Jihoon E. Joo

Neonatal DNA methylation profile in human twins is specified by a complex interplay between intrauterine environmental and genetic factors, subject to tissue-specific influence

Comparison between groups of monozygotic (MZ) and dizygotic (DZ) twins enables an estimation of the relative contribution of genetic and shared and nonshared environmental factors to phenotypic variability. Using DNA methylation profiling of ∼20,000 CpG sites as a phenotype, we have examined discordance levels in three neonatal tissues from 22 MZ and 12 DZ twin pairs. MZ twins exhibit a wide range of within-pair differences at birth, but show discordance levels generally lower than DZ pairs. Within-pair methylation discordance was lowest in CpG islands in all twins and increased as a function of distance from islands. Variance component decomposition analysis of DNA methylation in MZ and DZ pairs revealed a low mean heritability across all tissues, although a wide range of heritabilities was detected for specific genomic CpG sites. The largest component of variation was attributed to the combined effects of nonshared intrauterine environment and stochastic factors. Regression analysis of methylation on birth weight revealed a general association between methylation of genes involved in metabolism and biosynthesis, providing further support for epigenetic change in the previously described link between low birth weight and increasing risk for cardiovascular, metabolic, and other complex diseases. Finally, comparison of our data with that of several older twins revealed little evidence for genome-wide epigenetic drift with increasing age. This is the first study to analyze DNA methylation on a genome scale in twins at birth, further highlighting the importance of the intrauterine environment on shaping the neonatal epigenome.

Epigenome-wide association study reveals longitudinally stable DNA methylation differences in CD4+ T cells from children with IgE-mediated food allergy

Food allergy is mediated by a combination of genetic and environmental risk factors, potentially mediated by epigenetic mechanisms. CD4+ T-cells are key drivers of the allergic response, and may therefore harbor epigenetic variation in association with the disease phenotype. Here we retrospectively examined genome-wide DNA methylation profiles (~450 000 CpGs) from CD4+ T-cells on a birth cohort of 12 children with IgE-mediated food allergy diagnosed at 12-months, and 12 non-allergic controls. DNA samples were available at two time points, birth and 12-months. Case:control comparisons of CD4+ methylation profiles identified 179 differentially methylated probes (DMP) at 12-months and 136 DMP at birth (FDR-adjusted P value < 0.05, delta β > 0.1). Approximately 30% of DMPs were coincident with previously annotated SNPs. A total of 96 allergy-associated non-SNP DMPs were present at birth when individuals were initially disease-free, potentially implicating these loci in the causal pathway. Pathway analysis of differentially methylated genes identified several MAP kinase signaling molecules. Mass spectrometry was used to validate 15 CpG sites at 3 candidate genes. Combined analysis of differential methylation with gene expression profiles revealed gene expression differences at some but not all allergy associated differentially methylated genes. Thus, dysregulation of DNA methylation at MAPK signaling-associated genes during early CD4+ T-cell development may contribute to suboptimal T-lymphocyte responses in early childhood associated with the development of food allergy.

Expression discordance of monozygotic twins at birth: Effect of intrauterine environment and a possible mechanism for fetal programming

Within-pair comparison of monozygotic (MZ) twins provides an ideal model for studying factors that regulate epigenetic profile, by controlling for genetic variation. Previous reports have demonstrated epigenetic variability within MZ pairs, but the contribution of early life exposures to this variation remains unclear. As epigenetic marks govern gene expression, we have used gene expression discordance as a proxy measure of epigenetic discordance in MZ twins at birth in two cell types. We found strong evidence of expression discordance at birth in both cell types and some evidence for higher discordance in twin pairs with separate placentas. Genes previously defined as being involved in response to the external environment showed the most variable expression within pairs, independent of cell type, supporting the idea that even slight differences in intrauterine environment can influence expression profile. Focusing on birthweight, previously identified as a predisposing factor for cardiovascular, metabolic and other complex diseases, and using a statistical model that estimated association based on within-pair variation of expression and birthweight, we found some association between birthweight and expression of genes involved in metabolism and cardiovascular function. This study is the first to examine expression discordance in newborn twins. It provides evidence of a link between birthweight and activity of specific cellular pathways and, as evidence points to gene expression profiles being maintained through cell division by epigenetic factors, provides a plausible biological mechanism for the previously described link between low birthweight and increased risk of later complex disease.

