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Meningeal solitary fibrous tumors (SFT) are rare and have a high frequency of local recurrence and distant metastasis. In a cohort of 126 patients (57 female, 69 male; mean age at surgery 53.0 years) with pathologically confirmed meningeal SFTs with extended clinical follow-up (median 9.9 years; range 15 days-43 years), we performed extensive molecular characterization including genome-wide DNA methylation profiling (n = 80) and targeted TERT promoter mutation testing (n = 98). Associations were examined with NAB2::STAT6 fusion status (n = 101 cases; 51 = ex5-7::ex16-17, 26 = ex4::ex2-3; 12 = ex2-3::exANY/other and 12 = no fusion) and placed in the context of 2021 Central Nervous System (CNS) WHO grade. NAB2::STAT6 fusion breakpoints (fusion type) were significantly associated with metastasis-free survival (MFS) (p = 0.03) and, on multivariate analysis, disease-specific survival (DSS) when adjusting for CNS WHO grade (p = 0.03). DNA methylation profiling revealed three distinct clusters: Cluster 1 (n = 38), Cluster 2 (n = 22), and Cluster 3 (n = 20). Methylation clusters were significantly associated with fusion type (p

The insecticide dichlorodiphenyltrichloroethane (DDT) and its persistent metabolite, dichlorodiphenyldichloroethylene (DDE), have been associated with increased adiposity and obesity in multiple generations of rodents and humans. These lipophilic pollutants accumulate in adipose tissue and appear to decrease energy expenditure through the impairment of thermogenesis in brown adipose tissue (BAT). We hypothesized that impaired thermogenesis is due to persistent epigenetic modifications of BAT. To address this, we exposed C57BL/6 J mice to DDT or DDE from gestational day (GD) 11.5 to postnatal day (PND) 5, evaluated longitudinal body temperature, and performed reduced representation bisulfite sequencing and RNA sequencing of BAT from infant and adult offspring. Exposure to DDT or DDE reduced core body temperature in adult mice, and differential methylation at the pathway and gene level was persistent from infancy to adulthood. Furthermore, thermogenesis and biological pathways essential for thermogenic function, such as oxidative phosphorylation and mechanistic target of rapamycin kinase (mTOR) signaling, were enriched with differential methylation and RNA transcription in adult mice exposed to DDT or DDE. PAZ6 human brown preadipocytes were differentiated in the presence of DDT or DDE to understand the brown adipocyte-autonomous effect of these pollutants. In vitro exposure led to limited changes in RNA expression; however, mitochondrial membrane potential was decreased in vitro with 0.1 µM and 1 µM doses of DDT or DDE. These results demonstrate that concentrations of DDT and DDE relevant to human exposure have a significant effect on thermogenesis, the transcriptome, and DNA methylome of mouse BAT and the mitochondrial function of human brown adipocytes.

BACKGROUND: Incorporating genomic data into risk prediction has become an increasingly popular approach for rapid identification of individuals most at risk for complex disorders such as PTSD. Our goal was to develop and validate Methylation Risk Scores (MRS) using machine learning to distinguish individuals who have PTSD from those who do not. METHODS: Elastic Net was used to develop three risk score models using a discovery dataset (n = 1226; 314 cases, 912 controls) comprised of 5 diverse cohorts with available blood-derived DNA methylation (DNAm) measured on the Illumina Epic BeadChip. The first risk score, exposure and methylation risk score (eMRS) used cumulative and childhood trauma exposure and DNAm variables; the second, methylation-only risk score (MoRS) was based solely on DNAm data; the third, methylation-only risk scores with adjusted exposure variables (MoRSAE) utilized DNAm data adjusted for the two exposure variables. The potential of these risk scores to predict future PTSD based on pre-deployment data was also assessed. External validation of risk scores was conducted in four independent cohorts. RESULTS: The eMRS model showed the highest accuracy (92%), precision (91%), recall (87%), and f1-score (89%) in classifying PTSD using 3730 features. While still highly accurate, the MoRS (accuracy = 89%) using 3728 features and MoRSAE (accuracy = 84%) using 4150 features showed a decline in classification power. eMRS significantly predicted PTSD in one of the four independent cohorts, the BEAR cohort (beta = 0.6839, p=0.006), but not in the remaining three cohorts. Pre-deployment risk scores from all models (eMRS, beta = 1.92; MoRS, beta = 1.99 and MoRSAE, beta = 1.77) displayed a significant (p

Human cell line models, including the neuronal precursor line LUHMES, are important for investigating developmental transcriptional dynamics within imprinted regions, particularly the 15q11-q13 Angelman (AS) and Prader-Willi (PWS) syndrome locus. AS results from loss of maternal UBE3A in neurons, where the paternal allele is silenced by a convergent antisense transcript UBE3A-ATS, a lncRNA that terminates at PWAR1 in non-neurons. qRT-PCR analysis confirmed the exclusive and progressive increase in UBE3A-ATS in differentiating LUHMES neurons, validating their use for studying UBE3A silencing. Genome-wide transcriptome analyses revealed changes to 11 834 genes during neuronal differentiation, including the upregulation of most genes within the 15q11-q13 locus. To identify dynamic changes in chromatin loops linked to transcriptional activity, we performed a HiChIP validated by 4C, which identified two neuron-specific CTCF loops between MAGEL2-SNRPN and PWAR1-UBE3A. To determine if allele-specific differentially methylated regions (DMR) may be associated with CTCF loop anchors, whole genome long-read nanopore sequencing was performed. We identified a paternally hypomethylated DMR near the SNRPN upstream loop anchor exclusive to neurons and a paternally hypermethylated DMR near the PWAR1 CTCF anchor exclusive to undifferentiated cells, consistent with increases in neuronal transcription. Additionally, DMRs near CTCF loop anchors were observed in both cell types, indicative of allele-specific differences in chromatin loops regulating imprinted transcription. These results provide an integrated view of the 15q11-q13 epigenetic landscape during LUHMES neuronal differentiation, underscoring the complex interplay of transcription, chromatin looping, and DNA methylation. They also provide insights for future therapeutic approaches for AS and PWS.

We describe a framework that addresses concern that the rate of change in any aging biomarker displays a trivial inverse relation with maximum lifespan. We apply this framework to methylation data from the Mammalian Methylation Consortium. We study the relationship of lifespan with the average rate of change in methylation (AROCM) from two datasets: one with 90 dog breeds and the other with 125 mammalian species. After examining 54 chromatin states, we conclude three key findings: First, a reciprocal relationship exists between the AROCM in bivalent promoter regions and maximum mammalian lifespan: AROCM ∝ 1/MaxLifespan. Second, the correlation between average methylation and age bears no relation to maximum lifespan, Cor(Methyl,Age) ⊥ MaxLifespan. Third, the rate of methylation change in young animals is related to that in old animals: Young animals AROCM ∝ Old AROCM. These findings critically hinge on the chromatin context, as different results emerge in other chromatin contexts.

The World Health Organizations (WHO) classification of central nervous system (CNS) tumors is continually being refined to improve the existing diagnostic criteria for high-grade gliomas (HGGs), including glioblastoma. In 2021, advances in molecular analyses and DNA methylation profiling were incorporated to expand upon the diagnostic criteria for HGG, including the introduction of high-grade astrocytoma with piloid features (HGAP), a new tumor entity for which a match to the HGAP class in DNA methylation profiling is an essential criterion. We present an equivocal case of a 72-year-old male with an HGG exhibiting features of both HGAP and glioblastoma, but which did not conform to any existing 2021 WHO classification of CNS tumor entities. This no match in DNA methylation profiling resulted in a final diagnosis of HGG not elsewhere classified (NEC), for which standard treatment options do not exist.

