Epigenetics

BackgroundPregnancy in women with multiple sclerosis (MS) is associated with a reduction of long-term disability progression. The mechanism that drives this effect is unknown, but converging evidence suggests a role for epigenetic mechanisms altering immune and/or central nervous system function. ObjectivesWe aimed to identify whole blood and immune cell-specific DNA methylation patterns associated with parity in relapse-onset multiple sclerosis. MethodsWe compared whole-blood methylation patterns between 96 matched pairs of nulligravida and parous females with MS (n=192). Parity was defined as at least one term or pre-term birth, and nulligravida was defined as no prior pregnancies. Methylation was measured with Illumina EPIC arrays, and data was pre-processed and statistically analysed using the ChAMP package. Cell-type proportions were estimated using the EpiDISH package, and cell-specific analysis conducted using linear regression. Gene-set enrichment analysis (GSEA) was performed with ToppGene API and GOmeth. Methylation age was calculated with the methyAge package. Methylation age acceleration (MAA) was calculated by regressing methylation age on chronological age. FDR<0.05 was used to assess significance. ResultsThe median time from last pregnancy to blood collection was 16.66 years (range = 1.45 - 44.42 years). We identified 903 differentially methylated positions (DMPs) in whole blood; 365 were hypomethylated and 528 were hypermethylated in parous women. We further identified two differentially methylated regions (DMRs) in CRYGN on Chromosome 7 and an intergenic region on Chromosome 15. There were four and eight cell type specific DMPs in CD4+ and CD8+ cells, respectively. Differentially methylated genes were enriched in neuronal plasticity pathways. Parity was associated with reduced MAA by a mean of 1.44 to 2.27 years using the PhenoAge (p = 0.002) and GrimAge (p = 0.005) algorithms. ConclusionWhole-blood methylation patterns are associated with birth history in females with relapse-onset multiple sclerosis. We found enrichment of differentially methylated genes encoding neuronal processes and reduced MAA in parous women. These methylation changes could mediate the long-term benefit of pregnancy for disease progression in multiple sclerosis.

The underlying mechanism of the HIV-1 reservoir, a major barrier to an HIV cure, is largely unknown. The integration of HIV-1 DNA and immune defense mechanisms can disrupt the host epigenetic landscape, potentially silencing HIV-1 replication. Using bisulfite capture DNA methylation sequencing, we profiled approximately 3.2 million CpG sites in CD4+ T cells isolated from the blood of 427 virally suppressed women with HIV. The average total CD4+ T cell HIV-1 Reservoir (HRCD4) size was 1,409 copies per million cells. Most proviruses were defective with only a small proportion being intact. We found 245 differentially methylated positions (CpG sites) and 85 methylated regions associated with the total HRCD4 size. Notably, 52% of significant methylation sites were in intronic regions. HRCD4-associated genes were involved in viral replication (e.g., ISG15), HIV-1 latency (e.g., MBD2), and cell growth and apoptosis (e.g., IRF9). A subset of the identified genes with aberrant methylation was an established target of HIV-1 integration (e.g., NFIA, SPPL3, DLEU2, ELMSAN1). Overall, HRCD4 size was inversely associated with DNA methylation of interferon signaling genes and positively associated with methylation at established HIV-1 integration sites. HRCD4-associated genes were enriched in pathways including immune defense against the virus (i.e., interferon- response and interferon-{gamma} response), DNA binding transcription repression, and host-virus interaction such as Tau protein binding. Together, our results show that epigenomic alterations in CD4+ T cells are associated with total HIV-1 reservoir size, offering new insights into HIV-1 latency and may provide potential molecular targets for future HIV-1 eradication strategies.

