Differential CpG Methylation Research Articles

Methylome-proteome integration after late-life voluntary exercise training reveals regulation and target information for improved skeletal muscle health.

Exercise is a potent stimulus for combatting skeletal muscle ageing. To study the effects of exercise on muscle in a preclinical setting, we developed a combined endurance-resistance training stimulus for mice called progressive weighted wheel running (PoWeR). PoWeR improves molecular, biochemical, cellular and functional characteristics of skeletal muscle and promotes aspects of partial epigenetic reprogramming when performed late in life (22-24months of age). In this investigation, we leveraged pan-mammalian DNA methylome arrays and tandem mass-spectrometry proteomics in skeletal muscle to provide detailed information on late-life PoWeR adaptations in female mice relative to age-matched sedentary controls (n=7-10 per group). Differential CpG methylation at conserved promoter sites was related to transcriptional regulation genes as well as Nr4a3, Hes1 and Hox genes after PoWeR. Using a holistic method of -omics integration called binding and expression target analysis (BETA), methylome changes were associated with upregulated proteins related to global and mitochondrial translation after PoWeR (P=0.03). Specifically, BETA implicated methylation control of ribosomal, mitoribosomal, and mitochondrial complex I protein abundance after training. DNA methylation may also influence LACTB, MIB1 and UBR4 protein induction with exercise - all are mechanistically linked to muscle health. Computational cistrome analysis predicted several transcription factors including MYC as regulators of the exercise trained methylome-proteome landscape, corroborating prior late-life PoWeR transcriptome data. Correlating the proteome to muscle mass and fatigue resistance revealed positive relationships with VPS13A and NPL levels, respectively. Our findings expose differential epigenetic and proteomic adaptations associated with translational regulation after PoWeR that could influence skeletal muscle mass and function in aged mice. KEY POINTS: Late-life combined endurance-resistance exercise training from 22-24months of age in mice is shown to improve molecular, biochemical, cellular and in vivo functional characteristics of skeletal muscle and promote aspects of partial epigenetic reprogramming and epigenetic age mitigation. Integration of DNA CpG 36k methylation arrays using conserved sites (which also contain methylation ageing clock sites) with exploratory proteomics in skeletal muscle extends our prior work and reveals coordinated and widespread regulation of ribosomal, translation initiation, mitochondrial ribosomal (mitoribosomal) and complex I proteins after combined voluntary exercise training in a sizeable cohort of female mice (n=7-10 per group and analysis). Multi-omics integration predicted epigenetic regulation of serine β-lactamase-like protein (LACTB - linked to tumour resistance in muscle), mind bomb 1 (MIB1 - linked to satellite cell and type 2 fibre maintenance) and ubiquitin protein ligase E3 component N-recognin 4 (UBR4 - linked to muscle protein quality control) after training. Computational cistrome analysis identified MYC as a regulator of the late-life training proteome, in agreement with prior transcriptional analyses. Vacuolar protein sorting 13homolog A (VPS13A) was positively correlated to muscle mass, and the glycoprotein/glycolipid associated sialylation enzyme N-acetylneuraminate pyruvate lyase (NPL) was associated to in vivo muscle fatigue resistance.

Molecular subtypes explain lupus epigenomic heterogeneity unveiling new regulatory genetic risk variants

The heterogeneity of systemic lupus erythematosus (SLE) can be explained by epigenetic alterations that disrupt transcriptional programs mediating environmental and genetic risk. This study evaluated the epigenetic contribution to SLE heterogeneity considering molecular and serological subtypes, genetics and transcriptional status, followed by drug target discovery. We performed a stratified epigenome-wide association studies of whole blood DNA methylation from 213 SLE patients and 221 controls. Methylation quantitative trait loci analyses, cytokine and transcription factor activity - epigenetic associations and methylation-expression correlations were conducted. New drug targets were searched for based on differentially methylated genes. In a stratified approach, a total of 974 differential methylation CpG sites with dependency on molecular subtypes and autoantibody profiles were found. Mediation analyses suggested that SLE-associated SNPs in the HLA region exert their risk through DNA methylation changes. Novel genetic variants regulating DNAm in disease or in specific molecular contexts were identified. The epigenetic landscapes showed strong association with transcription factor activity and cytokine levels, conditioned by the molecular context. Epigenetic signals were enriched in known and novel drug targets for SLE. This study reveals possible genetic drivers and consequences of epigenetic variability on SLE heterogeneity and disentangles the DNAm mediation role on SLE genetic risk and novel disease-specific meQTLs. Finally, novel targets for drug development were discovered.

