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Background IgE-mediated sensitization may be epigenetically programmed in utero, but early childhood environment may further alter complex traits and disease phenotypes through epigenetic plasticity. However, the epigenomic footprint underpinning IgE-mediated type-I hypersensitivity has not been well-understood, especially under a longitudinal early-childhood life-course framework. Methods We used epigenome-wide DNA methylation (IlluminaHumanMethylation450 BeadChip) in cord blood and mid-childhood peripheral blood to investigate pre- and post-natal methylation marks associated with mid-childhood (age 6.7–10.2) total serum IgE levels in 217 mother-child pairs in Project Viva—a prospective longitudinal pre-birth cohort in eastern Massachusetts, USA. We identified methylation sites associated with IgE using covariate-adjusted robust linear regressions. Results Nineteen methylation marks in cord blood were associated with IgE in mid-childhood (FDR < 0.05) in genes implicated in cell signaling, growth, and development. Among these, two methylation sites ( C7orf50 , ZAR1 ) remained robust after the adjustment for the change in DNA methylation from birth to mid-childhood (FDR < 0.05). An analysis of the change in methylation between cord blood and mid-childhood DNA (Δ-DNAm) identified 395 methylation marks in 272 genes associated with mid-childhood IgE (FDR < 0.05), with multiple sites located within ACOT7 (4 sites), EPX (5 sites), EVL (3 sites), KSR1 (4 sites), ZFPM1 (3 sites), and ZNF862 (3 sites). Several of these methylation loci were previously associated with asthma ( ADAM19 , EPX , IL4 , IL5RA , and PRG2 ). Conclusion This study identified fetally programmed and mid-childhood methylation signals associated with mid-childhood IgE. Epigenetic priming during fetal development and early childhood likely plays an important role in IgE-mediated type-I hypersensitivity.

Background Although the etiology of primary biliary cirrhosis (PBC) remains enigmatic, there are several pieces of data supporting the thesis that a strong genetic predisposition and environmental factors interact to produce a selective loss of tolerance. The striking female predominance of PBC has suggested that this sex predisposition may be secondary to epigenetic alterations on the X chromosome. In the present study, we rigorously defined the X chromosome methylation profile of CD4, CD8, and CD14 cells from 30 PBC patients and 30 controls. Genomic DNA from sorted CD4, CD8, and CD14 subpopulations was isolated, sonicated, and immunoprecipitated for analysis of methylation. All products were hybridized to a custom-tiled four-plex array containing 27,728 CpG islands annotated by UCSC and 22,532 well-characterized RefSeq promoter regions. Furthermore, bisulfite sequencing was then used for validation on a subsequent group of independent samples from PBC patients and controls. Thence, expression levels of selected X-linked genes were evaluated by quantitative real-time PCR with cDNA samples from all subjects. Results We report herein that a total of 20, 15, and 19 distinct gene promoters reflected a significant difference in DNA methylation in CD4+ T, CD8+ T, and CD14+ cells in patients with PBC. Interestingly, there was hypermethylation of FUNDC2 in CD8+ T cells and a striking demethylation of CXCR3 in CD4+ T cells, which inversely correlated with CXCR3 expression levels in CD4+ T cells from early-stage PBC patients. Conclusions Our data provides a set of genes with epigenetic alteration likely to be indicators of autoimmunity and emphasizes the role of CXCR3 in the natural history of PBC.

Epigenetic pathways play a pivotal role in the development and function of the immune system. Over the last decade, a growing body of studies has been published out seeking to explain a correlation between epigenetic modifications and the development of autoimmune disorders. Epigenetic changes, such as DNA methylation, histone modifications, and noncoding RNAs, are involved in the pathogenesis of autoimmune diseases mainly by regulating gene expression. This paper reviews the importance of epigenetic alterations during the development of the most prevalent human autoimmune diseases, such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), systemic sclerosis (SSc), Sjogren’s syndrome (SS), autoimmune thyroid diseases (AITD), and type 1 diabetes (T1D), aiming to provide new insights in the pathogenesis of autoimmune diseases and the possibility to develop novel therapeutic approaches targeting the epigenome.