Long term associations of early life experiences with later life DNA methylation are understudied. In the U.S. Health and Retirement Study, participants self-reported early life experiences, including years in school, smoking during childhood, growing up in a rural area, and living with a grandparent. Later life DNA methylation was measured in blood for participants with a mean age of 69.6 years at 731,474 sites. We tested for associations between each early life experience with DNA methylation age acceleration, global and site-specific methylation, and enriched biological pathways. We compared results across early life experiences. Participants (N = 3,562) were 58.8% female and 68.1% non-Hispanic White. They reported 13 mean years in school, 18.3% smoked during childhood, 42.7% grew up in a rural area, and 26.8% lived with a grandparent. Fewer years in school (0.10, 95% CI: 0.06, 0.14) and smoking during childhood (0.65, 95% CI: 0.32, 0.97) were associated with accelerated GrimAge in later life, while living in a rural area and living with a grandparent were not associated. Early life experiences were associated (p<1x10-4) with distinct DNA methylation sites, specifically 574 sites for years in school, 20 for smoking during childhood, 49 for growing up in a rural area, and 23 for living with a grandparent. For example, one fewer year in school was associated with 0.47 (p-value = 4.61x10-15) lower percent methylation at cg07318158 in OTUD7B. Sites associated with our early life exposures were enriched for unique pathways. Years in school was enriched for embryonic development and cell structure pathways, smoking during childhood was enriched for nervous system development, exocytosis, and cell adhesion and structure pathways, growing up in a rural area was enriched for cell and vesicle processing pathways, and living with a grandparent was enriched for hormone regulation and protein breakdown pathways. Findings suggest our early life exposures are associated with unique DNA methylation patterns in later life, which can potentially allow for separate biomarker opportunities aimed at early intervention of adverse later life outcomes associated with these exposures.

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.

DNA methylation plays a critical role in the dynamics of gene expression regulation and the development of various disorders. Whole-genome bisulfite sequencing can provide single base resolution of CpG methylation levels and is the "gold standard" for DNA methylation quantification, but it also has a high cost. In contrast, targeted sequencing is optimal when focusing on specific candidate regions, while providing sufficient sequencing depth. Here, we present a targeted bisulfite sequencing approach to study the methylation status of regions of interest. We amplify selected regions from bisulfite-treated DNA and sequence them using Nanopore sequencing. In this work, we applied this workflow to candidate gene promoters for severe premature labor in a Latin American population. We successfully amplified fragments over 1 Kb in length using long PCR conditions for 12 genes that were barcoded per sample and pooled to be sequenced on MinION flow cells. This approach achieved high sequencing depths, ensuring reliable DNAm estimation. We found significant hypomethylation of the MIR155HG gene promoter in severe preterm birth samples, which is concordant with reported gene expression changes. We demonstrate that combining bisulfite DNA treatment with pooled long-read sequencing is a cost- and time-effective method to evaluate DNAm in several targeted regions and several samples in parallel. This study provides proof-of-concept for larger studies, demonstrating the applicability and high scalability of our assay to any locus of interest. Our experience suggests that this approach can be easily transferred to other diagnostic questions.

The benefits of breastfeeding for the health and wellbeing of both infants and mothers are well documented, yet global breastfeeding rates are low. One factor associated with low breastfeeding is maternal body mass index (BMI), which is used as a measure of obesity. The negative relationship between maternal obesity and breastfeeding is likely caused by a variety of social, psychological, and physiological factors. Maternal obesity may also have a direct biological association with breastfeeding through changes in maternal DNA methylation. Here, we investigate this potential biological association using data from a UK-based cohort study, the Avon Longitudinal Study of Parents and Children (ALSPAC). We find that pre-pregnancy body mass index (BMI) is associated with lower initiation to breastfeed and shorter breastfeeding duration. We conduct epigenome-wide association studies (EWAS) of maternal BMI and breastfeeding outcomes and candidate-gene analysis of methylation sites associated with BMI identified via previous meta-EWAS. We find that DNA methylation at cg11453712, annotated to PHTP1, is associated with maternal BMI. From our results, neither this association nor those at candidate-gene sites are likely to mediate the link between maternal BMI and breastfeeding.

BackgroundOsteoporosis is a prevalent bone metabolic disorder characterized by reduced bone mass, disruption of bone microarchitecture, and increased bone fragility, leading to a heightened risk of fracture. This condition significantly impairs patients quality of life and increases mortality risk. Emerging evidence suggests that DNA methylation may play a crucial role in regulating the expression of genes related to bone metabolism, thereby influencing the development of osteoporosis. However, the precise relationship between DNA methylation and osteoporosis remains unclear and warrants further investigation. ResultsOur study revealed significant differences in both the quantity and ratio of DNA methylation between individuals with osteoporosis and non-osteoporosis controls, with differences predominantly occurring in CpG islands. GO/KEGG enrichment analyses highlighted distinct osteoporosis-related gene pathways. Notably, we identified six genes, MSX1, HOXD4, AXIN2, WNT5A, TGFB1, and STAT3, respectively, that are potentially involved in the pathogenesis of osteoporosis and are broadly involved in various diseases and biological processes. ConclusionsThese findings indicate distinct methylation patterns between osteoporosis patients and healthy individuals, with differential methylation levels in genes associated with osteoporosis. This research offers new insights into the epigenetic mechanisms underlying osteoporosis.