A systematic review on the contribution of DNA methylation to hearing loss

BackgroundDNA methylation may have a regulatory role in monogenic sensorineural hearing loss and complex, polygenic phenotypic forms of hearing loss, including age-related hearing impairment or Meniere disease. The purpose of this systematic review is to critically assess the evidence supporting a functional role of DNA methylation in phenotypes associated with hearing loss.ResultsThe search strategy yielded a total of 661 articles. After quality assessment, 25 records were selected (12 human DNA methylation studies, 5 experimental animal studies and 8 studies reporting mutations in the DNMT1 gene). Although some methylation studies reported significant differences in CpG methylation in diverse gene promoters associated with complex hearing loss phenotypes (ARHI, otosclerosis, MD), only one study included a replication cohort that supported a regulatory role for CpG methylation in the genes TCF25 and POLE in ARHI. Conversely, several studies have independently confirmed pathogenic mutations within exon 21 of the DNMT1 gene, which encodes the DNA (cytosine-5)-methyltransferase 1 enzyme. This methylation enzyme is strongly associated with a rare disease defined by autosomal dominant cerebellar ataxia, deafness and narcolepsy (ADCA-DN). Of note, rare variants in DNMT1 and DNMT3A genes have also been reported in noise-induced hearing loss.ConclusionsEvidence supporting a functional role for DNA methylation in hearing loss is limited to few genes in complex disorders such as ARHI. Mutations in the DNMT1 gene are associated with ADCA-DN, suggesting the CpG methylation in hearing loss genes deserves further attention in hearing research.

P-008 Sperm DNA methylation landscape in infertile men with clinical varicocele before and after treatment

Abstract Study question As varicocele leads to poor semen parameters, does it also affect sperm DNA methylation levels? Does varicocele treatment help in epigenetics restoration in the sperm? Summary answer Varicocele leads to aberrant sperm DNA methylation and varicocele treatment helps in the partial restoration of sperm DNA methylation levels. What is known already Varicocele affects 15% to 20% of the adult male population and 20% of men with varicocele are reported to be infertile. Treatment modalities for varicocele improve semen parameters, yet more than 50% of cases remain infertile. High oxidative stress (OS) has been implicated as a key factor in varicocele-associated infertility. OS during spermatogenesis may lead to aberrant epigenetic modifications in spermatozoa. In view of the crucial role of sperm epigenome in its functionality, this study aims to evaluate sperm genome-wide DNA methylation by whole-genome bisulfite sequencing (WGBS) before and after varicocele treatment in men with varicocele. Study design, size, duration This is a case-control study, which includes (n = 28) healthy fertile men and (n = 35) infertile men with clinical varicocele. Study participants were recruited from ICMR-NIRRCH and, LTMG Hospital, Mumbai. According to the inclusion and exclusion criteria, study participants were recruited from December 2020 to November 2023. Varicocele patients were followed up, and semen samples (n = 18) were recollected after 3-6 months of varicocele treatment. Participants/materials, setting, methods WGBS was used to identify differentially methylated CpG (DMC) sites in sperm of varicocele group compared to controls. 5 samples from each group were pooled for WGBS. DMCs identified with ≥25% methylation change and ≤0.05 q-value. DMCs within genes relevant to sperm function were selected for validation in the study population by pyrosequencing. Differentially methylated Genes (DMGs) were identified based on the location of DMC and used for pathway analysis using KEGG and DAVID tools. Main results and the role of chance Varicocele group had significantly lower semen parameters compared to controls, which were found to be improved after varicocele treatment. WGBS of sperm DNA was done and DMC sites were obtained in varicocele group compared to controls. A total of 6414 DMCs were obtained, of which, 3730 were hypermethylated and 2684 were hypomethylated. Based on genomic context, differential methylation was observed in introns, exons, promoters and intergenic regions. WGBS analysis gave a total of 1484 DMGs. KEGG and DAVID pathway analysis revealed that the PI3K-Akt signaling, MAPK signaling, cAMP signaling, calcium signaling, signal transduction pathway, cell adhesion molecules, and tight junction, were affected in the varicocele group. The DMGs SPATA6L, H2AX, SOD2, BCL2, NF1, PRKN, CDKN1B, BCR, P2RX7, RNF212B obtained are involved in spermatogenesis, regulation of cell cycle, apoptotic signaling pathway, cell differentiation, regulation of ROS metabolic process, response to heat, mitochondria regulation, regulation of gene expression, etc. Pyrosequencing validation of a few selected DMCs reflects the same methylation difference (hypo or hyper) as obtained by the WGBS pooled sample. Significant hypermethylation and hypomethylation were observed in the H2AX DMC and CDKN1B DMC, respectively, in the varicocele population; however, after varicocele treatments, these methylation levels seem to be restored. Limitations, reasons for caution The limitations of this study are the small number of patients and pooled sperm DNA sample WGBS analysis in the study population. However, a pooling sample strategy is considered more efficient than a random sample selection strategy, as pooling minimizes the amount of information lost below the detection threshold. Wider implications of the findings This study identified differentially methylated genes relevant to spermatogenesis and sperm functions in the case of varicocele compared to fertile controls and gave deeper insights into the possibility of epigenetic restoration after varicocele treatment. This study provides insight into the molecular aetiology of varicocele associated infertility. Trial registration number Not Applicable