Systemic low-grade inflammation is a feature of chronic disease. C-reactive protein (CRP) is a common biomarker of inflammation and used as an indicator of disease risk; however, the role of inflammation in disease is not completely understood. Methylation is an epigenetic modification in the DNA which plays a pivotal role in gene expression. In this study we evaluated differential DNA methylation patterns associated with blood CRP level to elucidate biological pathways and genetic regulatory mechanisms to improve the understanding of chronic inflammation. The racially and ethnically diverse participants in this study were included as 50% White, 41% Black or African American, 7% Hispanic or Latino/a, and 2% Native Hawaiian, Asian American, American Indian, or Alaska Native (total n = 13,433) individuals. We replicated 113 CpG sites from 87 unique loci, of which five were novel (CADM3, NALCN, NLRC5, ZNF792, and cg03282312), across a discovery set of 1,150 CpG sites associated with CRP level (p

Cytomegalovirus (CMV) infection and reactivation in solid organ transplant (SOT) recipients increases the risk of viremia, graft failure and death. Clinical studies of CMV serostatus indicate that donor positive recipient negative (D+/R-) patients have greater viremia risk than D-/R-. The majority of patients are R+ having intermediate serologic risk. To characterize the long-term impact of CMV infection and assess viremia risk, we sought to measure the effects of CMV on the recipient immune epigenome. Specifically, we profiled DNA methylation in 156 individuals before lung or kidney transplant. We found that the methylome of CMV positive SOT recipients is hyper-methylated at loci associated with neural development and Polycomb group (PcG) protein binding, and hypo-methylated at regions critical for the maturation of lymphocytes. In addition, we developed a machine learning-based model to predict the recipient CMV serostatus after correcting for cell type composition and ancestry. This CMV episcore measured at baseline in R+ individual stratifies viremia risk accurately in the lung transplant cohort, and along with serostatus the CMV episcore could be a potential biomarker for identifying R+ patients at high viremia risk.

BACKGROUND: The effect of vaccination on the epigenome remains poorly characterized. In previous research, we identified an association between seroprotection against influenza and DNA methylation at sites associated with the RIG-1 signaling pathway, which recognizes viral double-stranded RNA and leads to a type I interferon response. However, these studies did not fully account for confounding factors including age, gender, and BMI, along with changes in cell-type composition. RESULTS: Here, we studied the influenza vaccine response in a longitudinal cohort vaccinated over two consecutive years (2019-2020 and 2020-2021), using peripheral blood mononuclear cells and a targeted DNA methylation approach. To address the effects of multiple factors on the epigenome, we designed a multivariate multiple regression model that included seroprotection levels as quantified by the hemagglutination-inhibition (HAI) assay test. CONCLUSIONS: Our findings indicate that 179 methylation sites can be combined as potential signatures to predict seroprotection. These sites were not only enriched for genes involved in the regulation of the RIG-I signaling pathway, as found previously, but also enriched for other genes associated with innate immunity to viruses and the transcription factor binding sites of BRD4, which is known to impact T cell memory. We propose a model to suggest that the RIG-I pathway and BRD4 could potentially be modulated to improve immunization strategies.

Functional crosstalk between DNA methylation, histone H3 lysine-9 trimethylation (H3K9me3) and heterochromatin protein 1 (HP1) is essential for proper heterochromatin assembly and genome stability. However, how repressive chromatin cues guide DNA methyltransferases for region-specific DNA methylation remains largely unknown. Here, we report structure-function characterizations of DNA methyltransferase Defective-In-Methylation-2 (DIM2) in Neurospora. The DNA methylation activity of DIM2 requires the presence of both H3K9me3 and HP1. Our structural study reveals a bipartite DIM2-HP1 interaction, leading to a disorder-to-order transition of the DIM2 target-recognition domain that is essential for substrate binding. Furthermore, the structure of DIM2-HP1-H3K9me3-DNA complex reveals a substrate-binding mechanism distinct from that for its mammalian orthologue DNMT1. In addition, the dual recognition of H3K9me3 peptide by the DIM2 RFTS and BAH1 domains allosterically impacts the DIM2-substrate binding, thereby controlling DIM2-mediated DNA methylation. Together, this study uncovers how multiple heterochromatin factors coordinately orchestrate an activity-switching mechanism for region-specific DNA methylation.

BACKGROUND AND AIMS: Extracellular vesicles (EVs) secreted by cardiosphere-derived cells exert immunomodulatory effects through the transmission of small non-coding RNAs. METHODS: The mechanism and role of yREX3, a small Y RNA abundant in EVs in myocardial injury, was investigated. RESULTS: yREX3 attenuates cardiac ischaemic injury by selective DNA methylation. Synthetic yREX3 encapsulated in lipid nanoparticles triggers broad transcriptomic changes in macrophages, localizes to the nucleus, and mediates epigenetic silencing of protein interacting with C kinase-1 (Pick1) through methylation of upstream CpG sites. Moreover, yREX3 interacts with polypyrimidine tract binding protein 3 (PTBP3) to methylate the Pick1 gene locus in a DNA methyltransferase-dependent manner. Suppression of Pick1 in macrophages potentiates Smad3 signalling and enhances efferocytosis, minimizing heart necrosis in rats with myocardial infarction. Adoptive transfer of Pick1-deficient macrophages recapitulates the cardioprotective effects of yREX3 in vivo. CONCLUSIONS: These findings highlight the role of a small Y RNA mined from EVs with a novel gene-methylating mechanism.

Worldwide trends to delay childbearing have increased parental ages at birth. Older parental age may harm offspring health, but mechanisms remain unclear. Alterations in offspring DNA methylation (DNAm) patterns could play a role as aging has been associated with methylation changes in gametes of older individuals. We meta-analyzed epigenome-wide associations of parental age with offspring blood DNAm of over 9500 newborns and 2000 children (5-10 years old) from the Pregnancy and Childhood Epigenetics consortium. In newborns, we identified 33 CpG sites in 13 loci with DNAm associated with maternal age (PFDR

The impact of variations in the three-dimensional structure of the genome has been recognized, but solid cancer tissue studies are limited. Here, we performed integrated deep Hi-C sequencing with matched whole-genome sequencing, whole-genome bisulfite sequencing, 5-hydroxymethylcytosine (5hmC) sequencing and RNA sequencing across a cohort of 80 biopsy samples from patients with metastatic castration-resistant prostate cancer. Dramatic differences were present in gene expression, 5-methylcytosine/5hmC methylation and in structural variation versus mutation rate between A and B (open and closed) chromatin compartments. A subset of tumors exhibited depleted regional chromatin contacts at the AR locus, linked to extrachromosomal circular DNA (ecDNA) and worse response to AR signaling inhibitors. We also identified topological subtypes associated with stark differences in methylation structure, gene expression and prognosis. Our data suggested that DNA interactions may predispose to structural variant formation, exemplified by the recurrent TMPRSS2-ERG fusion. This comprehensive integrated sequencing effort represents a unique clinical tumor resource.

Isoform 1 of DNA methyltransferase DNMT3A (DNMT3A1) specifically recognizes nucleosome monoubiquitylated at histone H2A lysine-119 (H2AK119ub1) for establishment of DNA methylation. Mis-regulation of this process may cause aberrant DNA methylation and pathogenesis. However, the molecular basis underlying DNMT3A1-nucleosome interaction remains elusive. Here we report the cryo-EM structure of DNMT3A1s ubiquitin-dependent recruitment (UDR) fragment complexed with H2AK119ub1-modified nucleosome. DNMT3A1 UDR occupies an extensive nucleosome surface, involving the H2A-H2B acidic patch, a surface groove formed by H2A and H3, nucleosomal DNA, and H2AK119ub1. The DNMT3A1 UDRs interaction with H2AK119ub1 affects the functionality of DNMT3A1 in cells in a context-dependent manner. Our structural and biochemical analysis also reveals competition between DNMT3A1 and JARID2, a cofactor of polycomb repression complex 2 (PRC2), for nucleosome binding, suggesting the interplay between different epigenetic pathways. Together, this study reports a molecular basis for H2AK119ub1-dependent DNMT3A1-nucleosome association, with important implications in DNMT3A1-mediated DNA methylation in development.