Changes in the sperm epigenome induced by age and/or stressors often follow common unexplained patterns affecting genes responsible for embryonic development and neurodevelopment. The stochastic epigenetic variation (SEV) hypothesis proposes that in response to stressors naturally variable methylation regions (VMRs) associated with morphogenic genes increase in methylation variation to diversify phenotypes and improve chances of survival of the genetic lineage. Here, we test predictions from the SEV hypothesis using mouse and rat sperm DNA methylation and other -omics data. We demonstrate that the context of DNA regions determines the response of sperm methylome to various factors rather than the stressors and/or timing of these factors. We propose a model explaining age/stress-dependent shifts in methylation in VMRs by an asymmetric increase in methylation variation of these regions. Because methylation variation in VMRs increases with age, sperm methylome response to stressors may be characterized as an acceleration of epigenetic aging.

BackgroundHumans and mice display elevated levels of IL-27, an immunosuppressive cytokine shown to increase during neonatal bacterial sepsis and compromise survival. This study explores two hypotheses for regulation of IL-27 expression: 1) decreased DNA methylation in newborns that contributes to increased expression of IL-27 genes; 2) neonatal hormones regulate IL-27 expression through upstream hormone response elements (HREs). MethodsWhole genome methyl-seq analysis of neonatal and adult blood-derived macrophages identified differentially methylated regions (DMRs) at steady-state. Quantitative PCR (qPCR) measured expression of IL-27 genes (IL27p28 and EBI3) in human and murine neonatal macrophages stimulated in vitro with synthetic glucocorticoid or progesterone. Confocal microscopy and chromatin immunoprecipitation (ChIP) of glucocorticoid receptor (GR) assessed translocation into the nucleus and binding to the EBI3 promoter. ResultsThe IL-27p28 promoter contained DMRs that were increased in the neonatal cohort. The analysis did not identify DMRs within the EBI3 promoter. Dexamethasone stimulation increased EBI3 gene expression in human and murine neonatal macrophages. GR localized to the nucleus in response to dexamethasone and was enriched at the EBI3 upstream regulatory region. ConclusionThese data suggest glucocorticoid (GC) signaling increases EBI3 expression. This has importance in the context of antenatal GC administration that may increase IL-27 levels. Impact Statement{blacksquare} Elevated expression of IL-27 in early life impairs the host response to invasive bacterial infection in neonates. {blacksquare}Understanding the regulatory mechanisms contributing to increased IL-27 during the neonatal period is necessary to reduce susceptibility to infection in this vulnerable population. {blacksquare}The methylation status of the IL-27 genes in macrophages from neonatal and adult blood donors does not suggest regulation of differential expression with age. {blacksquare}Glucocorticoids are a signal that can induce EBI3 gene expression in a GR-dependent manner. {blacksquare}Glucocorticoid therapy for premature infants may increase IL-27 expression and promote enhanced susceptibility to infection.

Infinium Methylation BeadChip arrays remain one of the most popular platforms for epigenome-wide association studies, but tools for downstream pathway analysis have their limitations. Functional class scoring (FCS) is a group of pathway enrichment techniques that involve the ranking of genes and evaluation of their collective regulation in biological systems, but the implementations described for Infinium methylation array data do not retain direction information, which is important for mechanistic understanding of genomic regulation. Here, we evaluate several candidate FCS methods that retain directional information. According to simulation results, the best-performing method involves the mean aggregation of probe limma t-statistics by gene followed by a rank-ANOVA enrichment test using the mitch package. This method, which we call "LAM", outperformed an existing over-representation analysis method in simulations, and showed higher sensitivity and robustness in an analysis of real lung tumour-normal paired datasets. Using matched RNA-seq data we examine the relationship of methylation differences at promoters and gene bodies with RNA expression at the level of pathways in lung cancer. To demonstrate the utility of our approach, we apply it to three other contexts where public data were available. Firstly, we examine differential pathway methylation associated with chronological age. Secondly, we investigate pathway methylation differences in infants conceived with in vitro fertilisation. Lastly, we analyse differential pathway methylation in 19 disease states, identifying hundreds of novel associations. These results show LAM is a powerful method for the detection of differential pathway methylation as compared to existing methods. A reproducible vignette is provided to illustrate how to implement this method.