Gestational exposure to environmental chemicals and epigenetic alterations in the placenta and cord blood mononuclear cells

BackgroundExposure to environmental chemicals such as phthalates, phenols, and polycyclic aromatic hydrocarbons (PAHs) during pregnancy can increase the risk of adverse newborn outcomes. We explored the associations between maternal exposure to select environmental chemicals and DNA methylation in cord blood mononuclear cells (CBMC) and placental tissue (maternal and fetal sides) to identify potential mechanisms underlying these associations.MethodThis study included 75 pregnant individuals who planned to give birth at the University of Cincinnati Hospital between 2014 and 2017. Maternal urine samples during the delivery visit were collected and analyzed for 37 biomarkers of phenols (12), phthalates (13), phthalate replacements (4), and PAHs (8). Cord blood and placenta tissue (maternal and fetal sides) were also collected to measure the DNA methylation intensities using the Infinium HumanMethylation450K BeadChip. We used linear regression, adjusting for potential confounders, to assess CpG-specific methylation changes in CBMC (n = 54) and placenta [fetal (n = 67) and maternal (n = 68) sides] associated with gestational chemical exposures (29 of 37 biomarkers measured in this study). To account for multiple testing, we used a false discovery rate q-values < 0.05 and presented results by limiting results with a genomic inflation factor of 1±0.5. Additionally, gene set enrichment analysis was conducted using the Kyoto Encyclopedia of Genes and Genomics pathways.ResultsAmong the 29 chemical biomarkers assessed for differential methylation, maternal concentrations of PAH metabolites (1-hydroxynaphthalene, 2-hydroxyfluorene, 4-hydroxyphenanthrene, 1-hydroxypyrene), monocarboxyisononyl phthalate, mono-3-carboxypropyl phthalate, and bisphenol A were associated with altered methylation in placenta (maternal or fetal side). Among exposure biomarkers associated with epigenetic changes, 1-hydroxynaphthalene, and mono-3-carboxypropyl phthalate were consistently associated with differential CpG methylation in the placenta. Gene enrichment analysis indicated that maternal 1-hydroxynaphthalene was associated with lipid metabolism and cellular processes of the placenta. Additionally, mono-3-carboxypropyl phthalate was associated with organismal systems and genetic information processing of the placenta.ConclusionAmong the 29 chemical biomarkers assessed during delivery, 1-hydroxynaphthalene and mono-3-carboxypropyl phthalate were associated with DNA methylation in the placenta.

Identification of DNA methylation characteristics associated with metastasis and prognosis in colorectal cancer

Colorectal cancer (CRC) is prone to metastasis and recurrence after surgery, which is one of the main causes for its poor treatment and prognosis. Therefore, it is essential to identify biomarkers associated with metastasis and recurrence in CRC. DNA methylation has a regulatory role in cancer metastasis, tumor immune microenvironment (TME), and prognosis and may be one of the most valuable biomarkers for predicting CRC metastasis and prognosis. We constructed a diagnostic model and nomogram that can effectively predict CRC metastasis based on the differential methylation CpG sites (DMCs) between metastatic and non-metastatic CRC patients. Then, we identified 17 DMCs associated with progression free survival (PFS) of CRC and constructed a prognostic model. The prognosis model based on 17 DMCs can predict the PFS of CRC with medium to high accuracy. The results of immunohistochemical analysis indicated that the protein expression levels of the genes involved in prognostic DMCs were different between normal and colorectal cancer tissues. According to the results of immune-related analysis, we found that the low-risk patients had better immunotherapy response. In addition, high risk scores were negatively correlated with high tumor mutation burden (TMB) levels, and patients with low TMB levels in the high-risk group had the worst PFS. Our work shows the clinical value of DNA methylation in predicting CRC metastasis and PFS, as well as their correlation with TME, immunotherapy, and TMB, which helps understand the changes of DNA methylation in CRC metastasis and improving the treatment and prognosis of CRC.

