Epigenomics

Abstract. Introduction: Preterm birth drives adverse perinatal maternal and infant health outcomes through heterogeneous symptoms, severity, and etiologies. Delivery prior to reaching 37 weeks of gestation may result from medically indicated intervention for pregnancy complications or spontaneously in the absence of prior symptoms. Placental tissue collected following preterm birth exhibits differential DNA methylation compared to full-term placentas and may indicate pregnancy health during gestation. Placental DNA currently has limited utility for assessing health of ongoing pregnancy, as sampling placental tissue during gestation increases the risk of infection and miscarriage. Risks associated with placental sampling during pregnancy limit the use of DNA methylation in clinical preterm birth prediction. Assessing preterm birth risk during gestation requires non-invasive methods for characterizing placental DNA methylation. Results: We quantified genome-wide DNA methylation patterns of hypermethylated cell-free DNA in pregnant (n = 99) and non-pregnant (n = 93) plasma using cell-free methylated DNA immunoprecipitation sequencing (cfMeDIP-seq). In each sample, we assessed DNA methylation status in 300-bp genomic windows, examining both sequencing read counts and calculated absolute molar DNA amount. Known hypermethylated placental regions, including RASSF1, STAT5A, and ERG promoters showed significantly increased odds of detection in pregnant samples, suggesting enrichment of cell-free placental DNA. Of the 536,444 300-bp windows examined, 173,071 (32%) showed significant enrichment in pregnant plasma. Linear modeling identified 107,505 differentially methylated regions (DMRs) associated with pregnancies later diagnosed with intrauterine growth restriction (IUGR) (n = 22). Alu elements showed increased representation in these DMRs than expected, while other repetitive elements exhibited underrepresentation. Discussion: These results demonstrate cfMeDIP-seq's ability to enrich for cell-free placental DNA and characterize cell-free DNA methylation signatures of pregnancies complicated by IUGR. Enrichment of cell-free placental DNA enables non-invasive profiling of placental DNA methylation from maternal plasma. Detectable epigenetic signatures in maternal plasma may identify pregnancies at elevated risk for preterm birth before clinical symptoms appear. Our findings further highlight the potential of cell-free placental DNA for monitoring pregnancy health.

Background. DNA methylation patterns in cord blood are robustly associated with birthweight in the general population. However, it remains unknown whether these associations extend to clinically relevant populations, such as preterm neonates or those born small for gestational age, and whether they directly reflect birthweight or are driven indirectly by genetic, familial, maternal, and obstetric factors. Methods. We calculated a birthweight methylation profile score (MPSBW) using weights of 835 CpGs previously associated with birthweight in the general population and evaluated its association with birthweight in 67 monochorionic (MC) twin pairs including 134 neonates (97% born preterm) from the Twinlife study. MC twin pairs are identical twins sharing a single placenta, often unequally, which can result in unequal resource distribution and differential fetal growth. Results. We examined the association between within-pair differences in birthweight and MPSBW, thereby estimating the association independent of factors shared equally by co-twins. A 500-gram increase in birthweight was associated with a 0.256 SD increase in MPSBW (p<0.005) in this population of preterm neonates. Adjustment for polygenic score for birthweight (PGSBW) confirmed that the observed epigenetic associations were not driven by common genetic variation underlying birthweight. Interestingly, a similar effect size (0.226 SD per 500 g birthweight increase; p<0.05) was observed in the within-pair analysis, which controls for all shared influences within a twin pair. Conclusion DNA methylation is associated with individual differences in birthweight in a high-risk clinical population of MC twins, independent of shared genetic, familial or maternal influences.

Background: Preeclampsia (PE) is a hypertensive pregnancy disorder with contributions from both maternal and fetal genetic factors, but how both genomes regulate placental transcription remains incompletely understood. Results: We analyzed placental 5'-end RNA sequencing data from 59 pregnancies in the FINNPEC cohort and performed separate maternal and fetal cis-eQTL analyses using gene-level expression and promoter-level expression defined as Transcript Far 5' Ends (TFE). Genotype-by-PE interaction models were used to identify genetic effects that differ according to PE status. No gene-level interaction eQTLs remained significant after multiple-testing correction, although suggestive signals included LILRB2 in the maternal and LPCAT3 in the fetal analyses. In contrast, we identified two significant syntenic promoter-level interaction eQTLs: rs12504642-TFE61820 at the FHDC1 locus in the maternal analysis and rs1557150-TFE9032 at the PPIAP31 locus in the fetal analysis. Conclusion: These findings suggest that PE-dependent genetic interaction effects may be detectable at specific transcript promoter regions even when they are not captured at annotated gene levels.