BackgroundResearching the murine epigenome in disease models has been hampered by the lack of an appropriate and cost-effective DNA methylation array. Until recently, investigators have been limited to the relatively expensive and analysis intensive bisulphite sequencing methods. Here, we performed a comprehensive, comparative analysis between the new Mouse Methylation BeadChip (MMB) and reduced representation bisulphite sequencing (RRBS) in two murine models of colorectal carcinogenesis, providing insight into the utility to each platforms in a real world environment. ResultsWe captured 1.47x106 CpGs by RRBS and 2.64x105 CpGs by MMB, mapping to 13,778 and 13,365 CpG islands, respectively. RRBS captured significantly more CpGs per island (median 41 for RRBS versus 2 for MMB). We found that 64.4% of intra-island CpG methylation variability can be captured by measuring approximately one quarter of CpG island (CGI) CpGs. MMB was more precise in measuring DNA methylation, especially at sites that had low RRBS coverage. This impacted differential methylation analysis, with more statistically significantly differentially methylated CpG sites identified by MMB in all experimental conditions, however the difference was minute when appropriate thresholding for the magnitude of methylation change (0.2 beta value difference) was applied, providing confidence that both techniques can identify similar differential DNA methylation. Gene ontology enrichment analysis of differentially hypermethylated gene promoters identified similar biological processes and pathways by both RRBS and MMB across two murine model systems. ConclusionMMB is an effective tool for profiling the murine methylome that performs comparably to RRBS, identifying similar differentially methylated pathways. Although MMB captures a similar proportion of CpG islands, it does so with fewer CpGs per island. We show that subsampling informative CpGs from CpG islands is an appropriate strategy to capture whole island variation. Choice of technology is experiment dependent and will be predicated on the underlying biology being probed.

Tobacco smoking induces DNA methylation (DNAm) changes in blood and other tissues, which may influence chronic health outcomes. However, the breadth of smoking-related DNAm changes remains unmapped, offering a space for employing novel technologies. To expand our understanding of smoking impacts on DNAm, we conducted an epigenome-wide association study (EWAS) comparing ever smokers to never smokers, using blood from a multiethnic U.S. study population (n=887). We employed the newly developed Illumina Methylation Screening Array (MSA) covering 269,094 unique sites, including 123,776 CpGs not assayed in previous EWAS. Trans-ethnic meta-analysis identified 152 differentially methylated positions (DMPs) associated with ever-smoking status (n=764); European-specific analysis yielded 129 DMPs (n=674), including 106 overlapping with trans-ethnic analysis. A separate, large-scale replication EWAS (n=2,190) confirmed 91 trans-ethnic and 77 European-specific DMPs. Among our findings, we identified 61 DMPs at CpGs novel to the MSA platform, including near both new and known smoking-associated genes. Most notably, we uncovered a dense cluster of 12 DMPs within a 1117 bp region of ECEL1P1, forming the most long-lasting, persistent smoking-associated DMR ever detected, even among former smokers who quit decades prior. We also detected new signals at AHRR, a well-known locus for smoking-related DNAm changes. eFORGE analysis revealed that detected smoking-associated DNAm changes are predominantly located in hematopoietic stem and progenitor cell (HSPC) DNase I hotspots, aligning with gene set enrichment analyses that highlighted pathways related to hematopoietic stem cell differentiation. Our findings suggest that HSPCs serve as a reservoir for an epigenetic memory of smoking. Additionally, we observed short-term cell-specific smoking-associated DNAm changes in myeloid cells. Our results demonstrate the utility of the MSA in expanding our knowledge of both transient and persistent environmental exposure-associated DNAm changes.

BackgroundAccumulating evidence suggests prenatal air pollution exposure alters DNA methylation (DNAm), which could go on to affect long-term health. However, it remains unclear whether prenatal DNAm alterations persist through early life. Identifying DNAm changes that persist from birth into childhood would provide greater insight into the molecular mechanisms that most likely contribute to the association of prenatal air pollution exposure with health outcomes such as atopic disease. ObjectivesThis study investigated the persistence of DNAm changes associated with prenatal NO2 exposure (a surrogate measure of traffic-related air pollution) at age one to begin characterizing which DNAm changes most likely to contribute to atopic disease. MethodsWe used an atopy-enriched subset of CHILD study participants (N=145) to identify individual and regional cord blood DNAm differences associated with prenatal NO2, followed by an investigation of persistence in age one peripheral blood. As we had repeated DNAm measures, we also isolated postnatal-specific DNAm changes and examined their association with NO2 exposure in the first year of life. MANOVA tests were used to examine the association between DNAm changes associated with NO2 and child wheeze and atopy. ResultsWe identified 24 regions of altered cord blood DNAm, with several annotated to HOX genes. Two regions annotated to MPDU1 and C5orf63 were significantly associated with age one wheeze. Further, we found the effect of prenatal NO2 exposure across CpGs within all altered regions remained similar at age one. A single region of postnatal-specific DNAm annotated to HOXB6 was associated with year one NO2 and age one atopy. DiscussionRegional cord blood DNAm changes associated with prenatal NO2 exposure persist through at least the first year of life, and some of these changes are associated with age one wheeze. The early-postnatal period remains a sensitive window to DNAm perturbations that may also influence child health.