Differential CpG methylation at Nnat in the early establishment of beta cell heterogeneity

Aims/hypothesisBeta cells within the pancreatic islet represent a heterogenous population wherein individual sub-groups of cells make distinct contributions to the overall control of insulin secretion. These include a subpopulation of highly connected ‘hub’ cells, important for the propagation of intercellular Ca2+ waves. Functional subpopulations have also been demonstrated in human beta cells, with an altered subtype distribution apparent in type 2 diabetes. At present, the molecular mechanisms through which beta cell hierarchy is established are poorly understood. Changes at the level of the epigenome provide one such possibility, which we explore here by focusing on the imprinted gene Nnat (encoding neuronatin [NNAT]), which is required for normal insulin synthesis and secretion.MethodsSingle-cell RNA-seq datasets were examined using Seurat 4.0 and ClusterProfiler running under R. Transgenic mice expressing enhanced GFP under the control of the Nnat enhancer/promoter regions were generated for FACS of beta cells and downstream analysis of CpG methylation by bisulphite sequencing and RNA-seq, respectively. Animals deleted for the de novo methyltransferase DNA methyltransferase 3 alpha (DNMT3A) from the pancreatic progenitor stage were used to explore control of promoter methylation. Proteomics was performed using affinity purification mass spectrometry and Ca2+ dynamics explored by rapid confocal imaging of Cal-520 AM and Cal-590 AM. Insulin secretion was measured using homogeneous time-resolved fluorescence imaging.ResultsNnat mRNA was differentially expressed in a discrete beta cell population in a developmental stage- and DNA methylation (DNMT3A)-dependent manner. Thus, pseudo-time analysis of embryonic datasets demonstrated the early establishment of Nnat-positive and -negative subpopulations during embryogenesis. NNAT expression is also restricted to a subset of beta cells across the human islet that is maintained throughout adult life. NNAT+ beta cells also displayed a discrete transcriptome at adult stages, representing a subpopulation specialised for insulin production, and were diminished in db/db mice. ‘Hub’ cells were less abundant in the NNAT+ population, consistent with epigenetic control of this functional specialisation.Conclusions/interpretationThese findings demonstrate that differential DNA methylation at Nnat represents a novel means through which beta cell heterogeneity is established during development. We therefore hypothesise that changes in methylation at this locus may contribute to a loss of beta cell hierarchy and connectivity, potentially contributing to defective insulin secretion in some forms of diabetes.Data availabilityThe mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD048465.Graphical

Computationally inferred cell-type specific epigenome-wide DNA methylation analysis unveils distinct methylation patterns among immune cells for HIV infection in three cohorts.

Epigenome-wide association studies (EWAS) have identified CpG sites associated with HIV infection in blood cells in bulk, which offer limited knowledge of cell-type specific methylation patterns associated with HIV infection. In this study, we aim to identify differentially methylated CpG sites for HIV infection in immune cell types: CD4+ T-cells, CD8+ T-cells, B cells, Natural Killer (NK) cells, and monocytes. Applying a computational deconvolution method, we performed a cell-type based EWAS for HIV infection in three independent cohorts (Ntotal = 1,382). DNA methylation in blood or in peripheral blood mononuclear cells (PBMCs) was profiled by an array-based method and then deconvoluted by Tensor Composition Analysis (TCA). The TCA-computed CpG methylation in each cell type was first benchmarked by bisulfite DNA methylation capture sequencing in a subset of the samples. Cell-type EWAS of HIV infection was performed in each cohort separately and a meta-EWAS was conducted followed by gene set enrichment analysis. The meta-analysis unveiled a total of 2,021 cell-type unique significant CpG sites for five inferred cell types. Among these inferred cell-type unique CpG sites, the concordance rate in the three cohorts ranged from 96% to 100% in each cell type. Cell-type level meta-EWAS unveiled distinct patterns of HIV-associated differential CpG methylation, where 74% of CpG sites were unique to individual cell types (false discovery rate, FDR <0.05). CD4+ T-cells had the largest number of unique HIV-associated CpG sites (N = 1,624) compared to any other cell type. Genes harboring significant CpG sites are involved in immunity and HIV pathogenesis (e.g. CD4+ T-cells: NLRC5, CX3CR1, B cells: IFI44L, NK cells: IL12R, monocytes: IRF7), and in oncogenesis (e.g. CD4+ T-cells: BCL family, PRDM16, monocytes: PRDM16, PDCD1LG2). HIV-associated CpG sites were enriched among genes involved in HIV pathogenesis and oncogenesis that were enriched among interferon-α and -γ, TNF-α, inflammatory response, and apoptotic pathways. Our findings uncovered computationally inferred cell-type specific modifications in the host epigenome for people with HIV that contribute to the growing body of evidence regarding HIV pathogenesis.

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.