Endometriosis is a chronic inflammatory disease associated with pelvic pain, infertility, and delayed diagnosis. Growing evidence suggests that altered DNA methylation contributes to disease development and could serve as a biomarker for disease. We developed a leakage-safe machine learning pipeline to classify endometriosis case-control status and menstrual cycle phase using genome-wide DNA methylation data from eutopic endometrial tissue. The dataset consisted of 984 samples profiled using the Illumina Infinium MethylationEPIC array, with measurements across approximately 759,000 CpG sites. Technical variation was corrected using SmartSVA batch correction. Ridge logistic regression models were trained using stratified 80/20 train-test splits, with regularization strength selected via stratified cross-validation. Feature selection approaches included ridge coefficient ranking, per-CpG t-tests, and univariate logistic regression with FDR correction. Model validity was evaluated using label-shuffling analyses. Menstrual cycle phase classification showed strong performance (mean cross-validation AUROC: 0.971, held-out test AUROC: 0.989), reflecting genome-wide hormonally driven methylation. Ridge regression produced lower but meaningful performance for endometriosis classification (mean cross-validation AUROC: 0.854, held-out test AUROC: 0.875). Ridge coefficient-based feature selection identified compact predictive CpG sets, supporting the hypothesis that endometriosis-associated methylation signal is distributed across many loci rather than a few highly predictive CpGs. Pathway enrichment analyses identified substantial enrichment for menstrual cycle phase but limited enrichment for disease status following FDR correction, consistent with a diffuse endometriosis-associated signal. These findings demonstrate that ridge regression can detect methylation patterns associated with both endometriosis and menstrual cycle phase, highlighting the importance of accounting for cycle-related epigenetic variation in endometrial DNA methylation studies.

DNA methylation is a key epigenetic mechanism regulating the expression of genes involved in numerous developmental and pathological processes. However, the contribution of DNA methylation of microRNA genes to atherosclerosis remains poorly understood. In this study, we profiled DNA methylation patterns of both the regulatory elements and gene bodies of five microRNA genes (MIR10B, MIR21, MIR100, MIR127, and MIR143) in vascular tissues and paired peripheral blood cells (PBC) of 92 patients with advanced carotid atherosclerosis and 32 PBC of control participants by targeted bisulfite sequencing. We identified distinct tissue-specific DNA methylation patterns for all five microRNA genes in patients with advanced carotid atherosclerosis. The regulatory regions of MIR10B, MIR127, and MIR100 were moderately hypomethylated in carotid atherosclerotic plaques compared with intact vascular tissues. We further integrated our findings with lab-internal and publicly available epigenome-wide methylation datasets and evaluated the influence of vascular and blood cell composition using computational deconvolution approaches. After adjustment for cellular heterogeneity in vascular tissues, DNA methylation at CpG sites in MIR100 and MIR127 remained independently associated with atherosclerosis. Increased DNA methylation at a single CpG site (chr11:122025143, GRCh37/hg19) located within the MIR100 E-box region was associated with metabolic syndrome. Moreover, DNA methylation levels of MIR10B, MIR21, and MIR127 in atherosclerotic plaques were linked with indicators of histological instability and history of acute cerebrovascular events. In peripheral blood, we observed moderate hypomethylation of the regulatory regions of MIR10B, MIR21, and the MIR100 E-box region in patients compared with the control group. However, only the MIR10B remained robust against blood cell composition. In blood, MIR10B and MIR143 methylation correlated with lipid metabolism and carotid stenosis, while the MIR21 CpG island showed strong blood-plaque concordance, confirming its potential as a surrogate biomarker. Overall, advanced carotid atherosclerosis is characterized by specific tissue-altering DNA methylation patterns of microRNA genes, where alterations mainly occur in the regulatory regions, predominantly featuring hypomethylation. The results underscore the complex, cell- and tissue-specific nature of DNA methylation of microRNA genes in both the regulatory elements and gene bodies in vascular tissue and blood, highlighting the critical need to decipher these intricate epigenetic landscapes to identify reliable, robust biomarkers for assessing plaque instability and cardiovascular risk.