BACKGROUND/AIMS: While DNA methylation and gastric microbiome are each associated with gastric cancer (GC), their combined role in predicting GC remains unclear. This study investigated the potential of a combined DNA methylation and gastric microbiome signature to predict Helicobacter pylori-negative GC. METHODS: In this case-control study, we conducted quantitative methylation-specific polymerase chain reaction to measure the methylation levels of DKK3, SFRP1, EMX1, NKX6-1, MIR124-3, and TWIST1 in the gastric mucosa from 75 H. pylori-negative patients, including chronic gastritis (CG), intestinal metaplasia (IM), and GC. A combined analysis of DNA methylation and gastric microbiome, using 16S rRNA gene sequencing, was performed in 30 of 75 patients. RESULTS: The methylation levels of DKK3, SFRP1, EMX1, MIR124-3, and TWIST1 were significantly higher in patients with GC than in controls (all q

The evaluation of epigenetic features such as DNA methylation is becoming increasingly important in many biochemical processes like gene expression and transcription as well as in several diseases like schizophrenia or diabetes. Here, we report that self-assembled nanomechanical resonators entirely composed of DNA molecules can be used to explore gross changes in DNA methylation levels (0-25-50%), while careful control of tensile stress is needed to reduce the variability of resonance frequency for rigorous quantification. The effect of the tensile stress retained by the suspended DNA nanoresonators on the application of the technique is extensively explored using a combination of laser Doppler vibrometry and atomic force spectroscopy. DNA nanoresonators are real-time, label-free sensors and could avoid chemical functionalization and sample amplification. Therefore, they may represent in the future a key complementary routine tool for global DNA methylation analysis needed to evaluate the consequences of environmental stresses on the human genome.

Epigenetic dysregulation has emerged as an important etiological mechanism of neurodevelopmental disorders (NDDs). Pathogenic variation in epigenetic regulators can impair deposition of histone post-translational modifications leading to aberrant spatiotemporal gene expression during neurodevelopment. The male-specific lethal (MSL) complex is a prominent multi-subunit epigenetic regulator of gene expression and is responsible for histone 4 lysine 16 acetylation (H4K16ac). Using exome sequencing, here we identify a cohort of 25 individuals with heterozygous de novo variants in MSL complex member MSL2. MSL2 variants were associated with NDD phenotypes including global developmental delay, intellectual disability, hypotonia, and motor issues such as coordination problems, feeding difficulties, and gait disturbance. Dysmorphisms and behavioral and/or psychiatric conditions, including autism spectrum disorder, and to a lesser extent, seizures, connective tissue disease signs, sleep disturbance, vision problems, and other organ anomalies, were observed in affected individuals. As a molecular biomarker, a sensitive and specific DNA methylation episignature has been established. Induced pluripotent stem cells (iPSCs) derived from three members of our cohort exhibited reduced MSL2 levels. Remarkably, while NDD-associated variants in two other members of the MSL complex (MOF and MSL3) result in reduced H4K16ac, global H4K16ac levels are unchanged in iPSCs with MSL2 variants. Regardless, MSL2 variants altered the expression of MSL2 targets in iPSCs and upon their differentiation to early germ layers. Our study defines an MSL2-related disorder as an NDD with distinguishable clinical features, a specific blood DNA episignature, and a distinct, MSL2-specific molecular etiology compared to other MSL complex-related disorders.

ImportanceResearch on fetal epigenetic programming suggests that the intrauterine environment can have long-term effects on offspring disease susceptibility.ObjectiveTo examine the association between prenatal maternal occupation and child epigenetic age acceleration (EAA) among a farmworker community.Design, setting, and participantsThis cohort study included participants in the Center for the Health Assessment of Mothers and Children of Salinas, a prospective, Latino, prebirth cohort. Pregnant women were recruited from October 1, 1999, to October 1, 2000, from 6 community clinics in California's Salinas Valley agricultural region. Participants were 18 years or older, English or Spanish speaking, Medicaid eligible, and at 20 weeks' gestation or earlier at enrollment. Mother-child pairs who had blood DNA methylation measured at the ages of 7, 9, and 14 years were included. Data were analyzed from July 2021 to November 2023.ExposuresPrenatal maternal occupation was ascertained through study interviews conducted during prenatal visits and shortly after delivery.Main outcomes and measuresChild EAA at 7, 9, and 14 years of age was estimated using DNA methylation-based epigenetic age biomarkers. Three EAA measures were calculated: the Horvath EAA, skin and blood EAA, and intrinsic EAA. Linear mixed-effects models were used to estimate longitudinal associations of prenatal maternal occupation and child EAA, adjusting for confounders and prenatal organophosphate pesticide exposure.ResultsAnalyses included 290 mother-child pairs (mean [SD] maternal age at delivery, 26.5 [5.2] years; 152 [52.4%] female infants); 254 mothers (87.6%) were born in Mexico, 33 (11.4%) in the US, and 3 (1.0%) in other countries; and 179 families (61.7%) were below the federal poverty line during pregnancy. Mothers reported engaging in several types of work during pregnancy, including agricultural fieldwork (90 [31.0%]), other agricultural work (40 [13.8%]), nonagricultural work (53 [18.3%]), or no work (107 [36.9%]). Children whose mothers worked in agricultural fields during pregnancy had a mean of 0.66 (95% CI, 0.17-1.15) years of greater Horvath EAA, 0.62 (95% CI, 0.31-0.94) years of greater skin and blood EAA, and 0.45 (95% CI, 0.07-0.83) years of greater intrinsic EAA compared with children whose mothers did not work during pregnancy.Conclusions and relevanceIn this cohort study, prenatal maternal agricultural fieldwork was associated with accelerated childhood epigenetic aging independent of organophosphate pesticide exposure. Future research on which factors related to agricultural fieldwork accelerate aging in the next generation can inform targeted prevention programs and policies that protect children's health.

Phenotypic plasticity can buffer organisms against short-term environmental fluctuations. For example, previous exposure to increased temperatures can increase thermal tolerance in many species. Prior studies have found that acclimation to higher temperature can influence the magnitude of transcriptional response to subsequent acute thermal stress (hereafter, transcriptional response modulation). However, mechanisms mediating this gene expression response and, ultimately, phenotypic plasticity remain largely unknown. Epigenetic modifications are good candidates for modulating transcriptional response, as they broadly correlate with gene expression. Here, we investigate changes in DNA methylation as a possible mechanism controlling shifts in gene expression plasticity and thermal acclimation in the reef-building coral Acropora nana. We find that gene expression response to acute stress is altered in corals acclimated to different temperatures, with many genes exhibiting a dampened response to heat stress in corals pre-conditioned to higher temperatures. At the same time, we observe shifts in methylation during both acclimation (11 days) and acute heat stress (24 h). We observed that the acute heat stress results in shifts in gene-level methylation and elicits an acute transcriptional response in distinct gene sets. Further, acclimation-induced shifts in gene expression plasticity and differential methylation also largely occur in separate sets of genes. Counter to our initial hypothesis no overall correlation between the magnitude of differential methylation and the change in gene expression plasticity. We do find a small but statistically significant overlap in genes exhibiting both dampened expression response and shifts in methylation (14 genes), which could be candidates for further inquiry. Overall, our results suggest transcriptional response modulation occurs independently from methylation changes induced by thermal acclimation.

Glioblastoma (GBM) is the most common malignant primary brain tumor and remains incurable. Previous work has shown that systemic administration of Decitabine (DAC) induces sufficient expression of cancer-testis antigens (CTA) in GBM for targeting by adoptive T-cell therapy in vivo. However, the mechanisms by which DAC enhances immunogenicity in GBM remain to be elucidated. Using New York esophageal squamous cell carcinoma 1 (NY-ESO-1) as a representative inducible CTA, we demonstrate in patient tissue, immortalized glioma cells, and primary patient-derived gliomaspheres that basal CTA expression is restricted by promoter hypermethylation in gliomas. DAC treatment of glioma cells specifically inhibits DNA methylation silencing to render NY-ESO-1 and other CTA into inducible tumor antigens at single-cell resolution. Functionally, NY-ESO-1 T-cell receptor-engineered effector cell targeting of DAC-induced antigen in primary glioma cells promotes specific and polyfunctional T-cell cytokine profiles. In addition to induction of CTA, DAC concomitantly reactivates tumor-intrinsic human endogenous retroviruses, interferon response signatures, and MHC-I. Overall, we demonstrate that DAC induces targetable tumor antigen and enhances T-cell functionality against GBM, ultimately contributing to the improvement of targeted immune therapies in glioma.SignificanceThis study dissects the tumor-intrinsic epigenetic and transcriptional mechanisms underlying enhanced T-cell functionality targeting decitabine-induced cancer-testis antigens in glioma. Our findings demonstrate concomitant induction of tumor antigens, reactivation of human endogenous retroviruses, and stimulation of interferon signaling as a mechanistic rationale to epigenetically prime human gliomas to immunotherapeutic targeting.