Detailed immune profiling in pediatric Crohn’s disease using methylation cytometry

ABSTRACT DNA methylation has been extensively utilized to study epigenetic patterns across many diseases as well as to deconvolve blood cell type proportions. This study builds upon previous studies examining methylation patterns in paediatric patients with varying stages of Crohn’s disease to extend the immune profiling of these patients using a novel deconvolution approach. Compared with control subjects, we observed significantly decreased levels of CD4 memory and naive, CD8 naive, and natural killer cells and elevated neutrophil levels in Crohn’s disease. In addition, Crohn’s patients had a significantly elevated neutrophil-to-lymphocyte ratio. Using an epigenome-wide association approach and adjusting for potential confounders, including cell type, we observed 397 differentially methylated CpG (DMC) sites associated with Crohn’s disease. The top genetic pathway associated with the DMCs was the regulation of arginine metabolic processes which are involved in the regulation of T cells.

Epigenome‐Wide‐Association‐Scan Reveals Epigenetic Profile Changes Using Neurofilament Light as a Neurodegeneration Proxy Biomarker in Mexican Americans

AbstractBackgroundRecent literature shows Alzheimer’s Disease (AD) has both genetic and epigenetic components resulting in disease manifestation. Furthering our prior work on AD epigenetics in the Health and Aging Brain Study – Health Disparities (HABS‐HD) participants, we seek to identify differential CpG methylation profiles of Neurofilament Light (NfL) as a proxy for neurodegeneration in Mexican‐American participants with Normal Cognition (NC) or Mild Cognitive Impairment (MCI).MethodUsing data from the Epigenome Wide Association Scan (EWAS) that our group already published for 90 Mexican Americans in HABS‐HD (formerly Healthy Aging in Latino Elders Study [HABLE]), we conducted preliminary analysis in 32 individuals (NC = 21, MCI = 11) to assess the role of epigenetic markers in neurodegeneration among Mexican Americans. We performed the EWAS using the AT(N) biomarker, Neurofilament Light (NfL), as the outcome in our pilot group of 32 Mexican Americans. We first tested if the NfL data was normalized using the Shapiro Test, then tested NfL differences between NC and MCI with a Wilcox Test. We further tested CpG sites with NfL using a disease‐stratified and unstratified model with linear regression modelling and adjusted for covariates, such as age, gender, and education.ResultResults of our analysis for the association between CpG methylation profiles and NfL showed 14 down‐regulated CpG sites and 1 up‐regulated CpG site with significant P values after multiple testing. These significant CpG sites residing in LRP4 (Chr11p11.2), ABCB9 (Chr12q24.31), and KIAA (Chr13q14.13) are implicated in the CpG methylation profile changes. These genes have been implicated in AD pathogenesis by other molecular studies. When we stratified NfL count by disease subtype, we did not find any significance in this dataset. We are expanding our ongoing work to identify methylation profiles of NfL in a larger cohort.ConclusionOur pilot findings are critical in understanding epigenetic profiles of biomarkers for neurodegeneration. Results suggest that epigenetics play a major role in the pathophysiology of AD development and manifestation, and that this effect may be different by ethnicity. Future studies will include a larger sample size, longitudinal analyses, and additional ethnic groups to help bridge the health disparities gap in the literature.

Allele-Specific Expression of Leukemia Genes Is Associated with Pathogenicity in Poor Risk AML