Background Although many genetic variants have been associated with multiple sclerosis (MS) risk, they do not explain all the disease risk and there remains uncertainty as to how these variants contribute to disease. DNA methylation is an epigenetic mechanism that can influence gene expression and has the potential to mediate the effects of environmental factors on MS. In a previous study, we found a differentially methylation region (DMR) at MHC HLA-DRB1 that was associated within relapsing-remitting MS (RRMS) patients in CD4 + T cells. This study aimed to confirm this earlier finding in an independent RRMS cohort of treatment-naïve female patients. Methods Total genomic DNA was extracted from CD4 + T cells of 28 female RRMS and 22 age-matched healthy controls subjects. DNA was bisulfite-converted and hybridised to Illumina 450K arrays. Beta values for all CpGs were analysed using the DMPFinder function in the MINFI program, and a follow-up prioritisation process was applied to identify the most robust MS-associated DMRs. Results This study confirmed our previous findings of a hypomethylated DMR at HLA-DRB1 and a hypermethylated DMR at HLA-DRB5 in this RRMS patient cohort. In addition, we identified a large independent DMR at MHC, whereby 11 CpGs in RNF39 were hypermethylated in MS cases compared to controls (max. ∆beta = 0.19, P  = 2.1 × 10 −4 ). We did not find evidence that SNP genotype was influencing the DMR in this cohort. A smaller MHC DMR was also identified at HCG4B , and two non-MHC DMRs at PM20D1 on chr1 and ERICH1 on chr8 were also identified. Conclusions The findings from this study confirm our previous results of a DMR at HLA-DRB1 and also suggest hypermethylation in an independent MHC locus, RNF39 , is associated with MS . Taken together, our results highlight the importance of epigenetic factors at the MHC locus in MS independent of treatment, age and sex. Prospective studies are now required to discern whether methylation at MHC is involved in influencing risk of disease onset or whether the disease itself has altered the methylation profile.

Background Asthma heritability has only been partially explained by genetic variants and is known to be sensitive to environmental factors, implicating epigenetic modifications such as DNA methylation in its pathogenesis. Methods Using data collected in the Avon Longitudinal Study of Parents and Children (ALSPAC), we assessed associations of asthma and wheeze with DNA methylation at 7.5 and 16.5 years, at over 450,000 CpG sites in DNA from the peripheral blood of approx. 1000 participants. We used Mendelian randomization (MR), a method of causal inference that uses genetic variants as instrumental variables, to infer the direction of association between DNA methylation and asthma. Results We identified 302 CpGs associated with current asthma status (FDR-adjusted P value < 0.05) and 445 with current wheeze status at 7.5 years, with substantial overlap between the two. Genes annotated to the 302 associated CpGs were enriched for pathways related to movement of cellular/subcellular components, locomotion, interleukin-4 production and eosinophil migration. All associations attenuated when adjusted for eosinophil and neutrophil cell count estimates. At 16.5 years, two sites were associated with current asthma after adjustment for cell counts. The CpGs mapped to the AP2A2 and IL5RA genes, with a − 2.32 [95% CI − 1.47, − 3.18] and − 2.49 [95% CI − 1.56, − 3.43] difference in percentage methylation in asthma cases respectively. Two-sample bi-directional MR indicated a causal effect of asthma on DNA methylation at several CpG sites at 7.5 years. However, associations did not persist after adjustment for multiple testing. There was no evidence of a causal effect of asthma on DNA methylation at either of the two CpG sites at 16.5 years. Conclusion The majority of observed associations are driven by higher eosinophil cell counts in asthma cases, acting as an intermediate phenotype, with important implications for future studies of DNA methylation in atopic diseases.