BackgroundMigration from one environment to another often causes marked changes in developmental conditions. Here we compare epigenetic ageing and stability of the epigenetic maintenance system among British-Bangladeshi women who grew up in Bangladesh (adult migrants), where there are higher pathogen loads and poorer health care, to second-generation Bangladeshis who grew up in the UK. In our previous studies of these migrants, those who spent their childhoods in Bangladesh also had lower levels of reproductive hormones and a shorter reproductive lifespan compared to those who grew up in the UK, suggesting life history trade-offs during development. In the present study, we hypothesised that women who grew up in Bangladesh would have i) an older epigenetic/biological age compared to the women with a childhood in the UK and ii) that differences in the pace of epigenetic ageing might also be reflected by altered stability of DNA methylation marks. ResultsIllumina EPIC array methylation data from buccal tissue was used to establish epigenetic age estimates from 15 adult migrants and 11 second-generation migrants, aged 18-35 years. Using residuals from linear regression of DNA methylation-based biological age (DNAm age) on the chronological age, the results showed significant differences (p=0.016) in epigenetic age estimates: women whose childhood was in Bangladesh are on average 6.02 ({+/-} 2.34) years older, than those who grew up in London. We further investigated the efficiency of the epigenetic maintenance system which purportedly is reflected by epigenetic clocks. Methylation states of CpGs at the LHCGR/LHR locus, which contributes to Horvaths multi tissue epigenetic clock were evaluated. Based on the Ratio of Concordance Preference (RCP) approach that uses double-stranded methylation data, we find that maintenance of epigenetic information is more stable in women who grew up in Bangladesh. ConclusionsThe work supports earlier findings that adverse childhood environments lead to phenotypic life history trade-offs. The data indicate that childhood environments can induce subtle changes to the epigenetic maintenance system that are detectable long after exposure occurred. The implication of such a finding warrants further investigation as it implies that a less flexible epigenetic memory system established early in life could reduce the capacity to respond to different environmental conditions in adult life.

Non-syndromic cleft lip/palate (NSCLP), the most common human craniofacial malformations, is a complex disorder given its genetic heterogeneity and multifactorial component revealed by genetic, epidemiological and epigenetic findings. Association of epigenetic variations with NSCLP has been made, however still of little functional investigation. Here we combined a reanalysis of NSCLP methylome data with genetic analysis and used both in vitro and in vivo approaches to dissect the functional effects of epigenetic changes. We found a frequent differentially methylated region in mir152, hypomethylated in NSCLP cohorts (21-26%), leading to mir152 overexpression. In vivo analysis using zebrafish embryos revealed that mir152 upregulation leads to craniofacial impairment analogue to palatal defects. Also, we demonstrated that zebrafish embryonic hypoxia leads to mir152 upregulation combined with mir152 hypomethylation and also analogue palatal alterations. We therefore suggest mir152 hypomethylation, potentially induced by hypoxia in early development, as a novel and frequent predisposing factor to NSCLP.

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.

The prevalence of many diseases differs by sex, potentially due to sex-specific patterns in DNA methylation. Autosomal sex-specific differences in DNA methylation have been observed in cord blood and placental tissue, but are not well studied in saliva or in diverse populations. We sought to characterize sex-specific DNA methylation on autosomal chromosomes in saliva samples from children in the Fragile Families and Child Wellbeing Study, a multi-ethnic prospective birth cohort containing an oversampling of Black, Hispanic and low-income families. DNA methylation from saliva samples were analyzed on 796 children at both ages 9 and 15 with DNA methylation measured using the Illumina HumanMethylation 450k array. An epigenome-wide association analysis of the age 9 samples identified 8,430 sex-differentiated autosomal DNA methylation sites at age 9 (P < 2.4x10-7), of which 76.2% had higher DNA methylation in female children. The strongest sex-difference was in the cg26921482 probe, in the AMDHD2 gene, with 30.6% higher DNA methylation in female compared to male children (P < 1x10-300). Treating the age 15 samples as an internal replication set, we observed highly consistent results between the age 9 and age 15 measurements, indicating stable and replicable sex-differentiation. Further, we directly compared our results to previously published DNA methylation sex differences in both cord blood and saliva and again found strong consistency. Our findings support widespread and robust sex-differential DNA methylation across age, human tissues, and populations. These findings help inform our understanding of potential biological processes contributing to sex differences in human physiology and disease.