Preeclampsia (PE) is a hypertensive pregnancy-specific disorder and a leading cause of maternal and fetal mortality. A common feature of PE placentas and maternal plasma is dyslipidemia, or abnormal lipid levels, which can increase oxidative stress and endothelial dysfunction. However, the precise transcriptional, post-transcriptional, and epigenetic mechanisms underlying these abnormalities remain poorly characterized. Identifying such changes may clarify disease mechanisms and identify lipid-related PE biomarkers. We conducted a large-scale meta-analysis integrating public placental datasets from NCBI GEO, comprising four DNA methylation (DNAm) datasets (n = 172), three RNA-sequencing datasets (n = 92), and an independent RNA microarray validation cohort (n =146). We evaluated differential DNAm (limma), gene expression (DESeq2), transcript-level shifts (Swish), and alternative splicing (rMATS) in PE versus control placentas, with all analyses stratified by fetal sex via an interaction term model. We also performed placental cell-type deconvolution to quantify PE-associated cell-type proportion changes. Our results demonstrated that lipid-related regulation changes in PE placentas occur primarily at the gene and transcript level, with DNAm showing no changes. We also identified significant isoform switching in PE that were undetected by differential gene expression analysis, and primarily driven by alternative transcription initiation and termination sites rather than alternative splicing. A subset of these isoform switches mapped to pathways dysregulated in PE and were predicted to cause functional protein changes. An interaction term model identified several sex-specific differentially expressed genes (DEGs) in PE, including a subset of male-specific downregulated genes involved in oxidative metabolism. However, many of the remaining sex-specific DEGs across both sexes were previously uncharacterized in the literature. These findings suggest that transcriptional and isoform-level regulation play a role in PE-associated dyslipidemia, with certain regulatory pathways displaying fetal sex-specific patterns. Highlights- Preeclampsia-associated dyslipidemia manifests at the gene and transcript level - Reciprocal isoform switches were missed by standard gene-level analyses - Alternative transcript initiation and termination drove isoform switching - Sex-interaction modeling identified sex-specific transcriptional shifts in PE

Background: Psychosocial stress is a key risk factor for coronary heart disease (CHD), particularly in postmenopausal women who face both a high stress burden and elevated cardiovascular risk. DNA methylation (DNAm), a critical epigenetic modification bridging environment and health, remains understudied as a contributor to stress-related CHD. Methods: We conducted an epigenome-wide association study (EWAS) of stress in the Women's Health Initiative (WHI), an ancestrally diverse cohort of postmenopausal women (n=3,857). At screening visit, participants completed a questionnaire assessing stressful life events and provided whole blood for DNAm. Incident CHD was then longitudinally ascertained (follow-up mean/SD: 16.7/8.4 years), and DNAm signatures were evaluated as CHD predictors using Cox regression. Predictive models were independently validated in the Jackson Heart Study (JHS; n=3,053) and Multi-Ethnic Study of Atherosclerosis (MESA; n=870). The bulk-level DNAm associations were computationally deconvolved at the cell-type-specific level using tensor composition analysis (TCA). Results: The EWAS in WHI identified 841 stress-related DNAm sites (99 hypermethylated, 742 hypomethylated with stress) after FDR correction, with 13 significant after Bonferroni correction, including sites located on immune and CHD-related genes (e.g., TNF, ALDH2). Methylation risk scores (MRSs) integrating the 841 FDR-significant sites (MRS841) and 13 Bonferroni-significant sites (MRS13) predicted incident CHD (HR=1.33-1.37; p[&le;]0.0008) and mediated 16.5-17.7% of the association between stress and CHD. In JHS and MESA, MRS13 independently predicted CHD (HR=1.34; p=0.036), whereas MRS841 was suggestively associated with CHD (HR=1.27; p=0.087). TCA indicated that the greatest number of stress-related sites predictive of CHD was specifically in monocytes (133 total), with directions consistent with bulk-level associations (9 hypermethylated, 124 hypomethylated with stress). Conclusion: Our study supports methylation risk scores as novel biomarkers of stress-related CHD and uncovers epigenetic regulation in monocytes as a potential underlying mechanism. These findings highlight biological pathways linking stress and disease and may promote personalized interventions in high-risk populations.