Background Genome-wide DNA methylation (DNAm) studies have proven extremely useful to understand human hematopoiesis. Due to their active DNA content, nucleated red blood cells (nRBCs) contribute to epigenetic and transcriptomic studies derived from whole cord blood. Genomic studies of cord blood hematopoietic cells isolated by fluorescence-activated cell sorting (FACS) may be significantly altered by heterotopic interactions with nRBCs during conventional cell sorting. Results We report that cord blood T cells, and to a lesser extent monocytes and B cells, physically engage with nRBCs during FACS. These heterotopic interactions resulted in significant cross-contamination of genome-wide epigenetic and transcriptomic data. Formal exclusion of erythroid lineage-specific markers yielded DNAm profiles (measured by the Illumina 450K array) of cord blood CD4 and CD8 T lymphocytes, B lymphocytes, natural killer (NK) cells, granulocytes, monocytes, and nRBCs that were more consistent with expected hematopoietic lineage relationships. Additionally, we identified eight highly differentially methylated CpG sites in nRBCs (false detection rate <5 %, |Δ β | >0.50) that can be used to detect nRBC contamination of purified hematopoietic cells or to assess the impact of nRBCs on whole cord blood DNAm profiles. Several of these erythroid markers are located in or near genes involved in erythropoiesis ( ZFPM1 , HDAC4 ) or immune function ( MAP3K14 , IFIT1B ), reinforcing a possible immune regulatory role for nRBCs in early life. Conclusions Heterotopic interactions between erythroid cells and white blood cells can result in contaminated cell populations if not properly excluded during cell sorting. Cord blood nRBCs have a distinct DNAm profile that can significantly skew epigenetic studies. Our findings have major implications for the design and interpretation of genome-wide epigenetic and transcriptomic studies using human cord blood.

Background Systemic sclerosis (SSc) is a rare autoimmune fibrosing disease with an incompletely understood genetic and non-genetic etiology. Defining its etiology is important to allow the development of effective predictive, preventative, and therapeutic strategies. We conducted this epigenomic study to investigate the contributions of DNA methylation to the etiology of SSc while minimizing confounding due to genetic heterogeneity. Methods Genomic methylation in whole blood from 27 twin pairs discordant for SSc was assayed over 450 K CpG sites. In silico integration with reported differentially methylated cytosines, differentially expressed genes, and regulatory annotation was conducted to validate and interpret the results. Results A total of 153 unique cytosines in limited cutaneous SSc (lcSSc) and 266 distinct sites in diffuse cutaneous SSc (dcSSc) showed suggestive differential methylation levels in affected twins. Integration with available data revealed 76 CpGs that were also differentially methylated in blood cells from lupus patients, suggesting their role as potential epigenetic blood biomarkers of autoimmunity. It also revealed 27 genes with concomitant differential expression in blood from SSc patients, including IFI44L and RSAD2 . Regulatory annotation revealed that dcSSc-associated CpGs (but not lcSSc) are enriched at Encyclopedia of DNA Elements-, Roadmap-, and BLUEPRINT-derived regulatory regions, supporting their potential role in disease presentation. Notably, the predominant enrichment of regulatory regions in monocytes and macrophages is consistent with the role of these cells in fibrosis, suggesting that the observed cellular dysregulation might be, at least partly, due to altered epigenetic mechanisms of these cells in dcSSc. Conclusions These data implicate epigenetic changes in the pathogenesis of SSc and suggest functional mechanisms in SSc etiology.

Background We have previously demonstrated a Toll-like receptor (TLR)-mediated hyper-responsive phenotype in our cohort of localized aggressive periodontitis (LAP) individuals. However, mechanisms related to this phenotype are still not clear in the literature. The objective of this cross-sectional study is to examine the role of epigenetic regulation, specifically DNA methylation status of genes in the TLR pathway in this cohort. Peripheral blood was collected from 20 LAP patients and 20 healthy unrelated controls. Whole blood was stimulated with 1 μl (100 ng/μl) of purified Escherichia coli lipopolysaccharide (LPS) for 24 h and cyto/chemokines in the supernatants analyzed by Luminex multiplex assays. Genomic DNA extracted from buffy coats prepared from a second tube of whole blood was used for DNA methylation analysis by pyrosequencing of seven TLR signaling genes (FADD, MAP3K7, MYD88, IL6R, PPARA, IRAK1BP1, RIPK2). Results Significant differences in the methylation status were observed at specific CpG positions in LAP patients compared to healthy controls and interestingly also between severe and moderate LAP. Specifically, subjects with moderate LAP presented hypermethylation of both the upregulating (MAP3K7, MYD88, IL6R, and RIPK2) and downregulating (FADD, IRAK, and PPARA) genes, while severe LAP presented hypomethylation of these genes. Further analysis on CpG sites with significant differences in methylation status correlates with an increased pro-inflammatory cytokine profile for LAP patients. Conclusions Our findings suggest that epigenetic modifications of genes in the TLR pathway may orchestrate the thresholds for balancing induction and prevention of tissue destruction during the course of disease, and thus differ significantly at different stages of the disease, where moderate LAP shows hypermethylation and severe LAP shows hypomethylation of several genes. Trial registration https://clinicaltrials.gov , NCT01330719