Introduction: An imbalance in gene expression of the different alleles of a gene (allele-specific expression or ASE) is an intriguing genome-wide and dynamic phenomenon that could have an important role in tumorigenesis. Little is known about the causes and consequences of the differential cis- and trans-regulation on resultant ASE, aside from the impact of copy number alterations (CNAs) or imprinting. Acute myeloid leukemia (AML) is an aggressive clonal hematopoietic stem cell malignancy, with a low mutational burden in comparison with other cancers, where the impact of ASE may constitute a novel mechanism of pathogenesis. Aim: Application of Whole Genome Sequencing (WGS), RNA-seq and Oxford Nanopore methylation sequencing to study the ASE landscape in a cohort of poor risk AML patients and shed light on the mechanisms triggering this phenomenon as well as its temporal dynamics. Methods: ASE landscape was explored using paired germline and tumour WGS and RNA-seq on diagnostic bone marrow or peripheral blood samples from 33 cytogenetically poor risk AML patients, and additional RNA-seq from 18 samples corresponding to follow-up timepoints (including remission, relapse and refractory samples) from eight of the patients.Heterozygous SNPs were identified in the genomic DNA, and allele frequencies in RNA-seq reads were used to quantify ASE, after filtering for CNAs in the tumor DNA. Cas9-enriched Oxford Nanopore sequencing was then performed on available patient samples at diagnosis and subsequent time points. Allele-specific methylation was determined on the Nanopore sequencing using Guppy v6.3. Results: In the diagnostic cohort, we identified 4,802 protein-coding genes exhibiting ASE in at least one sample, with a median of 169 genes per sample (range 119-468, with one outlier sample showing ASE in 1,705 genes). Overall, 283 protein-coding genes showed ASE in ≥ 20% patients across the 33 samples. These genes included known imprinted genes and the transcription factor GATA2, where ASE was previously associated with AML, validating our ASE pipeline and filtering criteria. We also identified for the first time recurrent ASE in other myeloid genes such as KMT2A (20%), the homeobox genes PBX2 (41%), PBX3 (20%), HOXA6 (20%) and HOXB cluster ( HOXB2 (31%), HOXB3 (27%), HOXB5 (40%), HOXB6 (30%)); and the myeloid differentiation genes CDKN2A (40%), PDGFRB (25%) and PRTN3 (57%). Eighteen primary AML samples from 8/33 patients had paired diagnostic, remission and relapse or refractory material, allowing us to track the dynamic ASE pattern. RNA-seq and Sanger sequencing of cDNA in these samples showed that the ASE observed at diagnosis in HOXB2, HOXB3, HOXB6, PBX3 and GATA2 was lost in the corresponding remission sample (bi-allelic expression), with ASE returning at relapse and maintained in the refractory samples. In order to explore the epigenetic mechanisms underlying these allele-specific changes in expression, Cas9-enriched Oxford Nanopore sequencing was performed in 2 patients with available DNA at diagnosis and sequential time points, to determine the pattern of allele-specific methylation of the promoter region of GATA2 (patient 1) and HOXB2 (patient 2). In patient 1 at diagnosis, 90% of CpG sites were methylated at the GATA2 promoter region on one allele versus 61% in the other allele, with the hypomethylated allele corresponding to the allele that is overexpressed in the ASE sample. However, in the remission sample, the proportion of methylated CpG sites on the two alleles was similar (83% and 73%, respectively), in agreement with the expression of both GATA2 alleles at this stage. Likewise, in the diagnostic sample of patient 2, 6% of CpG sites were methylated in the HOXB2 promoter region of one allele, corresponding to the overexpressed allele in the ASE sample, while the other promoter allele had 17% of CpG sites methylated; with no differences in CpG methylation between alleles in the remission samples (9% versus 9%), again correlating with the bi-allelic expression of HOXB2 at remission. Conclusions: Combining WGS and RNA-seq in AML primary samples, we have identified key leukemia genes showing recurrent dynamic ASE associated with disease evolution: since it is found at diagnosis, lost in remission, but recovered in relapse and persisting in refractory stages. Our results also suggest allele-specific methylation as the potential mechanism orchestrating this phenomenon.

Defining high confidence targets of differential CpG methylation in response to in utero arsenic exposure and implications for cancer risk

Arsenic is a relatively abundant metalloid that impacts DNA methylation and has been implicated in various adverse health outcomes including several cancers and diabetes. However, uncertainty remains about the identity of genomic CpGs that are sensitive to arsenic exposure, in utero or otherwise. Here we identified a high confidence set of CpG sites whose methylation is sensitive to in utero arsenic exposure. To do so, we analyzed methylation of infant CpGs as a function of maternal urinary arsenic in cord blood and placenta from geographically and ancestrally distinct human populations. Independent analyses of these distinct populations were followed by combination of results across sexes and populations/tissue types. Following these analyses, we concluded that both sex and tissue type are important drivers of heterogeneity in methylation response at several CpGs. We also identified 17 high confidence CpGs that were hypermethylated across sex, tissue type and population; 11 of these were located within protein coding genes. This pattern is consistent with hypotheses that arsenic increases cancer risk by inducing the hypermethylation of genic regions. This study represents an opportunity to understand consistent, reproducible patterns of epigenomic responses after in utero arsenic exposure and may aid towards novel biomarkers or signatures of arsenic exposure. Identifying arsenic-responsive sites can also contribute to our understanding of the biological mechanisms by which arsenic exposure can affect biological function and increase risk of cancer and other age-related diseases.