IntroductionFetal development represents a critical window during which genetic and environmental influences shape lifelong health. DNA methylation (DNAm) is a candidate underlying mechanism. While individual prenatal exposures have been related to DNAm, no studies have investigated the broader prenatal exposome, nor incorporated genetics with the exposome. Here, we integrated the prenatal exposome and genetics as predictors of DNAm at birth. MethodsWe used data from the Dutch Generation R (n=2282) and English Avon Longitudinal Study of Parents and Children (ALSPAC; n=809) cohorts. We performed epigenome-wide elastic net regression, using Generation R for model development/internal validation and ALSPAC for external validation, to predict DNAm at each CpG site. We used three models: Model 1 included 42 prenatal exposures, Model 2 additionally included child sex, gestational age and birth weight, and Model 3 further included meQTLs. ResultsIn Model 1, the prenatal exposome explained on average 0.7% of DNAm variation across 347 validated CpGs (0.1% of tested CpGs). This increased to 40,044 CpGs (10.2%) with 1.3% of variation explained in Model 2, and 91,305 CpGs (23.2%) with 3.0% of variation explained in Model 3. In Model 1, prenatal smoking was the largest predictor, followed by delivery characteristics, among which meconium-stained amniotic fluid was a novel finding. In Model 3, typically both SNPs and multiple prenatal exposures were selected. DiscussionWe find that genomic associations with cord blood DNAm are stronger and more widespread than prenatal exposures, although typically, the prenatal exposome explains additional variation in DNAm beyond genetic influences.

Prostate cancer remains one of the most common cancers among men globally. While significant strides have been made in diagnosis and treatment, understanding the complex genetic and epigenetic underpinnings of the disease remains crucial for guiding intervention and developing more personalized and effective therapies. The importance of DNA methylation in prostate cancer has been known for some time, but important facets of the modulation of the epigenome during carcinogenesis remain obscure, partly because the bulk of cancer methylation data have been produced using microarray technologies. Here we utilise the TruSeq methyl capture method (EPICseq) to profile the, previously defined, UK Prostate ICGC cohort of well-annotated primary prostate cancers. To this we add methylation sequencing of benign tissue from the same men. These data allow us to identify differentially methylated regions distinguishing cancerous and non-cancerous prostate tissue, while identifying numerous genes whose methylation profiles can perform that task as well as distinguishing between classes of prostate cancer. We describe a describe a methylation-based control mechanism for prostate-cancer-associated SNPs, and show that this seems a likely mechanism of action for a SNP near the MMP7 gene. We describe three novel molecular signatures that arise from different aspects of the biology of prostate cancer revealed by sequencing. Each is shown to be an independent classifier of cancers into groups with different expected times to relapse. These consist of patterns in driver gene methylation, strand-specific methylation, and signal arising in mitochondrial reads. We show that these signatures, combined with existing molecular tools, provide a powerful predictor of time to recurrence. By substantially enhancing understanding of prostate cancer risk, detection, and prognosis, we pave the way for the development of clinical practices that will benefit patients and improve outcomes.

Progesterone is essential for the establishment and maintenance of pregnancy, primarily through the classical nuclear progesterone receptor (PGR). However, progesterone also exerts rapid, non-genomic effects through membrane progesterone receptors, including members of the progestin and adipoQ receptor (PAQR) family, whose cellular distribution and potential roles at the human maternal-fetal interface remain poorly understood. We therefore sought to define the cell-type-specific expression landscape of classical and membrane progesterone receptors during early human pregnancy. We analyzed publicly available single-cell RNA-sequencing datasets of the first-trimester human maternal-fetal interface to characterize the expression of PGR and PAQR family members across trophoblast, immune, stromal, and vascular cell populations. Major expression patterns were examined across independent datasets and further assessed using spatial transcriptomic data. PGR expression was predominantly localized to decidual stromal and perivascular populations, whereas individual PAQR family members showed distinct cellular distributions. PAQR6 was enriched in decidual natural killer cells, PAQR7 was broadly expressed in trophoblast populations, PAQR8 was prominently expressed in fetal stromal and macrophage populations, including Hofbauer cells, and PAQR9 showed prominent enrichment in syncytiotrophoblasts. Selected cell-type-specific patterns were further supported by spatial transcriptomic analysis. These findings provide a cell-type-resolved framework for investigating how distinct progesterone receptor systems mediate progesterone signaling across maternal and fetal cellular compartments during early human placentation.