Antenatal corticosteroids (ACS) are provided to improve perinatal survival when there is risk of preterm birth. Though evidence suggests increased risk of developing neurobehavioural disorders in exposed offspring, the mechanisms that mediate this relationship remain largely unknown. Here, we investigated the DNA methylation patterns in the prefrontal cortex (PFC) of exposed offspring. We hypothesized that differential methylation will be evident at both newborn and juvenile ages. Pregnant guinea pigs were administered saline or betamethasone (1mg/kg) on gestational days 50/51 to mimic a single course of ACS. gDNA was isolated from the PFC of term-born offspring on postnatal day 1 (PND1) and PND14 to identify differentially methylated CpG sites (DMCs) using reduced representative bisulfite sequencing. In the PND1 PFC, 1521 DMCs, annotating to 145 genes were identified following ACS. Identified genes were involved in pathways regulating developmental cellular process. In the PND14 PFC, 776 DMCs representing 46 genes were identified, and were enriched in synaptic signalling pathways. Though no individual DMCs were identified at both PND1 and PND14, differential methylation was consistently observed at the binding sites of transcription factors PLAGL1, TFAP2C, ZNF263, and SP1 at both ages. In this study, we identified an altered DNA methylome in the PFC of ACS-exposed guinea pig offspring at both newborn and juvenile ages. Notably, a unique methylation signature was consistently observed at four key transcription factor binding sites at multiple post-natal time points, indicating a persistent change which may predispose the development of altered neurobehavioural phenotypes that have been described in exposed offspring.

BackgroundHuman DNA is known to exhibit an overall tendency toward demethylation with aging. However, assuming a simple linear relationship between DNA methylation and age does not align with the phenotype of human development and the aging process. This study aimed to investigate the existence of DNA methylation patterns with peaks or troughs at specific ages in addition to simple linear changes. MethodsA large-scale dataset of genome-wide DNA methylation data from 10,420 individuals was analyzed. Hierarchical multiple regression models were applied to detect patterns of the association between age and DNA methylation: linear increase, linear decrease, U-shaped curve, and inverse U-shaped curve. ResultsAmong the 864,627 CpG sites analyzed, 8.4% exhibited an increase in DNA methylation with age, 23.9% showed a decrease, and 5.5% were better explained by a quadratic model (P < 5.7815x10 ). Within the non-linear subset, inverse U-shaped CpG sites peaking in methylation during middle age were predominant. Genes exhibiting quadratic association patterns between DNA methylation and age, and those linked to diseases with common onset during middle age, were also detected. ConclusionsNon-linear age-related DNA methylation patterns, with peaks or troughs occurring at specific ages, were detected. This suggests that humans do not simply age linearly, but that programmed mechanisms or cascade-like processes may exist to promote or suppress the expression of specific genes at certain ages, contributing onset of certain diseases at specific timings.

The existence and functional relevance of mitochondrial DNA methylation remain controversial. Here, we systematically profiled cytosine methylation and hydroxymethylation across human brain and blood tissues spanning healthy and malignant states using orthogonal sequencing approaches that avoid chemical conversion during library preparation. While nuclear DNA exhibited canonical methylation patterns, mitochondrial DNA consistently showed negligible signal, indistinguishable from background technical noise. By mapping cytosine-guanine sites between mitochondrial DNA and nuclear-embedded mitochondrial sequences, we demonstrate the potential of these nuclear counterparts to confound not only cytosine methylation but also hydroxymethylation measurements, corroborating and extending prior findings implicating nuclear contamination as a potential source of apparent mitochondrial epigenetic signals. Additional technical factors that inflate apparent mtDNA methylation signals were identified, including sequence context biases, flow cell chemistries, and coverage-dependent discrepancies between the heavy and light strands. Collectively, these results provide convergent evidence against the presence of biologically meaningful cytosine methylation or hydroxymethylation in mitochondrial DNA. These findings caution against interpreting apparent mtDNA methylation signals in human adult tissues as meaningful without rigorous orthogonal validation and comprehensive consideration of technical and analytical confounding factors.