Maternal adverse childhood experiences (ACEs) and DNA methylation of newborns in cord blood

BackgroundAdverse childhood experiences (ACEs) increase the risk of poor health outcomes later in life. Psychosocial stressors may also have intergenerational health effects by which parental ACEs are associated with mental and physical health of children. Epigenetic programming may be one mechanism linking parental ACEs to child health. This study aimed to investigate epigenome-wide associations of maternal preconception ACEs with DNA methylation patterns of children. In the Center for the Health Assessment of Mothers and Children of Salinas study, cord blood DNA methylation was measured using the Illumina HumanMethylation450 BeadChip. Preconception ACEs, which occurred during the mothers’ childhoods, were collected using a standard ACE questionnaire including 10 ACE indicators. Maternal ACE exposures were defined in this study as (1) the total number of ACEs; (2) the total number of ACEs categorized as 0, 1–3, and > 4; and (3) individual ACEs. Associations of ACE exposures with differential methylated positions, regions, and CpG modules determined using weighted gene co-expression network analysis were evaluated adjusting for covariates.ResultsData on maternal ACEs and cord blood DNA methylation were available for 196 mother/newborn pairs. One differential methylated position was associated with maternal experience of emotional abuse (cg05486260/FAM135B gene; q value < 0.05). Five differential methylated regions were significantly associated with the total number of ACEs, and 36 unique differential methylated regions were associated with individual ACEs (Šidák p value < 0.05). Fifteen CpG modules were significantly correlated with the total number of ACEs or individual ACEs, of which 8 remained significant in fully adjusted models (p value < 0.05). Significant modules were enriched for pathways related to neurological and immune development and function.ConclusionsMaternal ACEs prior to conception were associated with cord blood DNA methylation of offspring at birth. Although there was limited overlap between differential methylated regions and CpGs in modules associated with ACE exposures, statistically significant regions and networks were related to genes involved in neurological and immune function. Findings may provide insights to pathways linking psychosocial stressors to health. Further research is needed to understand the relationship between changes in DNA methylation and child health.

Intergenerational effects of early life-stage temperature modulation on gene expression and DNA methylation in Atlantic cod (Gadus morhua)

ABSTRACT After suffering several collapses, the cod farming industry is now in the process of trying to re-establish itself. We have used material from Norway’s National Cod Breeding Program to study how different early life-stage temperature regimes affect DNA methylation and gene expression. Long-term effects were detected by sampling fish several weeks after the end of differential treatments, and offspring from the different exposure groups was also sampled. Many overlapping genes were found between the different exposure groups and generations, coupled with genes associated with differential CpG methylation levels. Genes involved in muscle fibre development, general metabolic processes and formation of deformities were significantly affected, and genes relevant for intergenerational transfer of epigenetic marks were also detected. We believe the use of environmental cues can be a useful strategy for improving the production of Atlantic cod.

In Cis Effect of DMPK Expanded Alleles in Myotonic Dystrophy Type 1 Patients Carrying Variant Repeats at 5' and 3' Ends of the CTG Array.

Myotonic dystrophy type 1 (DM1) is an autosomal dominant multisystemic disease caused by a CTG repeat expansion in the 3'-untranslated region (UTR) of DMPK gene. DM1 alleles containing non-CTG variant repeats (VRs) have been described, with uncertain molecular and clinical consequences. The expanded trinucleotide array is flanked by two CpG islands, and the presence of VRs could confer an additional level of epigenetic variability. This study aims to investigate the association between VR-containing DMPK alleles, parental inheritance and methylation pattern of the DM1 locus. The DM1 mutation has been characterized in 20 patients using a combination of SR-PCR, TP-PCR, modified TP-PCR and LR-PCR. Non-CTG motifs have been confirmed by Sanger sequencing. The methylation pattern of the DM1 locus was determined by bisulfite pyrosequencing. We characterized 7 patients with VRs within the CTG tract at 5' end and 13 patients carrying non-CTG sequences at 3' end of the DM1 expansion. DMPK alleles with VRs at 5' end or 3' end were invariably unmethylated upstream of the CTG expansion. Interestingly, DM1 patients with VRs at the 3' end showed higher methylation levels in the downstream island of the CTG repeat tract, preferentially when the disease allele was maternally inherited. Our results suggest a potential correlation between VRs, parental origin of the mutation and methylation pattern of the DMPK expanded alleles. A differential CpG methylation status could play a role in the phenotypic variability of DM1 patients, representing a potentially useful diagnostic tool.

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.

Integrated analysis reveals common DNA methylation patterns of alcohol-associated cancers: A pan-cancer analysis.