DNA methylation may link early-life exposures to later obesity risk; however, most studies examine individual CpG sites using commercial arrays that cover <5% of the genome and often overlook biologically-relevant regulatory regions. Imprinting control regions (ICRs) regulate parent-of-origin gene expression and are enriched for growth and metabolic genes, representing strong candidates for developmental programming of obesity. In 586 participants of the Newborn Epigenetics STudy (NEST), we leveraged the novel Human Imprintome Array to measure methylation at >1,000 characterized and putative ICRs genome-wide in umbilical cord blood. We applied principal component and kernel machine regression to identify ICR methylation at birth associated with sustained childhood obesity, defined as BMI [&ge;]95th percentile during most of childhood. Sustained childhood obesity was associated with differential methylation at seven ICRs in cord blood, with 2-10% mean differences between obesity and normal weight groups, including novel regions localized to chromosome 20q11.1-20q11.21 (ICR_1182, ICR_1179, ICR_1181, ICR_1177, ICR_1180, and ICR_1165), mapping to or near CDC27P4, LINC01597, DUX4L37, and DUX4L34, and ICR_927 mapping to previously characterized ZNF597/NAA60. Most ICRs overlapped multiple transcriptional regulator binding sites. Associations persisted in peripheral blood collected in later childhood at ages 8-16 years. Among offspring of mothers with pre-pregnancy obesity, an additional 50 associations were identified. Sex-stratified analyses revealed no further insights. These findings identify novel ICRs-particularly on chromosome 20q-as potential early-life epigenetic biomarkers of childhood obesity. If replicated, these stable ICR methylation patterns may identify children at risk for obesity at birth, informing targeted prevention before obesity develops and guiding novel therapeutic strategies.

Males experience elevated mortality and morbidity during the perinatal period and infancy; however, the biological origins of this vulnerability remain unclear. Emerging evidence suggests that sex-dependent placental development may contribute to differences in fetal growth and outcomes. Vascular networks are central to placental function, established early in the first trimester to support growth and mitigate harmful exposures, and maturing by term to sustain late fetal development. Although these processes are tightly regulated by gene-expression programmes, the role of sex-specific vascular signalling in placental development remains poorly understood. To address this, we analysed raw, publicly available RNA-sequencing transcriptomics data from first trimester and term placental samples. We conducted parallel differential gene expression and functional enrichment analyses across the datasets to ensure direct comparability and to disentangle the specific contribution of sex chromosomes. We identified multiple differentially expressed genes and enriched pathways between male and female placentas. In the first trimester, male placentas exhibited significant enrichment of vascular signalling pathways, particularly Notch signalling, cell adhesion, and migration, consistent with heightened proliferative and remodelling activity. At the end of gestation, sex differences remained detectable; however differentially expressed genes was clearly sex-chromosome driven, with autosomal single-gene sex differences and autosomal-sex-specific pathways markedly reduced. This reflected a mature placental vascular system with minimal sex-based differences outside of the sex chromosome genes. These results reveal gestational stage-dependent sex differences in placental vascular development, with early male fetuses exhibiting increased angiogenic activity and immature vascular signalling, potentially contributing to accelerated growth and increased vulnerability in male neonates.

Purpose Long non-coding RNAs (lncRNAs) are important regulators of tumor biology, but their dependence on major oncogenic drivers and cancer specificity remain poorly characterized. We aimed to identify lncRNAs regulated by MYC, mutant KRAS, and mutant TP53 across colorectal, lung, and pancreatic cancer and to determine their functional relevance and molecular mechanism. Methods We performed an integrative analysis of lncRNA expression profiles driven by MYC, mutant KRAS, and mutant TP53 across colorectal, lung, and pancreatic cancer cell lines. Candidate lncRNAs were functionally characterized using assays of cancer cell viability, migration, and clonogenicity. Expression in patient-derived tumor and normal tissues and downstream molecular pathways were investigated. Results MYC was associated with the most extensive lncRNA regulation, with expression patterns largely independent of tissue origin. MYC-dependent LINC00997 and the previously uncharacterized AC104447.1, as well as specifically KRAS-dependent RAKRAR (RASSF3 antisense KRAS regulated lncRNA) promoted cancer cell viability, migration, and clonogenicity. LINC00997 and AC104447.1 expression was elevated in specific tumor types compared to normal tissues. We identified CAMOR (CARNMT1 Antisense Multiple-Oncoprotein Regulated lncRNA), a pan-cancer lncRNA upregulated by all three oncogenic drivers. CAMOR promoted viability and migration specifically in cancer cells across analyzed cell lines and tumor types. Mechanistically, CAMOR and neighboring CARNMT1 form a negative feedback regulatory loop affecting CAMOR oncogenic activity. Distinct downstream gene expression signatures suggested that the studied lncRNAs act through different regulatory processes. Conclusion Our findings expand the repertoire of oncogenic lncRNAs and demonstrate that integrating multiple oncogenic contexts enables identification of both driver-specific and broadly acting lncRNAs, providing a framework for identifying lncRNAs that may contribute to cancer development and represent potential diagnostic or therapeutic targets.