Cohort Profile: The Peri/post-natal Epigenetic Twins Study

Cancer and Disease Epigenetics Group, Murdoch Childrens Research Institute (MCRI), Royal Children’s Hospital, Parkville, Victoria, Australia, Early Life Epigenetics Group, MCRI, Royal Children’s Hospital, Parkville, Victoria, Australia, Clinical Epidemiology and Biostatistics Unit, MCRI, Royal Children’s Hospital, Parkville, Victoria, Australia, Department of Paediatrics, University of Melbourne, Victoria, Australia, The Ritchie Centre, Monash Institute of Medical Research, Victoria, Australia, Department of Obstetrics and Gynaecology, University of Melbourne, Royal Women’s Hospital, Melbourne, Victoria, Australia, Department of Obstetrics and Gynaecology,University of Melbourne, Mercy Hospital for Women, Heidelberg, Victoria, Australia Present address: Department of Public Health, Twin Study, Hjelt Institute, University of Helsinki, Finland

DNA methylation changes measured in pre-diagnostic peripheral blood samples are associated with smoking and lung cancer risk.

DNA methylation changes are associated with cigarette smoking. We used the Illumina Infinium HumanMethylation450 array to determine whether methylation in DNA from pre‐diagnostic, peripheral blood samples is associated with lung cancer risk. We used a case‐control study nested within the EPIC‐Italy cohort and a study within the MCCS cohort as discovery sets (a total of 552 case‐control pairs). We validated the top signals in 429 case‐control pairs from another 3 studies. We identified six CpGs for which hypomethylation was associated with lung cancer risk: cg05575921 in the AHRR gene (p‐valuepooled = 4 × 10−17), cg03636183 in the F2RL3 gene (p‐valuepooled = 2 × 10 − 13), cg21566642 and cg05951221 in 2q37.1 (p‐valuepooled = 7 × 10−16 and 1 × 10−11 respectively), cg06126421 in 6p21.33 (p‐valuepooled = 2 × 10−15) and cg23387569 in 12q14.1 (p‐valuepooled = 5 × 10−7). For cg05951221 and cg23387569 the strength of association was virtually identical in never and current smokers. For all these CpGs except for cg23387569, the methylation levels were different across smoking categories in controls (p‐valuesheterogeneity ≤ 1.8 x10 − 7), were lowest for current smokers and increased with time since quitting for former smokers. We observed a gain in discrimination between cases and controls measured by the area under the ROC curve of at least 8% (p‐values ≥ 0.003) in former smokers by adding methylation at the 6 CpGs into risk prediction models including smoking status and number of pack‐years. Our findings provide convincing evidence that smoking and possibly other factors lead to DNA methylation changes measurable in peripheral blood that may improve prediction of lung cancer risk.

Genome-scale case-control analysis of CD4+ T-cell DNA methylation in juvenile idiopathic arthritis reveals potential targets involved in disease

BackgroundJuvenile Idiopathic Arthritis (JIA) is a complex autoimmune rheumatic disease of largely unknown cause. Evidence is growing that epigenetic variation, particularly DNA methylation, is associated with autoimmune disease. However, nothing is currently known about the potential role of aberrant DNA methylation in JIA. As a first step to addressing this knowledge gap, we have profiled DNA methylation in purified CD4+ T cells from JIA subjects and controls. Genomic DNA was isolated from peripheral blood CD4+ T cells from 14 oligoarticular and polyarticular JIA cases with active disease, and healthy age- and sex-matched controls. Genome-scale methylation analysis was carried out using the Illumina Infinium HumanMethylation27 BeadChip. Methylation data at >25,000 CpGs was compared in a case-control study design.ResultsMethylation levels were significantly different (FDR adjusted p<0.1) at 145 loci. Removal of four samples exposed to methotrexate had a striking impact on the outcome of the analysis, reducing the number of differentially methylated loci to 11. The methotrexate-naive analysis identified reduced methylation at the gene encoding the pro-inflammatory cytokine IL32, which was subsequently replicated using a second analysis platform and a second set of case-control pairs.ConclusionsOur data suggests that differential T cell DNA methylation may be a feature of JIA, and that reduced methylation at IL32 is associated with this disease. Further work in larger prospective and longitudinal sample collections is required to confirm these findings, assess whether the identified differences are causal or consequential of disease, and further investigate the epigenetic modifying properties of therapeutic regimens.