Background: The role of alcohol in carcinogenesis has received increasing attention in recent years. Evidence shows its impacts on various aspects, including epigenetics alteration. The DNA methylation patterns underlying alcohol-associated cancers are not fully understood. Methods: We investigated the aberrant DNA methylation patterns in four alcohol-associated cancers based on the Illumina HumanMethylation450 BeadChip. Pearson coefficient correlations were identified between differential methylated CpG probes and annotated genes. Transcriptional factor motifs were enriched and clustered using MEME Suite, and a regulatory network was constructed. Results: In each cancer, differential methylated probes (DMPs) were identified, and 172 hypermethylated and 21 hypomethylated pan-cancer DMPs (PDMPs) were examined further. Annotated genes significantly regulated by PDMPs were investigated and enriched in transcriptional misregulation in cancers. The CpG island chr19:58220189-58220517 was hypermethylated in all four cancers and silenced in the transcription factor ZNF154. Various biological effects were exerted by 33 hypermethylated and seven hypomethylated transcriptional factor motifs grouped into five clusters. Eleven pan-cancer DMPs were identified to be associated with clinical outcomes in the four alcohol-associated cancers, which might provide a potential point of view for clinical outcome prediction. Conclusion: This study provides an integrated insight into DNA methylation patterns in alcohol-associated cancers and reveals the corresponding features, influences, and potential mechanisms.

Abstract IA011: mTOR pathway gene expression in association with race and clinicopathological characteristics in Black and White breast cancer patients

Abstract Background Aberrant activation of the mammalian Target of Rapamycin (mTOR) pathway has been linked to obesity and endocrine therapy resistance, factors that may contribute to Black White disparities in breast cancer outcomes. We evaluated associations of race and clinicopathological characteristics with mRNA expression of key mTOR pathway genes in breast tumors. Methods Surgical tumor tissue blocks were collected from 367 newly diagnosed breast cancer patients (190 Black and 177 White). Gene expression of AKT1, EIF4EBP1, MTOR, RPS6KB2, and TSC1 were quantified by NanoString nCounter. Differential gene expression was assessed using linear regression on log2-transformed values. Gene expression and DNA methylation data from TCGA were used for validation and investigation of race-related differences. Results Compared to White women, Black women had relative under-expression of AKT1 (log2 fold-change = - 0.31, 95% CI - 0.44, - 0.18) and RPS6KB2 (log2 fold-change = - 0.11, 95% CI - 0.19, - 0.03). Higher vs. lower tumor grade was associated with relative over-expression of EIF4EBP1 and RPS6KB2, but with lower expression of TSC1. Compared to luminal tumors, triple-negative tumors had relative under-expression of TSC1 (log2 fold-change = - 0.42, 95% CI - 0.22, - 0.01). The results were similar in the TCGA breast cancer dataset. Post-hoc analyses identified differential CpG methylation within the AKT1 and RPS6KB2 locus between Black and White women. Conclusions Over-expression of RPS6KB2 and EIF4EBP1 and under-expression of TSC1 might be indicators of more aggressive breast cancer phenotypes. Differential expression of AKT1 and RPS6KB2 by race warrants further investigation to elucidate their roles in racial disparities of treatment resistance and outcomes between Black and White women with breast cancer. Citation Format: Mmadili N. Ilozumba, Song Yao, Adana A. M. Llanos, Angela R. Omilian, Weizhou Zhang, Susmita Datta, Chi Chen Hong, Warren Davis, Thaer Khoury, Elisa V. Bandera, Michael Higgins, Christine B. Ambrosone, Ting Yuan David Cheng. mTOR pathway gene expression in association with race and clinicopathological characteristics in Black and White breast cancer patients [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr IA011.

Consistent DNA Hypomethylations in Prostate Cancer.

With approximately 1.4 million men annually diagnosed with prostate cancer (PCa) worldwide, PCa remains a dreaded threat to life and source of devastating morbidity. In recent decades, a significant decrease in age-specific PCa mortality has been achieved by increasing prostate-specific antigen (PSA) screening and improving treatments. Nevertheless, upcoming, augmented recommendations against PSA screening underline an escalating disproportion between the benefit and harm of current diagnosis/prognosis and application of radical treatment standards. Undoubtedly, new potent diagnostic and prognostic tools are urgently needed to alleviate this tensed situation. They should allow a more reliable early assessment of the upcoming threat, in order to enable applying timely adjusted and personalized therapy and monitoring. Here, we present a basic study on an epigenetic screening approach by Methylated DNA Immunoprecipitation (MeDIP). We identified genes associated with hypomethylated CpG islands in three PCa sample cohorts. By adjusting our computational biology analyses to focus on single CpG-enriched 60-nucleotide-long DNA probes, we revealed numerous consistently differential methylated DNA segments in PCa. They were associated among other genes with NOTCH3, CDK2AP1, KLK4, and ADAM15. These can be used for early discrimination, and might contribute to a new epigenetic tumor classification system of PCa. Our analysis shows that we can dissect short, differential methylated CpG-rich DNA fragments and combinations of them that are consistently present in all tumors. We name them tumor cell-specific differential methylated CpG dinucleotide signatures (TUMS).