gene-environment interactions are poorly studied. Herein, we measure genome-wide DNA methylation profiles of post-mortem brain tissue (Broadmann area 7) from 15 pathologically confirmed DLB brains and compare them with 16 cognitively normal controls using Illumina MethylationEPIC arrays. We identify 17 significantly differentially methylated CpGs (DMCs) and 17 differentially methylated regions (DMRs) between the groups. The DMCs are mainly located at the CpG islands, promoter and first exon regions. Genes associated with the DMCs are linked to “Parkinson’s disease” and “metabolic pathway”, as well as the diseases of “severe intellectual disability” and “mood disorders”. Overall, our study highlights previously unreported DMCs offering insights into DLB pathogenesis with the possibility that some of these could be used as biomarkers of DLB in the future.

Epigenetic Alterations of Repeated Relapses in Patient-matched Childhood Ependymomas

Recurrence is frequent in pediatric ependymoma (EPN). Our longitudinal integrated analysis of 30 patient-matched repeated relapses (3.67 ± 1.76 times) over 13 years (5.8 ± 3.8) reveals stable molecular subtypes (RELA and PFA) and convergent DNA methylation reprogramming during serial relapses accompanied by increased orthotopic patient derived xenograft (PDX) (13/27) formation in the late recurrences. A set of differentially methylated CpGs (DMCs) and DNA methylation regions (DMRs) are found to persist in primary and relapse tumors (potential driver DMCs) and are acquired exclusively in the relapses (potential booster DMCs). Integrating with RNAseq reveals differentially expressed genes regulated by potential driver DMRs (CACNA1H, SLC12A7, RARA in RELA and HSPB8, GMPR, ITGB4 in PFA) and potential booster DMRs (PLEKHG1 in RELA and NOTCH, EPHA2, SUFU, FOXJ1 in PFA tumors). DMCs predicators of relapse are also identified in the primary tumors. This study provides a high-resolution epigenetic roadmap of serial EPN relapses and 13 orthotopic PDX models to facilitate biological and preclinical studies.

EPCO-39. ADAPTIVE CONVERGENCE OF METHYLOMES REVEALS EPIGENETIC DRIVERS AND BOOSTERS OF REPEATED RELAPSES IN PATIENT-MATCHED CHILDHOOD EPENDYMOMAS AND IDENTIFIES TARGETS FOR ANTI-RECURRENCE THERAPIES

Abstract Ependymoma (EPN) is the third most common brain tumor in children and frequently recurs. Here, we report an integrated longitudinal analysis of epigenetic, genetic and tumorigenic changes in 30 patient-matched repeated relapses obtained from 10 pediatric patients to understand the mechanism of recurrences. Genome-wide DNA methylation analysis revealed stable molecular subtypes and convergent epigenetic reprogramming during serial relapses of the 5 RELA and 5 PFA EPNs that paralleled with elevated patient-derived orthotopic xenograft (PDOX) (13/27) formation in the late relapses. Differentially methylated CpGs (DMCs) preexisted in the primary tumors and persisted in the relapses (driver DMCs) were detected, ranging from 51 hypo-methylated in RELA to 148 hyper-methylated DMCs in PFA tumors; while newly acquired DMCs sustained in all the relapses but was absent in the primary tumors (booster DMCs) ranged from 38- 323 hyper-methylated DMCs in RELA and PFA EPNs, respectively. Integrated analysis of these DMC associated DNA methylation regions (DMRs) and RNAseq in both patient and PDOX tumors identified a small fraction of the differentially expressed genes (4.6±4.4% in RELA and 4.5±1.1% in PFA) as regulated by driver DMRs (e.g., up-regulated CACNA1H, SLC12A7, RARA in RELA and HSPB8, GMPR, ITGB4 in PFA) and booster DMRs (including the sole upregulated PLEKHG1 in RELA and NOTCH, EPHA2, SUFU, FOXJ1 in PFA tumors). Most these genes were novel to EPN relapses. Seven DMCs in RELA and 22 in PFA tumors were also identified as potential relapse predictors. Finally, integrating DNA methylation with histone modification identified LSD1 as a relapse driver gene. Combined treatment of a novel inhibitor SYC-836 with radiation significantly prolonged survival times in two PDOX models of recurrent PFA. This high-resolution epigenetic and genetic roadmap of EPN relapse and our 13 new PDOX models should significantly facilitate biological and preclinical studies of pediatric EPN recurrences.

Methylome Profiling of PD-L1-Expressing Glioblastomas Shows Enrichment of Post-Transcriptional and RNA-Associated Gene Regulation.