Adverse early-life experiences (ELA) affect a majority of the worlds children. Whereas the enduring impact of ELA on cognitive and emotional health is established, there are no tools to predict vulnerability to ELA consequences in an individual child. Epigenetic markers including peripheral-cell DNA-methylation profiles may encode ELA and provide predictive outcome markers, yet the interindividual variance of the human genome and rapid changes in DNA methylation in childhood pose significant challenges. Hoping to mitigate these challenges we examined the relation of several ELA dimensions to DNA methylation changes and outcome using a within-subject longitudinal design and a high methylation-change threshold. DNA methylation was analyzed in buccal swab/saliva samples collected twice (neonatally and at 12 months) in 110 infants. We identified CpGs differentially methylated across time for each child and determined whether they associated with ELA indicators and executive function at age 5. We assessed sex differences and derived a sex-dependent impact score based on sites that most contributed to methylation changes. Changes in methylation between two samples of an individual child reflected age-related trends and correlated with executive function years later. Among tested ELA dimensions and life factors including income to needs ratios, maternal sensitivity, body mass index and infant sex, unpredictability of parental and household signals was the strongest predictor of executive function. In girls, high early-life unpredictability interacted with methylation changes to presage executive function. Thus, longitudinal, within-subject changes in methylation profiles may provide a signature of ELA and a potential predictive marker of individual outcome.

Accurate diagnosis of lung cancer is important for treatment decision-making. Tumor biopsy and histologic examination are the standard for determining histologic lung cancer subtypes. Liquid biopsy, particularly cell-free DNA (cfDNA), has recently shown promising results in cancer detection and classification. In this study, we investigate the potential of cfDNA methylome for the noninvasive classification of lung cancer histologic subtypes. We focused on the two most prevalent lung cancer subtypes, lung adenocarcinoma and lung squamous cell carcinoma. Using a fragment-based marker discovery approach, we identified robust subtype-specific methylation markers from tumor samples. These markers were successfully validated in independent cohorts and associated with subtype-specific transcriptional activity. Leveraging these markers, we constructed a subtype classification model using cfDNA methylation profiles, achieving an AUC of 0.808 in cross-validation and an AUC of 0.747 in the independent validation. Tumor copy-number alterations inferred from cfDNA methylome analysis revealed potential for treatment selection. In summary, our study demonstrates the potential of cfDNA methylome analysis for noninvasive lung cancer subtyping, offering insights for cancer monitoring and early detection.SignificanceThis study explores the use of cfDNA methylomes for the classification of lung cancer subtypes, vital for effective treatment. By identifying specific methylation markers in tumor tissues, we developed a robust classification model achieving high accuracy for noninvasive subtype detection. This cfDNA methylome approach offers promising avenues for early detection and monitoring.

IntroductionIdentifying reliable biomarkers that reflect cancer survivorship symptoms remains a challenge for researchers. DNA methylation (DNAm) measurements reflecting epigenetic changes caused by anti-cancer therapy may provide needed insights. Given lack of consensus describing utilization of DNAm data to predict survivorship issues, a review evaluating the current landscape is warranted.ObjectiveProvide an overview of current studies examining associations of DNAm with survivorship burdens in cancer survivors.MethodsA literature review was conducted including studies if they focused on cohorts of cancer survivors, utilized peripheral blood cell DNAm data, and evaluated the associations of DNAm and survivorship issues.ResultsA total of 22 studies were identified, with majority focused on breast (n = 7) or childhood cancer (n = 9) survivors, and half studies included less than 100 patients (n = 11). Survivorship issues evaluated included those related to neurocognition (n = 5), psychiatric health (n = 3), general wellness (n = 9), chronic conditions (n = 5), and treatment specific toxicities (n = 4). Studies evaluated epigenetic age metrics (n = 10) and DNAm levels at individual CpG sites or regions (n = 12) for their associations with survivorship issues in cancer survivors along with relevant confounding factors. Significant associations of measured DNAm in the peripheral blood samples of cancer survivors and survivorship issues were identified.Discussion/conclusionStudies utilizing epigenetic age metrics and differential methylation analysis demonstrated significant associations of DNAm measurements with survivorship burdens. Associations were observed encompassing diverse survivorship outcomes and timeframes relative to anti-cancer therapy initiation. These findings underscore the potential of these measurements as useful biomarkers in survivorship care and research.

BackgroundRestraining or slowing ageing hallmarks at the cellular level have been proposed as a route to increased organismal lifespan and healthspan. Consequently, there is great interest in anti-ageing drug discovery. However, this currently requires laborious and lengthy longevity analysis. Here, we present a novel screening readout for the expedited discovery of compounds that restrain ageing of cell populations in vitro and enable extension of in vivo lifespan.MethodsUsing Illumina methylation arrays, we monitored DNA methylation changes accompanying long-term passaging of adult primary human cells in culture. This enabled us to develop, test, and validate the CellPopAge Clock, an epigenetic clock with underlying algorithm, unique among existing epigenetic clocks for its design to detect anti-ageing compounds in vitro. Additionally, we measured markers of senescence and performed longevity experiments in vivo in Drosophila, to further validate our approach to discover novel anti-ageing compounds. Finally, we bench mark our epigenetic clock with other available epigenetic clocks to consolidate its usefulness and specialisation for primary cells in culture.ResultsWe developed a novel epigenetic clock, the CellPopAge Clock, to accurately monitor the age of a population of adult human primary cells. We find that the CellPopAge Clock can detect decelerated passage-based ageing of human primary cells treated with rapamycin or trametinib, well-established longevity drugs. We then utilise the CellPopAge Clock as a screening tool for the identification of compounds which decelerate ageing of cell populations, uncovering novel anti-ageing drugs, torin2 and dactolisib (BEZ-235). We demonstrate that delayed epigenetic ageing in human primary cells treated with anti-ageing compounds is accompanied by a reduction in senescence and ageing biomarkers. Finally, we extend our screening platform in vivo by taking advantage of a specially formulated holidic medium for increased drug bioavailability in Drosophila. We show that the novel anti-ageing drugs, torin2 and dactolisib (BEZ-235), increase longevity in vivo.ConclusionsOur method expands the scope of CpG methylation profiling to accurately and rapidly detecting anti-ageing potential of drugs using human cells in vitro, and in vivo, providing a novel accelerated discovery platform to test sought after anti-ageing compounds and geroprotectors.

Whole-genome bisulfite sequencing (BS-Seq) measures cytosine methylation changes at single-base resolution and can be used to profile cell-free DNA (cfDNA). In plasma, ultrashort single-stranded cfDNA (uscfDNA, ∼50 nt) has been identified together with 167 bp double-stranded mononucleosomal cell-free DNA (mncfDNA). However, the methylation profile of uscfDNA has not been described. Conventional BS-Seq workflows may not be helpful because bisulfite conversion degrades larger DNA into smaller fragments, leading to erroneous categorization as uscfDNA. We describe the '5mCAdpBS-Seq' workflow in which pre-methylated 5mC (5-methylcytosine) single-stranded adapters are ligated to heat-denatured cfDNA before bisulfite conversion. This method retains only DNA fragments that are unaltered by bisulfite treatment, resulting in less biased uscfDNA methylation analysis. Using 5mCAdpBS-Seq, uscfDNA had lower levels of DNA methylation (∼15%) compared to mncfDNA and was enriched in promoters and CpG islands. Hypomethylated uscfDNA fragments were enriched in upstream transcription start sites (TSSs), and the intensity of enrichment was correlated with expressed genes of hemopoietic cells. Using tissue-of-origin deconvolution, we inferred that uscfDNA is derived primarily from eosinophils, neutrophils, and monocytes. As proof-of-principle, we show that characteristics of the methylation profile of uscfDNA can distinguish non-small cell lung carcinoma from non-cancer samples. The 5mCAdpBS-Seq workflow is recommended for any cfDNA methylation-based investigations.