Most established DNA methylation (DNAm) biomarkers were developed on legacy Illumina EPIC arrays. The Infinium Methylation Screening Array (MSA) offers a lower-cost, higher-throughput alternative with reduced probe content, but EPIC-trained algorithms cannot be assumed to transfer directly. Here we present a reproducibility-based framework for developing and transferring DNAm algorithms on the MSA. Using paired biological replicates profiled on EPICv1 and MSA (1,764 EPICv1-MSA sample pairs, plus within-array MSA replicates on the same and different beadchips), we quantified probe-level agreement using mean absolute error (MAE) and intraclass correlation coefficients (ICC). Of 140,150 CpG sites shared between EPICv1 and MSA, 40,786 (29.1%) met both stability criteria (MAE < 0.05 and ICC(2,k) > 0.6). This stable feature space supported two modelling streams. First, we trained 134 epigenetic biomarker proxies (EBPs) natively on MSA, with and without kernel principal component analysis (kPCA) for sample-level harmonisation. All 134 reached same-beadchip ICC(2,1) >= 0.80 (median 0.97) and 96.3% reached different-beadchip ICC(2,1) >= 0.60 (median 0.81), with a median Spearman correlation of 0.48 against observed values. Among the 72 kPCA-selected models with a comparable stable-probe baseline, 70 (97%) showed higher cross-beadchip ICC (median improvement +0.18). Second, we transferred three established clocks using model-specific strategies: OMICmAge and SystemsAge were retrained to estimate their EPICv1-derived values (held-out test-set rho = 0.944 and 0.912-0.949), whereas DunedinPACE required stable-probe normalisation and robust linear calibration, which raised cross-array ICC(2,1) from 0.784-0.810 to 0.891-0.925 and reduced MAE from 0.085-0.089 to 0.041-0.050 across three sample sets. Reduced probe content does not preclude reproducible DNAm biomarker measurement, and transfer strategy must be matched to model architecture.

Colorectal cancer (CRC) disproportionately affects Hispanic individuals, who remain underrepresented in genomic discovery and lack ancestry-matched functional resources. Many CRC risk variants are non-coding, and those in 3' untranslated regions (3' UTRs) can alter microRNA (miRNA) binding sites and reprogram post-transcriptional gene regulation. Here, we identify allele-specific miRNA-binding variants at CRC risk loci within the Hispanic population using a discovery pipeline anchored to Hispanic-relevant resources. CRC-associated 3' UTR variants were compiled from the GWAS Catalog, further verified in Hispanic/Latino individuals using the All of Us Research Program cohort (n = 453), expanded into proxy sets using Admixed American-specific linkage disequilibrium, annotated against Ensembl transcripts, and evaluated with TargetScan and RNAhybrid. Screening of 38 CRC-associated 3' UTR SNPs identified 19 variants that alter miRNA binding across nine genes, with each gene represented by a variant confirmed in the Hispanic cohort. Three oncogenic loci, DCBLD2 (lead SNP rs11552978), GREM1 (lead SNP rs10318), and SMC1B (rs3747239, prioritized as an AMR-specific proxy in near-complete LD with confirmed lead rs6007600), met all prioritization criteria. In each case, the risk allele disrupts a strong miRNA-binding site, while the gene shows increased tumor expression, weak correlation between expression and methylation, and an absence of local active transcriptional chromatin marks (cCREs/H3K27Ac). These results suggest that the variants promote gene overexpression through loss of miRNA-mediated repression rather than changes in promoter methylation. Overall, the study identifies non-coding CRC susceptibility variants in the Hispanic population and highlights DCBLD2, GREM1, and SMC1B as strong candidates for functional validation.