Glioblastomas are the most frequent primary brain tumors in adults. They show highly malignant behavior and devastating outcomes. Since there are still no targeted therapies available, median survival remains in the range of 12 to 15 months for glioblastoma patients. Programmed Cell Death Ligand 1 (PD-L1) is a promising novel candidate in precision medicine. Here, we performed integrated epigenome-wide methylation profiling of 866,895 methylation-specific sites in 20 glioblastoma samples comparing PD-L1 high- (i.e., TPS (tumor proportion score) &gt; 30%) and PD-L1 low-expressing glioblastomas (i.e., TPS &lt; 10%). We found 12,597 significantly differentially methylated CpGs (DMCG) (Δβ ≥ 0.1 and p-value &lt; 0.05) in PD-L1 high- compared with PD-L1 low-expressing glioblastomas. These DMCGs were annotated to 2546 tiling regions, 139 promoters, 107 genes, and 107 CpG islands. PD-L1 high-expressing glioblastomas showed hypomethylation in 68% of all DMCGs. Interestingly, the list of the top 100 significantly differentially methylated genes showed the enrichment of regulatory RNAs with 19 DMCGs in miRNA, snoRNAs, lincRNAs, and asRNAs. Gene Ontology analysis showed the enrichment of post-transcriptional and RNA-associated pathways in the hypermethylated gene regions. In summary, dissecting the methylomes depending on PD-L1 status revealed significant alterations in RNA regulation and novel molecular targets in glioblastomas.

The Role of Dynamic DNA Methylation in Liver Transplant Rejection in Children.

Background.Transcriptional regulation of liver transplant (LT) rejection may reveal novel predictive and therapeutic targets. The purpose of this article is to test the role of differential DNA methylation in children with biopsy-proven acute cellular rejection after LT.Methods.Paired peripheral blood DNA samples were obtained before and after LT from 17 children, including 4 rejectors (Rs) and 13 nonrejectors (NRs), and assayed with MethylC capture sequencing approach covering 5 million CpGs in immune-cell–specific regulatory elements. Differentially methylated CpGs (DMCs) were identified using generalized linear regression models adjusting for sex and age and merged into differentially methylated regions (DMRs) comprising 3 or more DMCs.Results.Contrasting Rs versus NRs, we identified 2238 DMCs in post-LT and 2620 DMCs in pre-LT samples, which clustered in 216 and 282 DMRs, respectively. DMCs associated with R were enriched in enhancers and depleted in promoters. Among DMRs, the proportion of hypomethylated DMRs increased from 61/282 (22%) in pre-LT to 103/216 (48%, P < 0.0001) in post-LT samples. The highest-ranked biological processes enriched in post-LT DMCs were antigen processing and presentation via major histocompatibility complex (MHC) class I, MHC class I complex, and peptide binding (P < 7.92 × 10−17), respectively. Top-ranked DMRs mapped to genes that mediate B-cell receptor signaling (ADAP1) or regulate several immune cells (ARRB2) (P < 3.75 × 10−08). DMRs in MHC class I genes were enriched for single nucleotide polymorphisms (SNPs), which bind transcription factors, affect gene expression and splicing, or alter peptide-binding amino acid sequences.Conclusions.Dynamic methylation in distal regulatory regions reveals known transplant-relevant MHC-dependent rejection pathways and identifies novel loci for future mechanistic evaluations in pediatric transplant subcohorts.

Gestational diabetes mellitus placentas exhibit epimutations at placental development genes

ABSTRACT Gestational diabetes mellitus (GDM) is a maternal metabolic disorder that perturbs placental development and increases the risk of offspring short- and long-term metabolic disorders. The mechanisms by which GDM impairs placental development remain poorly understood. Here, we defined the DNA methylome of GDM placentas and determined whether GDM perturbs methylation at genes important for placental development. We conducted an epigenome-wide association study of 42 placentas from pregnancies in the South African Soweto First 1000 days cohort (S1000). Using genome-wide bisulfite sequencing, we compared non-GDM placentas to GDM placentas with similar proportions from obese and non-obese mothers. Compared to non-GDM, GDM placentas exhibited a distinct methylation profile consisting of 12,210 differentially methylated CpGs (DMCs) that mapped to 3,875 genes. Epigenetically altered genes were enriched in Wnt and cadherin signalling pathways, both critical in placentation and embryogenesis. We also defined regional DNA methylation perturbation in GDM placentas at 11 placental development genes. These findings reveal extensive changes to the placental epigenome of GDM pregnancies and highlight perturbation enriched at important placental development genes. These molecular changes represent potential mechanisms for GDM-induced placental effects that may serve as candidate biomarkers for placental, maternal, and foetal health. Using a study design that used similar proportions of obese and non-obese mothers in our case and control pregnancies, we minimized the detection of changes due to obesity alone. Further work will be necessary to investigate the extent of the influence of obesity on these GDM-related placental epigenetic changes.