BackgroundMaternal cigarette smoking during pregnancy (MSDP) is associated with numerous adverse health outcomes in infants and children with potential lifelong consequences. Negative effects of MSDP on placental DNA methylation (DNAm), placental structure, and function are well established.ObjectiveOur aim was to develop biomarkers of MSDP using DNAm measured in placentas (N=96), collected as part of the Vitamin C to Decrease the Effects of Smoking in Pregnancy on Infant Lung Function double-blind, placebo-controlled randomized clinical trial conducted between 2012 and 2016. We also aimed to develop a digital polymerase chain reaction (PCR) assay for the top ranking cytosine-guanine dinucleotide (CpG) so that large numbers of samples can be screened for exposure at low cost.MethodsWe compared the ability of four machine learning methods [logistic least absolute shrinkage and selection operator (LASSO) regression, logistic elastic net regression, random forest, and gradient boosting machine] to classify MSDP based on placental DNAm signatures. We developed separate models using the complete EPIC array dataset and on the subset of probes also found on the 450K array so that models exist for both platforms. For comparison, we developed a model using CpGs previously associated with MSDP in placenta. For each final model, we used model coefficients and normalized beta values to calculate placental smoking index (PSI) scores for each sample. Final models were validated in two external datasets: the Extremely Low Gestational Age Newborn observational study, N=426; and the Rhode Island Children's Health Study, N=237.ResultsLogistic LASSO regression demonstrated the highest performance in cross-validation testing with the lowest number of input CpGs. Accuracy was greatest in external datasets when using models developed for the same platform. PSI scores in smokers only (n=72) were moderately correlated with maternal plasma cotinine levels. One CpG (cg27402634), with the largest coefficient in two models, was measured accurately by digital PCR compared with measurement by EPIC array (R2=0.98).DiscussionTo our knowledge, we have developed the first placental DNAm-based biomarkers of MSDP with broad utility to studies of prenatal disease origins. https://doi.org/10.1289/EHP13838.

Altered transcriptional and epigenetic regulation of brain cell types may contribute to cognitive changes with advanced age. Using single-nucleus multi-omic DNA methylation and transcriptome sequencing (snmCT-seq) in frontal cortex from young adult and aged donors, we found widespread age- and sex-related variation in specific neuron types. The proportion of inhibitory SST- and VIP-expressing neurons was reduced in aged donors. Excitatory neurons had more profound age-related changes in their gene expression and DNA methylation than inhibitory cells. Hundreds of genes involved in synaptic activity, including EGR1, were less expressed in aged adults. Genes located in subtelomeric regions increased their expression with age and correlated with reduced telomere length. We further mapped cell-type-specific sex differences in gene expression and X-inactivation escape genes. Multi-omic single-nucleus epigenomes and transcriptomes provide new insight into the effects of age and sex on human neurons.

Few neuropsychiatric disorders have replicable biomarkers, prompting high-resolution and large-scale molecular studies. However, we still lack consensus on a more foundational question: whether quantitative shifts in cell types-the functional unit of life-contribute to neuropsychiatric disorders. Leveraging advances in human brain single-cell methylomics, we deconvolve seven major cell types using bulk DNA methylation profiling across 1270 postmortem brains, including from individuals diagnosed with Alzheimers disease, schizophrenia, and autism. We observe and replicate cell-type compositional shifts for Alzheimers disease (endothelial cell loss), autism (increased microglia), and schizophrenia (decreased oligodendrocytes), and find age- and sex-related changes. Multiple layers of evidence indicate that endothelial cell loss contributes to Alzheimers disease, with comparable effect size to APOE genotype among older people. Genome-wide association identified five genetic loci related to cell-type composition, involving plausible genes for the neurovascular unit (P2RX5 and TRPV3) and excitatory neurons (DPY30 and MEMO1). These results implicate specific cell-type shifts in the pathophysiology of neuropsychiatric disorders.

PURPOSE: Advancing age is one of the strongest risk factors for osteoarthritis (OA). DNA methylation-based measures of epigenetic age acceleration may provide insights into mechanisms underlying OA. METHODS: We analyzed data from the Multicenter Osteoarthritis Study in a subset of 671 participants ages 45-69 years with no or mild radiographic knee OA. DNA methylation was assessed with the Illumina Infinium MethylationEPIC 850K array. We calculated predicted epigenetic age according to Hannum, Horvath, PhenoAge, and GrimAge epigenetic clocks, then regressed epigenetic age on chronological age to obtain the residuals. Associations between the residuals and knee, hand, and multi-joint OA were assessed using logistic regression, adjusted for chronological age, sex, clinical site, smoking status, and race. RESULTS: Twenty-three percent met criteria for radiographic hand OA, 25% met criteria for radiographic knee OA, and 8% met criteria for multi-joint OA. Mean chronological age (SD) was 58.4 (6.7) years. Mean predicted epigenetic age (SD) according to Horvath, Hannum, PhenoAge, and GrimAge epigenetic clocks was 64.9 (6.4), 68.6 (5.9), 50.5 (7.7), and 67.0 (6.2), respectively. Horvath epigenetic age acceleration was not associated with an increased odds of hand OA, odds ratio (95% confidence intervals) = 1.03 (0.99-1.08), with similar findings for knee and multi-joint OA. We found similar magnitudes of associations for Hannum epigenetic age, PhenoAge, and GrimAge acceleration compared to Horvath epigenetic age acceleration. CONCLUSIONS: Epigenetic age acceleration as measured by various well-validated epigenetic clocks based on DNA methylation was not associated with increased risk of knee, hand, or multi-joint OA independent of chronological age.

Plants rely on Nucleotide-binding, Leucine-rich repeat Receptors (NLRs) for pathogen recognition. Highly variable NLRs (hvNLRs) show remarkable intraspecies diversity, while their low-variability paralogs (non-hvNLRs) are conserved between ecotypes. At a population level, hvNLRs provide new pathogen-recognition specificities, but the association between allelic diversity and genomic and epigenomic features has not been established. Our investigation of NLRs in Arabidopsis Col-0 has revealed that hvNLRs show higher expression, less gene body cytosine methylation, and closer proximity to transposable elements than non-hvNLRs. hvNLRs show elevated synonymous and nonsynonymous nucleotide diversity and are in chromatin states associated with an increased probability of mutation. Diversifying selection maintains variability at a subset of codons of hvNLRs, while purifying selection maintains conservation at non-hvNLRs. How these features are established and maintained, and whether they contribute to the observed diversity of hvNLRs is key to understanding the evolution of plant innate immune receptors.

A regulated stress response is essential for healthy child growth and development trajectories. We conducted a cluster-randomized trial in rural Bangladesh (funded by the Bill & Melinda Gates Foundation, ClinicalTrials.gov NCT01590095) to assess the effects of an integrated nutritional, water, sanitation, and handwashing intervention on child health. We previously reported on the primary outcomes of the trial, linear growth and caregiver-reported diarrhea. Here, we assessed additional prespecified outcomes: physiological stress response, oxidative stress, and DNA methylation (N = 759, ages 1-2 years). Eight neighboring pregnant women were grouped into a study cluster. Eight geographically adjacent clusters were block-randomized into the control or the combined nutrition, water, sanitation, and handwashing (N + WSH) intervention group (receiving nutritional counseling and lipid-based nutrient supplements, chlorinated drinking water, upgraded sanitation, and handwashing with soap). Participants and data collectors were not masked, but analyses were masked. There were 358 children (68 clusters) in the control group and 401 children (63 clusters) in the intervention group. We measured four F2-isoprostanes isomers (iPF(2α)-III; 2,3-dinor-iPF(2α)-III; iPF(2α)-VI; 8,12-iso-iPF(2α)-VI), salivary alpha-amylase and cortisol, and methylation of the glucocorticoid receptor (NR3C1) exon 1F promoter including the NGFI-A binding site. Compared with control, the N + WSH group had lower concentrations of F2-isoprostanes isomers (differences ranging from -0.16 to -0.19 log ng/mg of creatinine, P

Duckweeds are among the fastest reproducing plants, able to clonally divide at exponential rates. However, the genetic and epigenetic impact of clonality on plant genomes is poorly understood. 5-methylcytosine (5mC) is a modified base often described as necessary for the proper regulation of certain genes and transposons and for the maintenance of genome integrity in plants. However, the extent of this dogma is limited by the current phylogenetic sampling of land plant species diversity. Here we analyzed DNA methylomes, small RNAs, mRNA-seq, and H3K9me2 histone modification for Spirodela polyrhiza. S. polyrhiza has lost highly conserved genes involved in de novo methylation of DNA at sites often associated with repetitive DNA, and within genes, however, symmetrical DNA methylation and heterochromatin are maintained during cell division at certain transposons and repeats. Consequently, small RNAs that normally guide methylation to silence repetitive DNA like retrotransposons are diminished. Despite the loss of a highly conserved methylation pathway, and the reduction of small RNAs that normally target repetitive DNA, transposons have not proliferated in the genome, perhaps due in part to the rapid, clonal growth lifestyle of duckweeds.