Background Intrauterine inflammation is associated with increased lifetime cardiovascular disease (CVD) risk in offspring, yet long-term molecular mechanisms remain underexplored. We investigated the lasting cardiometabolic, hemodynamic, and vascular effects of prenatal inflammatory exposure in adult offspring using a mouse model. Methods On day 15 of gestation, pregnant CD-1 mice were randomized to receive intraperitoneal (IP) injections of either lipopolysaccharide (LPS group) or saline (SAL group). Blood samples were collected before and after injection to measure IL-6 concentrations. Blood pressure (BP) in offspring was measured at 6-8 months of age using the tail-cuff method, and tissues were collected. Protein was extracted from the brain cortex to measure cytokines utilizing the xMAP Luminex assay. The C-reactive protein (CRP) was measured in serum. Gene expression of IL-6, NOS3, NFkB, AT-1, and Trp53 was determined by real time RT-qPCR in the aorta. The experimental unit was a mother. Results were analyzed using the appropriate statistical methods. Results Maternal LPS administration induced significant acute systemic inflammation (P<0.05 for IL-6). At 6-8 months after delivery, maternal circulating CRP was significantly higher in LPS mice as compared to SAL (P=0.02). In offspring at 6-8 months of age, systolic BP was significantly elevated in LPS groups compared with SAL animals (P=0.04). LPS-exposed offspring exhibited higher total body and visceral adipose tissue weights. Aortic expression of the angiotensin II type 1 receptor (AT1) and NFkB were significantly upregulated (P=0.04 and P=0.03, respectively). Cortices of LPS offspring displayed a pro-inflammatory profile with increased total Inflammatory score, elevated pro-inflammatory IL-1{beta}, and reduced anti-inflammatory IL-4 and IL-10. Furthermore, protective Trp53 gene expression was significantly downregulated in both the aorta (P=0.01) and heart (P=0.04). Conclusion A single mid-gestation inflammatory insult programs long-term cardio-inflammatory perturbations in offspring and results in long-term systemic inflammation in mothers. Prenatal LPS exposure induces persistent visceral obesity, hypertension, vascular RAS upregulation, central neuroinflammation, and cardiac/vascular Trp53 suppression in adult offspring, offering a mechanistic link between prenatal inflammation and offspring CVD risk.

Retinoblastoma (RB), the most common pediatric intraocular malignancy, is initiated by biallelic inactivation of the RB1 tumor suppressor gene. Although LINE-1 (L1) retrotransposon activation has been linked to genomic instability and tumor evolution in several adult cancers, its contribution to pediatric malignancies remains poorly understood. Here, we investigated L1 expression in retinoblastoma samples from an Indian pediatric cohort by integrating transcriptomic, locus-specific, and immunohistochemistry. Transcriptomic profiling revealed increased L1 expression in RB tumors compared with control retinal tissues, which was validated by ORF1p immunostaining in tumor sections. Locus-specific analysis identified a subset of transcriptionally active L1 loci, suggesting selective activation of young L1 elements. We also observed altered expression of genes associated with proliferation, chromatin regulation, and oncogenic signaling, including MYCN, MDM2, E2F3, SOX4 and CREBBP. Our findings identify previously underexplored L1 activation as a molecular feature associated with retinoblastoma and suggest that disruption of transposon silencing accompanies the RB transcriptional landscape. Validation in larger patient cohorts will be important; however, this study provides a foundation for exploring L1 activity as a potential molecular marker and future therapeutic vulnerability in retinoblastoma. HighlightsO_LITranscriptomic profiling reveals increased LINE-1 expression in retinoblastoma. C_LIO_LIORF1p protein is robustly expressed in retinoblastoma. C_LIO_LIA subset of LINE-1 loci exhibits transcriptional activation, suggesting locus-specific derepression. C_LI In BriefRetinoblastoma is initiated by RB1 loss, but the contribution of transposable elements to its molecular landscape remains poorly understood. Here, we show that retinoblastoma is associated with LINE-1 promoter hypomethylation, increased LINE-1 transcript expression, ORF1p accumulation, and preferential activation of selected LINE-1 loci. These changes occur together with altered expression of oncogenic signalling pathways and host factors involved in transposable element repression, identifying dysregulated LINE-1 control as a molecular feature of this paediatric cancer. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=115 SRC="FIGDIR/small/740688v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@10dcf8forg.highwire.dtl.DTLVardef@1b5d07forg.highwire.dtl.DTLVardef@18b5116org.highwire.dtl.DTLVardef@1788145_HPS_FORMAT_FIGEXP M_FIG C_FIG Retinoblastoma exhibits epigenetic activation of LINE-1 characterized by promoter hypomethylation, increased expression of young LINE-1 elements, and ORF1p accumulation. This is accompanied by coordinated transcriptomic remodeling and disruption of host mechanisms that normally restrain transposable elements, linking LINE-1 activation to the molecular landscape of retinoblastoma.