Background The genetic risk associated with rheumatoid arthritis (RA) includes genes regulating DNA methylation, one of the hallmarks of epigenetic re-programing, as well as many T-cell genes, with a strong MHC association, pointing to immunogenetic mechanisms as disease triggers leading to chronicity. The aim of our study was to explore DNA methylation in early, drug-naïve RA patients, towards a better understanding of early events in pathogenesis. Result Monocytes, naïve and memory CD4 + T-cells were sorted from 6 healthy controls and 10 RA patients. DNA methylation was assessed using a genome-wide Illumina 450K CpG promoter array. Differential methylation was confirmed using bisulfite sequencing for a specific gene promoter, ELISA for several cytokines and flow cytometry for cell surface markers. Differentially methylated (DM) CpGs were observed in 1047 genes in naïve CD4 + T-cells, 913 in memory cells and was minimal in monocytes with only 177 genes. Naive CD4 + T-cells were further investigated as presenting differential methylation in the promoter of > 500 genes associated with several disease-relevant pathways, including many cytokines and their receptors. We confirmed hypomethylation of a region of the TNF-alpha gene in early RA and differential expression of 3 cytokines (IL21, IL34 and RANKL). Using a bioinformatics package (DMRcate) and an in-house analysis based on differences in β values, we established lists of DM genes between health and RA. Publicly available gene expression data were interrogated to confirm differential expression of over 70 DM genes. The lists of DM genes were further investigated based on a functional relationship database analysis, which pointed to an IL6/JAK1/STAT3 node, related to TNF-signalling and engagement in Th17 cell differentiation amongst many pathways. Five DM genes for cell surface markers (CD4, IL6R, IL2RA/CD25, CD62L, CXCR4) were investigated towards identifying subpopulations of CD4 + T-cells undergoing these modifications and pointed to a subset of naïve T-cells, with high levels of CD4, IL2R, and CXCR4, but reduction and loss of IL6R and CD62L, respectively. Conclusion Our data provided novel conceptual advances in the understanding of early RA pathogenesis, with implications for early treatment and prevention.

Background The autoimmune disease systemic lupus erythematosus (SLE) has a modified epigenome with modified tri-methylation of histone H3 lysine 4 (H3K4me3) at specific loci across the genome. H3K4me3 is a canonical chromatin mark of active transcription. Recent studies have suggested that H3K4me3 breadth has an important regulatory role in cell identity. This project examined H3K4me3 breadth at transcription start sites (TSS) in primary monocytes and its association with differential gene transcription in SLE. Results Integrative analysis was applied to chromatin immunoprecipitation sequencing (ChIP-seq) and RNA sequencing (RNA-seq) data generated from primary monocytes as well as genomic data available in public repositories. Four distinctive H3K4me3 patterns of ChIP-seq peaks were identified at 8399 TSSs. Narrow peaks were highly enriched with genes related to housekeeping functions. The broader peaks with extended H3K4me3 immediately upstream and/or downstream of TSS were associated with immune response genes. Many TSSs had downstream H3K4me3 extended to ~650 bp, where the transition of H3K4me3 to H3K36me3, a transcriptional elongation mark, is often found. The H3K4me3 pattern was strongly associated with transcription in SLE. Genes with narrow peaks were less likely (OR = 0.14, p  = 2 × 10 −4 ) while genes with extended downstream H3K4me3 were more likely (OR = 2.37, p  = 1 × 10 −11 ) to be overexpressed in SLE. Of the genes significantly overexpressed in SLE, 78.8 % had increased downstream H3K4me3 while only 47.1 % had increased upstream H3K4me3. Gene transcription sensitively and consistently responded to H3K4me3 change downstream of TSSs. Every 1 % increase of H3K4me3 in this region leads to ~1.5 % average increase of transcription. Conclusions We identified the immediate TSS downstream nucleosome as a crucial regulator responsible for transcription changes in SLE. This study applied a unique method to study the effect of H3K4me3 breadth on diseases and revealed new insights about epigenetic modifications in SLE, which could lead to novel treatments.