BACKGROUND: MGMT (O 6 -methylguanine-DNA methyltransferase) promoter methylation is a commonly assessed prognostic marker in glioblastoma (GBM). Epigenetic silencing of the MGMT gene by promoter methylation is associated with greater overall and progression free survival with alkylating agent regimens. To date, there is marked heterogeneity in how MGMT promoter methylation is tested and which CpG sites are interrogated. METHODS: To further elucidate which MGMT promoter CpG sites are of greatest interest, we performed comprehensive searches in PubMed, Web of Science, and Embase and reviewed 2,925 article abstracts. We followed the GRADE scoring system to assess risk of bias and the quality of the studies we included. RESULTS: We included articles on adult glioblastoma that examined significant sites or regions within MGMT promoter for the outcomes: overall survival, progression free survival, and/or MGMT expression. We excluded systemic reviews and articles on lower grade glioma. fifteen articles met inclusion criteria with variable overlap in laboratory and statistical methods employed, as well as CpG sites interrogated. Pyrosequencing or BeadChip arrays were the most popular methods utilized, and CpG sites between CpGs 70-90 were most frequently investigated. Overall, there was moderate concordance between the CpG sites that the studies reported to be highly predictive of prognosis. Combinations or means of sites between CpGs 73-89 were associated with improved OS and PFS. Six studies identified CpG sites associated with prognosis that were closer to the transcription start site: CpGs 8, 19, 22, 25, 27, 32,38, and CpG sites 21-37, as well as low methylation level of the enhancer regions. CONCLUSION: The following systematic review details a comprehensive investigation of the current literature and highlights several potential key CpG sites that demonstrate significant association with OS, PFS, and MGMT expression. However, the relationship between extent of MGMT promoter methylation and survival may be non-linear and could be influenced by potential CpG hotspots, the extent of methylation at each CpG site, and MGMT enhancer methylation status. There were several limitations within the studies such as smaller sample sizes, variance between methylation testing methods, and differences in the various statistical methods to test for association to outcome. Further studies of high impact CpG sites in MGMT methylation is warranted.

DNA methylation provides a crucial epigenetic mark linking genetic variations to environmental influence. We have analyzed array-based DNA methylation profiles of 160 human retinas with co-measured RNA-seq and >8 million genetic variants, uncovering sites of genetic regulation in cis (37,453 methylation quantitative trait loci and 12,505 expression quantitative trait loci) and 13,747 DNA methylation loci affecting gene expression, with over one-third specific to the retina. Methylation and expression quantitative trait loci show non-random distribution and enrichment of biological processes related to synapse, mitochondria, and catabolism. Summary data-based Mendelian randomization and colocalization analyses identify 87 target genes where methylation and gene-expression changes likely mediate the genotype effect on age-related macular degeneration. Integrated pathway analysis reveals epigenetic regulation of immune response and metabolism including the glutathione pathway and glycolysis. Our study thus defines key roles of genetic variations driving methylation changes, prioritizes epigenetic control of gene expression, and suggests frameworks for regulation of macular degeneration pathology by genotype-environment interaction in retina.

Background: The osteoclastogenesis RANKL gene plays a key role in bone remodeling. The hypomethylation of its promoter region may cause osteoporosis. The present study aimed to elucidate the influence of physical activity on DNA methylation changes of RANKL promoter cytosine-phosphate-guanine (CpG)-rich region in active and sedentary adults and to assess the effect of aerobic and strength training on RANKL DNA methylation changes among Tunisian-North African adults. Methods: A total of 104 participants including 52 adults (58% males and 42% females) and 52 adults (31% males and 69% females) were recruited for the observational and interventional part of the study, respectively. The intervention consisted of 12 weeks of aerobic training (30 min/session) followed by 10 minutes of strengthening exercises. All participants completed the International Physical Activity Questionnaire and provided blood samples for quantitative methylation-specific polymerase chain reaction (PCR) analysis. Results: The study revealed a significant difference (P = 6 × 10−10) in the methylation level of the RANKL promoter region between active and sedentary adults, with a 6.68-fold increase observed in the active group. After the intervention, both the trained (P = 41 × 10−5) and untrained (P =.002) groups displayed high methylation levels in the RANKL promoter region. In addition, the trained group exhibited significant improvements in heart rate (P = 2.2 × 10−16), blood pressure (P = 39 × 10−3), maximal oxygen uptake (P = 1.5 × 10−7), and fat mass (P = 7 × 10−4). Conclusion: Exploring epigenetic modifications in the RANKL promoter region may contribute to a more comprehensive understanding of the complexity of osteoporosis. This suggests that aerobic/strength training could potentially improve the bone system, reducing its vulnerability to osteoporosis by increasing RANKL DNA methylation. [ABSTRACT FROM AUTHOR]

In this study, the effect of cumulative ACEs experienced on human maternal DNA methylation (DNAm) was estimated while accounting for interaction with domains of ACEs in prenatal peripheral blood mononuclear cell samples from the Maternal and Developmental Risks from Environmental Stressors (MADRES) pregnancy cohort. The intergenerational transmission of ACE-associated DNAm was also explored used paired maternal (N = 120) and neonatal cord blood (N = 69) samples. Replication in buccal samples was explored in the Childrens Health Study (CHS) among adult parental (N = 31) and pediatric (N = 114) samples. We used a four-level categorical indicator variable for ACEs exposure: none (0 ACEs), low (1-3 ACEs), moderate (4-6 ACEs), and high (>6 ACEs). Effects of ACEs on maternal DNAm (N = 240) were estimated using linear models. To evaluate evidence for intergenerational transmission, mediation analysis (N = 60 mother-child pairs) was used. Analysis of maternal samples displayed some shared but mostly distinct effects of ACEs on DNAm across low, moderate, and high ACEs categories. CLCN7 and PTPRN2 was associated with maternal DNAm in the low ACE group and this association replicated in the CHS. CLCN7 was also nominally significant in the gene expression correlation analysis among maternal profiles (N = 35), along with 11 other genes. ACE-associated methylation was observed in maternal and neonatal profiles in the COMT promoter region, with some evidence of mediation by maternal COMT methylation. Specific genomic loci exhibited mutually exclusive maternal ACE effects on DNAm in either maternal or neonatal population. There is some evidence for an intergenerational effect of ACEs, supported by shared DNAm signatures in the COMT gene across maternal-neonatal paired samples.

Maternal educational attainment (MEA) shapes offspring health through multiple potential pathways. Differential DNA methylation may provide a mechanistic understanding of these long-term associations. We aimed to quantify the associations of MEA with offspring DNA methylation levels at birth, in childhood and in adolescence. Using 37 studies from high-income countries, we performed meta-analysis of epigenome-wide association studies (EWAS) to quantify the associations of completed years of MEA at the time of pregnancy with offspring DNA methylation levels at birth (n = 9 881), in childhood (n = 2 017), and adolescence (n = 2 740), adjusting for relevant covariates. MEA was found to be associated with DNA methylation at 473 cytosine-phosphate-guanine sites at birth, one in childhood, and four in adolescence. We observed enrichment for findings from previous EWAS on maternal folate, vitamin-B12 concentrations, maternal smoking, and pre-pregnancy BMI. The associations were directionally consistent with MEA being inversely associated with behaviours including smoking and BMI. Our findings form a bridge between socio-economic factors and biology and highlight potential pathways underlying effects of maternal education. The results broaden our understanding of bio-social associations linked to differential DNA methylation in multiple early stages of life. The data generated also offers an important resource to help a more precise understanding of the social determinants of health.