Prenatal alcohol exposure (PAE) can produce persistent alterations in mammary development and increase susceptibility to mammary tumorigenesis, but the early molecular events underlying these effects remain poorly understood. Here, we examined whether PAE alters the molecular program of the mammary gland at birth. Pregnant MMTV-ErbB2 mice received control or ethanol-containing liquid diets during gestation, and mammary tissues were collected from newborn female offspring. RNA sequencing revealed exposure-level-dependent transcriptional reprogramming, with substantially broader alterations following moderate compared with lower ethanol exposure. Pathway analyses identified prominent changes in metabolic and growth-regulatory networks, including insulin signaling, with coordinated alterations in components of the insulin/IGF-IRS-PI3K/AKT pathway. Biochemical analyses further demonstrated exposure-dependent changes in IGFBP expression and IRS1 abundance and phosphorylation, supporting remodeling of insulin/IGF-associated signaling. Transcriptomic analyses also revealed enrichment of RNA metabolic and translational processes and, particularly following moderate exposure, enhanced DNA-replication, cell-cycle, and mitotic programs. These molecular signatures were accompanied by increased proliferative features in newborn mammary tissue. Together, these findings demonstrate that PAE establishes substantial molecular reprogramming of the mammary gland by birth and identify altered insulin/metabolic signaling and proliferative programming as prominent features of this early developmental response.

Background: Infants <30 weeks gestational age (very preterm (VPT)), are at risk of experiencing multiple serious neonatal morbidities. These morbidities impart serious biological stress, which may be reflected in dysregulated protein expression even after the morbidities have resolved. Recent studies suggest that DNA methylation-based predictors of circulating proteins may reflect the molecular effects of neonatal adversity. The objective of this study is to examine associations between DNA methylation-derived EpiScores, which are externally trained methylation signatures that proxy circulating protein levels, and neonatal morbidity burden in children born VPT. Buccal DNA methylation was obtained at NICU discharge in 537 infants from the NOVI cohort. A total of 111 EpiScores were calculated and tested for association with multimorbidity (0-1 vs. [&ge;] 2 morbidities) using adjusted linear regression models. Cross-tissue analyses in an external paired blood-buccal dataset (n=21) were used to identify EpiScores with moderate-to-strong correlations across tissues. Results: Eighteen EpiScores were nominally associated with multimorbidity (p < 0.05), with Melanoma-derived growth regulatory protein (MIA) with FDR < 0.05 (beta = 0.0023, p = 0.0004). In cross-tissue analyses, 35 EpiScores yielded r > 0.50 (p<0.05), 11 of which showed nominal associations with multimorbidity, including MIA. Overall, the morbidity-associated EpiScores reflected immune, inflammatory, metabolic, and growth-related pathways. Conclusions: Buccal DNA methylation-derived EpiScores capture biological signatures associated with neonatal morbidity burden in very preterm infants.

Mammalian preimplantation development requires precisely coordinated lineage decisions to establish the trophectoderm (TE), inner cell mass (ICM), epiblast (EPI), and primitive endoderm (PrE). Glucose metabolism and epigenetic regulation are increasingly recognised as key determinants of lineage specification during preimplantation development. However, how glucose-dependent metabolic cues interface with epigenetic mechanisms to regulate embryonic cell fate remains poorly understood. Here, we investigated the role of glucose in regulating protein methylation by protein arginine methyltransferases (PRMT) in bovine preimplantation development. PRMT1 and its associated histone mark H4R3me2a were detected throughout bovine oocyte maturation and embryo development. Pharmacological inhibition of Type I PRMTs using two structurally distinct inhibitors, GSK3368715 and MS023, markedly reduced global protein asymmetric dimethylarginine (ADMA) and H4R3me2a levels. PRMT inhibition impaired blastocyst cell proliferation, reduced total cell number, and disrupted both first and second lineage decisions, as demonstrated by decreased CDX2- and SOX2-positive TE and ICM cells and reduced NANOG- and GATA6-positive EPI and PrE cell allocation. Mechanistically, Type I PRMT inhibition downregulated key components of the Hippo-associated TE programme, including YAP, TEAD4, and TFAP2C. Consistent effects were observed in mouse embryos, where MS023 treatment reduced ADMA, CDX2, YAP, and TFAP2C expression and impaired TE and ICM allocation. Collectively, our findings identify Type I PRMT-mediated ADMA as an essential epigenetic regulator of early mammalian lineage specification and support a conserved ADMA-Hippo regulatory axis linking arginine methylation to embryonic cell fate decisions. In briefType I protein arginine methyltransferase (PRMT)-mediated asymmetric dimethylarginine (ADMA) is required for proper lineage specification during mammalian preimplantation development. ADMA depletion disrupts Hippo signalling, cell proliferation, and trophectoderm and inner cell mass allocation in bovine and mouse embryos.