Integrated Analyses of DNA Methylation and Gene Expression of Rainbow Trout Muscle under Variable Ploidy and Muscle Atrophy Conditions.

Rainbow trout, Oncorhynchus mykiss, is an important cool, freshwater aquaculture species used as a model for biological research. However, its genome reference has not been annotated for epigenetic markers affecting various biological processes, including muscle growth/atrophy. Increased energetic demands during gonadogenesis/reproduction provoke muscle atrophy in rainbow trout. We described DNA methylation and its associated gene expression in atrophying muscle by comparing gravid, diploid females to sterile, triploid females. Methyl Mini-seq and RNA-Seq were simultaneously used to characterize genome-wide DNA methylation and its association with gene expression in rainbow trout muscle. Genome-wide enrichment in the number of CpGs, accompanied by depleted methylation levels, was noticed around the gene transcription start site (TSS). Hypermethylation of CpG sites within ±1 kb on both sides of TSS (promoter and gene body) was weakly/moderately associated with reduced gene expression. Conversely, hypermethylation of the CpG sites in downstream regions of the gene body +2 to +10 kb was weakly associated with increased gene expression. Unlike mammalian genomes, rainbow trout gene promotors are poor in CpG islands, at <1% compared to 60%. No signs of genome-wide, differentially methylated (DM) CpGs were observed due to the polyploidy effect; only 1206 CpGs (0.03%) were differentially methylated, and these were primarily associated with muscle atrophy. Twenty-eight genes exhibited differential gene expression consistent with methylation levels of 31 DM CpGs. These 31 DM CpGs represent potential epigenetic markers of muscle atrophy in rainbow trout. The DM CpG-harboring genes are involved in apoptosis, epigenetic regulation, autophagy, collagen metabolism, cell membrane functions, and Homeobox proteins. Our study also identified genes explaining higher water content and modulated glycolysis previously shown as characteristic biochemical signs of rainbow trout muscle atrophy associated with sexual maturation. This study characterized DNA methylation in the rainbow trout genome and its correlation with gene expression. This work also identified novel epigenetic markers associated with muscle atrophy in fish/lower vertebrates.

Abstract 5698: Genome-wide DNA methylation profiling for differentiating synchronous endometrial and ovarian cancer

Abstract Background: Synchronous endometrial and ovarian cancer (SEOC) refers to simultaneously discovered endometrial and ovarian tumours on the same patient. SEOC may represent double primary (DP) tumours that arise independently on the two sites, or clonally related (CR) tumours that arise either on the endometrium or ovary and metastasized to the other site. Accurate differential diagnosis between these two scenarios can implicate on the treatment options and prognostication of SEOC. However, it is difficult to achieve as the histopathological features of DP and CR tumours are often ambiguous. DNA methylation landscape of SEOC may provide markers for differentiating DP and CR tumours but remain largely unexplored. Methods: The archival tissues of six pairs of SEOC and their accompanied normal uterine tissues were retrieved. The tumours were accessed by conventional histopathological criteria and mitochondrial DNA (mtDNA) sequencing to categorize them into DP or CR. We then analyzed the methylation profiles of &amp;gt;850,000 CpG sites of the tumor samples using the Illumina Infinium MethylationEPIC BeadChip array. Downstream methylation analysis was performed in the R/Bioconductor package ChAMP. Gene ontology (GO) enrichment analysis on the methylation driven genes was done using PANTHER. Results: mtDNA sequencing revealed that among the 6 cases of SEOC, there are 3 DP and 3 CR. Clustering analysis of differentially methylated CpGs (DMPs) between tumours of the same tissue type effectively distinguished endometrial and ovarian tumours into DP or CR. Thus, DP and CR endometrial tumour showed 50 DMPs and 15 differentially methylated regions (DMRs), but no significantly enriched signaling pathway was identified. On the other hand, there were 1,105 DMPs and 44 DMRs that mapped to 601 known genes between DP and CR ovarian tumours. GO analysis revealed that the hypomethylated genes in CR ovarian tumour were enriched in several metastatic-related processes, including cellular response to stimulus, regulation of cell projection organisation, cell migration and negative regulation of voltage-gated potassium channel activity. Conclusions: Taken together, our findings demonstrated that DNA methylation profiling can be used for SEOC patient stratification and may enhance our understanding of epigenetic regulation of SEOC pathogenesis. Citation Format: Yusanne Yung Sing Chan, Oscar Gee Wan Wong, Annie Nga Yin Cheung. Genome-wide DNA methylation profiling for differentiating synchronous endometrial and ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5698.