Young blood plasma is known to confer beneficial effects on various organs in mice and rats. However, it was not known whether plasma from young adult pigs rejuvenates old rat tissues at the epigenetic level; whether it alters the epigenetic clock, which is a highly accurate molecular biomarker of aging. To address this question, we developed and validated six different epigenetic clocks for rat tissues that are based on DNA methylation values derived from n = 613 tissue samples. As indicated by their respective names, the rat pan-tissue clock can be applied to DNA methylation profiles from all rat tissues, while the rat brain, liver, and blood clocks apply to the corresponding tissue types. We also developed two epigenetic clocks that apply to both human and rat tissues by adding n = 1366 human tissue samples to the training data. We employed these six rat clocks to investigate the rejuvenation effects of a porcine plasma fraction treatment in different rat tissues. The treatment more than halved the epigenetic ages of blood, heart, and liver tissue. A less pronounced, but statistically significant, rejuvenation effect could be observed in the hypothalamus. The treatment was accompanied by progressive improvement in the function of these organs as ascertained through numerous biochemical/physiological biomarkers, behavioral responses encompassing cognitive functions. An immunoglobulin G (IgG) N-glycosylation pattern shift from pro- to anti-inflammatory also indicated reversal of glycan aging. Overall, this study demonstrates that a young porcine plasma-derived treatment markedly reverses aging in rats according to epigenetic clocks, IgG glycans, and other biomarkers of aging.

Improvements in survival have been made over the past two decades for childhood acute myeloid leukemia (AML), but the approximately 40% of patients who relapse continue to have poor outcomes. A combination of checkpoint-inhibitor nivolumab and azacitidine has demonstrated improvements in median survival in adults with AML. This phase I/II study with nivolumab and azacitidine in children with relapsed/refractory AML (NCT03825367) was conducted through the Therapeutic Advances in Childhood Leukemia & Lymphoma consortium. Thirteen patients, median age 13.7 years, were enrolled. Patients had refractory disease with multiple reinduction attempts. Twelve evaluable patients were treated at the recommended phase II dose (established at dose level 1, 3 mg/kg/dose). Four patients (33%) maintained stable disease. This combination was well tolerated, with no dose-limiting toxicities observed. Grade 3-4 adverse events (AEs) were primarily hematological. Febrile neutropenia was the most common AE ≥ grade 3. A trend to improved quality of life was noted. Increases in CD8+ T cells and reductions in CD4+/CD8+ T cells and demethylation were observed. The combination was well tolerated and had an acceptable safety profile in pediatric patients with relapsed/refractory AML. Future studies might explore this combination for the maintenance of remission in children with AML at high risk of relapse.

Influenza virus infection alters the promoter DNA methylation of key immune response-related genes, including type-1 interferons and proinflammatory cytokines. However, less is known about the effect of the influenza vaccine on the epigenome. We utilized a targeted DNA methylation approach to study the longitudinal effects (day 0 pre-vaccination and day 28 post-vaccination) on influenza vaccination responses in peripheral blood mononuclear cells. We found that baseline, pre-vaccination methylation profiles are associated with pre-existing, protective serological immunity. Additionally, we identified 481 sites that were differentially methylated between baseline and day 28 post-vaccination. These were enriched for genes involved in the regulation of the RIG-I signaling pathway, an important regulator of viral responses. Our results suggest that DNA methylation changes to components of the RIG-I pathway are associated with vaccine effectiveness. Therefore, immunization strategies that target this pathway may improve serological responses to influenza vaccination.

Dementia with Lewy bodies (DLB) is a common form of dementia in the elderly population. We performed genome-wide DNA methylation mapping of cerebellar tissue from pathologically confirmed DLB cases and controls to study the epigenetic profile of this understudied disease. After quality control filtering, 728,197 CpG-sites in 278 cases and 172 controls were available for the analysis. We undertook an epigenome-wide association study, which found a differential methylation signature in DLB cases. Our analysis identified seven differentially methylated probes and three regions associated with DLB. The most significant CpGs were located in ARSB (cg16086807), LINC00173 (cg18800161), and MGRN1 (cg16250093). Functional enrichment evaluations found widespread epigenetic dysregulation in genes associated with neuron-to-neuron synapse, postsynaptic specialization, postsynaptic density, and CTCF-mediated synaptic plasticity. In conclusion, our study highlights the potential importance of epigenetic alterations in the pathogenesis of DLB and provides insights into the modified genes, regions and pathways that may guide therapeutic developments.

Maternal use of e-cigarette (e-cig) aerosols poses significant risks to fetal brain development, potentially increasing susceptibility to neurodevelopmental disorders in later life. However, the underlying mechanisms remain incompletely understood. This study aimed to understand the effects of fetal e-cig exposure on DNA methylome and transcriptomic changes in the neonatal brain. Pregnant rats were exposed to e-cig aerosols, and neonatal brains (5 males and 5 females/group) from both control and e-cig-exposed groups were used for experimental analysis. Results indicated that prenatal e-cig exposure altered site-specific DNA methylation patterns at both CpG and CH (non-CpG) sites, predominantly in intergenic and intronic regions, with sex dimorphism in methylation and gene expression changes. Gene ontology analysis revealed that e-cig exposure not only affected neuron projection development and axonogenesis but also altered pathways related to neurodegeneration and long-term depression. These findings provide novel insights into the dynamic changes of CpG and CH methylation induced by e-cig exposure, underscoring the susceptibility of the developing brain to maternal e-cig exposure and its potential implications for developmental disorders and neurodegenerative diseases later in life.

BackgroundEpigenome-wide association studies have identified multiple DNA methylation sites (CpGs) associated with alcohol consumption, an important lifestyle risk factor for cardiovascular diseases. This study aimed to test the hypothesis that an alcohol consumption epigenetic risk score (ERS) is associated with blood pressure (BP) traits.ResultsWe implemented an ERS based on a previously reported epigenetic signature of 144 alcohol-associated CpGs in meta-analysis of participants of European ancestry. We found a one-unit increment of ERS was associated with eleven drinks of alcohol consumed per day, on average, across several cohorts (p

Aim: This study addresses the challenge of predicting the response of head and neck squamous cell carcinoma (HNSCC) patients to immunotherapy.Methods: Using DNA methylation cytometry, we analyzed the immune profiles of six HNSCC patients who showed a positive response to immunotherapy over a year without disease progression.Results: There was an initial increase in CD8 T memory cells and natural killer cells during the first four cycles of immunotherapy, which then returned to baseline levels after a year. Baseline CD8 T cell levels were lower in HNSCC immunotherapy responders but became similar to those in healthy subjects after immunotherapy.Conclusion: These findings suggest that monitoring fluctuations in immune profiles could potentially identify biomarkers for immunotherapy response in HNSCC patients.