Variable promoter methylation contributes to differential expression of key genes in human placenta-derived venous and arterial endothelial cells

BackgroundThe endothelial compartment, comprising arterial, venous and lymphatic cell types, is established prenatally in association with rapid phenotypic and functional changes. The molecular mechanisms underpinning this process in utero have yet to be fully elucidated. The aim of this study was to investigate the potential for DNA methylation to act as a driver of the specific gene expression profiles of arterial and venous endothelial cells.ResultsPlacenta-derived venous and arterial endothelial cells were collected at birth prior to culturing. DNA methylation was measured at >450,000 CpG sites in parallel with expression measurements taken from 25,000 annotated genes. A consistent set of genomic loci was found to show coordinate differential methylation between the arterial and venous cell types. This included many loci previously not investigated in relation to endothelial function. An inverse relationship was observed between gene expression and promoter methylation levels for a limited subset of genes implicated in endothelial function, including NOS3, encoding endothelial Nitric Oxide Synthase.ConclusionEndothelial cells derived from the placental vasculature at birth contain widespread methylation of key regulatory genes. These are candidates involved in the specification of different endothelial cell types and represent potential target genes for environmentally mediated epigenetic disruption in utero in association with cardiovascular disease risk later in life.

Reliability of DNA methylation measures from dried blood spots and mononuclear cells using the HumanMethylation450k BeadArray

The reliability of methylation measures from the widely used HumanMethylation450 (HM450K) microarray has not been assessed for DNA from dried blood spots (DBS) or peripheral blood mononuclear cells (PBMC), nor for combined data from different studies. Repeated HM450K methylation measures in DNA from DBS and PBMC samples were available from participants in six case-control studies nested within the Melbourne Collaborative Cohort Study. Reliability was assessed for individual CpGs by calculating the intraclass correlation coefficient (ICC) based on technical replicates (samples repeated in a single study; 126 PBMC, 136 DBS) and study duplicates (samples repeated across studies; 280 PBMC, 769 DBS) using mixed-effects models. Reliability based on technical replicates was moderate for PBMC (median ICC = 0.42), but lower for DBS (median ICC = 0.20). Study duplicates gave lower ICCs than technical replicates. CpGs that were either highly methylated or unmethylated generally had lower ICCs, which appeared to be mostly related to their lower variability. The ICCs for global methylation measures were high, typically greater than 0.70. The reliability of methylation measures determined by the HM450K microarray is wide-ranging and depends primarily on the variability in methylation at individual CpG sites. The power of association studies is low for a substantial proportion of CpGs in the HM450K assay.

Global measures of peripheral blood-derived DNA methylation as a risk factor in the development of mature B-cell neoplasms

AIM To examine whether peripheral blood methylation is associated with risk of developing mature B-cell neoplasms (MBCNs). MATERIALS & METHODS We conducted a case-control study nested within a large prospective cohort. Peripheral blood was collected from healthy participants. Cases of MBCN were identified by linkage to cancer registries. Methylation was measured using the Infinium(®) HumanMethylation450. RESULTS During a median of 10.6-year follow-up, 438 MBCN cases were evaluated. Global hypomethylation was associated with increased risk of MBCN (odds ratio: 2.27, [95% CI: 1.59-3.25]). Within high CpG promoter regions, hypermethylation was associated with increased risk (odds ratio: 1.76 [95% CI: 1.25-2.48]). Promoter hypermethylation was observed in HOXA9 and CDH1 genes. CONCLUSION Aberrant global DNA methylation is detectable in peripheral blood collected years before diagnosis and is associated with increased risk of MBCN, suggesting changes to DNA methylation are an early event in MBCN development.

Human active X-specific DNA methylation events showing stability across time and tissues

The phenomenon of X chromosome inactivation in female mammals is well characterised and remains the archetypal example of dosage compensation via monoallelic expression. The temporal series of events that culminates in inactive X-specific gene silencing by DNA methylation has revealed a ‘patchwork’ of gene inactivation along the chromosome, with approximately 15% of genes escaping. Such genes are therefore potentially subject to sex-specific imbalance between males and females. Aside from XIST, the non-coding RNA on the X chromosome destined to be inactivated, very little is known about the extent of loci that may be selectively silenced on the active X chromosome (Xa). Using longitudinal array-based DNA methylation profiling of two human tissues, we have identified specific and widespread active X-specific DNA methylation showing stability over time and across tissues of disparate origin. Our panel of X-chromosome loci subject to methylation on Xa reflects a potentially novel mechanism for controlling female-specific X inactivation and sex-specific dimorphisms in humans. Further work is needed to investigate these phenomena.