Genetic variants and epigenetic features both contribute to the risk of Alzheimer's disease (AD). We studied the AD association of CpG-related single nucleotide polymorphisms (CGS), which act as a hub of both the genetic and epigenetic effects, in Caribbean Hispanics (CH) and generalized the findings to Non-Hispanic Whites (NHW). First, we conducted a genome-wide, sliding-window-based association with AD, in 7,155 CH and 1,283 NHW participants. Next, using data from the dorsolateral prefrontal cortex in 179 CH brains, we tested the cis- and trans-effects of AD-associated CGS on brain DNA methylation to mRNA expression. For the genes with significant cis- and trans-effects, we investigated their enriched pathways. We identified six genetic loci in CH with CGS dosage associated with AD at genome-wide significance levels: ADAM20 (Score = 55.19, P = 4.06 × 10-8), the intergenic region between VRTN and SYNDIG1L (Score = - 37.67, P = 2.25 × 10-9), SPG7 (16q24.3) (Score = 40.51, P = 2.23 × 10-8), PVRL2 (Score = 125.86, P = 1.64 × 10-9), TOMM40 (Score = - 18.58, P = 4.61 × 10-8), and APOE (Score = 75.12, P = 7.26 × 10-26). CGSes in PVRL2 and APOE were also significant in NHW. Except for ADAM20, CGSes in the other five loci were associated with CH brain methylation levels (mQTLs) and CGSes in SPG7, PVRL2, and APOE were also mQTLs in NHW. Except for SYNDIG1L (P = 0.08), brain methylation levels in the other five loci affected downstream mRNA expression in CH (P

Background: Early adversity increases the risk for mental and physical disorders as well as premature death. Epigenetic processes, and altered epigenetic aging in particular, might mediate these effects. While the literature that examined links between early adversity and epigenetic aging is growing, results have been heterogeneous.Objective: In the current work, we explored the link between early adversity and epigenetic aging in a sample of formerly out-of-home placed young adults.Method: A total of N = 117 young adults (32% women, age mean = 26.3 years, SD = 3.6 years) with previous youth residential care placements completed the Childhood Trauma Questionnaire (CTQ) and the Life Events Checklist (LEC-R) and provided blood samples for the analysis of DNA methylation using the Illumina Infinium MethylationEPIC BeadChip Microarray. Epigenetic age was estimated using Hovarths and Hannums epigenetic clocks. Furthermore, Hovarths and Hannums epigenetic age residuals were calculated as a proxy of epigenetic aging by regressing epigenetic age on chronological age. The statistical analysis plan was preregistered (https://osf.io/b9ev8).Results: Childhood trauma (CTQ) was negatively associated with Hannums epigenetic age residuals, β = -.23, p = .004 when controlling for sex, BMI, smoking status and proportional white blood cell type estimates. This association was driven by experiences of physical neglect, β = -.25, p = .001. Lifetime trauma exposure (LEC-R) was not a significant predictor of epigenetic age residuals.Conclusion: Childhood trauma, and physical neglect in particular, was associated with decelerated epigenetic aging in our sample. More studies focusing on formerly institutionalized at-risk populations are needed to better understand which factors affect stress-related adaptations following traumatic experiences.

DNA N6-adenine methylation (6mA) has recently gained importance as an epigenetic modification in eukaryotes. Its function in lineages with high levels, such as early-diverging fungi (EDF), is of particular interest. Here, we investigated the biological significance and evolutionary implications of 6mA in EDF, which exhibit divergent evolutionary patterns in 6mA usage. The analysis of two Mucorales species displaying extreme 6mA usage reveals that species with high 6mA levels show symmetric methylation enriched in highly expressed genes. In contrast, species with low 6mA levels show mostly asymmetric 6mA. Interestingly, transcriptomic regulation throughout development and in response to environmental cues is associated with changes in the 6mA landscape. Furthermore, we identify an EDF-specific methyltransferase, likely originated from endosymbiotic bacteria, as responsible for asymmetric methylation, while an MTA-70 methylation complex performs symmetric methylation. The distinct phenotypes observed in the corresponding mutants reinforced the critical role of both types of 6mA in EDF.

BACKGROUND: Children from families with low socioeconomic status (SES), as determined by income, experience several negative outcomes, such as higher rates of newborn mortality and behavioral issues. Moreover, associations between DNA methylation and low income or poverty status are evident beginning at birth, suggesting prenatal influences on offspring development. Recent evidence suggests neighborhood opportunities may protect against some of the health consequences of living in low income households. The goal of this study was to assess whether neighborhood opportunities moderate associations between household income (HI) and neonate developmental maturity as measured with DNA methylation. METHODS: Umbilical cord blood DNA methylation data was available in 198 mother-neonate pairs from the larger CANDLE cohort. Gestational age acceleration was calculated using an epigenetic clock designed for neonates. Prenatal HI and neighborhood opportunities measured with the Childhood Opportunity Index (COI) were regressed on gestational age acceleration controlling for sex, race, and cellular composition. RESULTS: Higher HI was associated with higher gestational age acceleration (B = .145, t = 4.969, p = 1.56x10-6, 95% CI [.087, .202]). Contrary to expectation, an interaction emerged showing higher neighborhood educational opportunity was associated with lower gestational age acceleration at birth for neonates with mothers living in moderate to high HI (B = -.048, t = -2.08, p = .03, 95% CI [-.092, -.002]). Female neonates showed higher gestational age acceleration at birth compared to males. However, within males, being born into neighborhoods with higher social and economic opportunity was associated with higher gestational age acceleration. CONCLUSION: Prenatal HI and neighborhood qualities may affect gestational age acceleration at birth. Therefore, policy makers should consider neighborhood qualities as one opportunity to mitigate prenatal developmental effects of HI.

Chronic kidney disease (CKD) impacts about 1 in 7 adults in the United States, but African Americans (AAs) carry a disproportionately higher burden of disease. Epigenetic modifications, such as DNA methylation at cytosine-phosphate-guanine (CpG) sites, have been linked to kidney function and may have clinical utility in predicting the risk of CKD. Given the dynamic relationship between the epigenome, environment, and disease, AAs may be especially sensitive to environment-driven methylation alterations. Moreover, risk models incorporating CpG methylation have been shown to predict disease across multiple racial groups. In this study, we developed a methylation risk score (MRS) for CKD in cohorts of AAs. We selected nine CpG sites that were previously reported to be associated with estimated glomerular filtration rate (eGFR) in epigenome-wide association studies to construct a MRS in the Hypertension Genetic Epidemiology Network (HyperGEN). In logistic mixed models, the MRS was significantly associated with prevalent CKD and was robust to multiple sensitivity analyses, including CKD risk factors. There was modest replication in validation cohorts. In summary, we demonstrated that an eGFR-based CpG score is an independent predictor of prevalent CKD, suggesting that MRS should be further investigated for clinical utility in evaluating CKD risk and progression.

BACKGROUND: Type 1 diabetes (T1D) is preceded by a heterogenous pre-clinical phase, islet autoimmunity (IA). We aimed to identify pre vs. post-IA seroconversion (SV) changes in DNAm that differed across three IA progression phenotypes, those who lose autoantibodies (reverters), progress to clinical T1D (progressors), or maintain autoantibody levels (maintainers). METHODS: This epigenome-wide association study (EWAS) included longitudinal DNAm measurements in blood (Illumina 450K and EPIC) from participants in Diabetes Autoimmunity Study in the Young (DAISY) who developed IA, one or more islet autoantibodies on at least two consecutive visits. We compared reverters - individuals who sero-reverted, negative for all autoantibodies on at least two consecutive visits and did not develop T1D (n=41); maintainers - continued to test positive for autoantibodies but did not develop T1D (n=60); progressors - developed clinical T1D (n=42). DNAm data were measured before (pre-SV visit) and after IA (post-SV visit). Linear mixed models were used to test for differences in pre- vs post-SV changes in DNAm across the three groups. Linear mixed models were also used to test for group differences in average DNAm. Cell proportions, age, and sex were adjusted for in all models. Median follow-up across all participants was 15.5 yrs. (interquartile range (IQR): 10.8-18.7). RESULTS: The median age at the pre-SV visit was 2.2 yrs. (IQR: 0.8-5.3) in progressors, compared to 6.0 yrs. (IQR: 1.3-8.4) in reverters, and 5.7 yrs. (IQR: 1.4-9.7) in maintainers. Median time between the visits was similar in reverters 1.4 yrs. (IQR: 1-1.9), maintainers 1.3 yrs. (IQR: 1.0-2.0), and progressors 1.8 yrs. (IQR: 1.0-2.0). Changes in DNAm, pre- vs post-SV, differed across the groups at one site (cg16066195) and 11 regions. Average DNAm (mean of pre- and post-SV) differed across 22 regions. CONCLUSION: Differentially changing DNAm regions were located in genomic areas related to beta cell function, immune cell differentiation, and immune cell function.