Epigenetic events are crucial for early development, but can be influenced by environmental factors, potentially programming the genome for later adverse health outcomes. The insulin-like growth factor 2 (IGF2)/H19 locus is crucial for prenatal growth and the epigenetic state at this locus is environmentally labile. Recent studies have implicated maternal factors, including folate intake and smoking, in the regulation of DNA methylation at this locus, although data are often conflicting in the direction and magnitude of effect. Most studies have focused on single tissues and on one or two differentially-methylated regions (DMRs) regulating IGF2/H19 expression. In this study, we investigated the relationship between multiple shared and non-shared gestational/maternal factors and DNA methylation at four IGF2/H19 DMRs in five newborn cell types from 67 pairs of monozygotic and 49 pairs of dizygotic twins. Data on maternal and non-shared supply line factors were collected during the second and third trimesters of pregnancy and DNA methylation was measured via mass spectrometry using Sequenom MassArray EpiTyper analysis. Our exploratory approach showed that the site of umbilical cord insertion into the placenta in monochorionic twins has the strongest positive association with methylation in all IGF2/H19 DMRs (p < 0.05). Further, evidence for tissue- and locus-specific effects were observed, emphasizing that responsiveness to environmental exposures in utero cannot be generalized across genes and tissues, potentially accounting for the lack of consistency in previous findings. Such complexity in responsiveness to environmental exposures in utero has implications for all epigenetic studies investigating the developmental origins of health and disease.
DNA methylation; imprinted genes; maternal factors; twins; developmental origins of health and disease (DOHaD)
Despite extensive research, a direct correlation between low to moderate prenatal alcohol exposure (PAE) and Fetal Alcohol Spectrum Disorders has been elusive. Conflicting results are attributed to a lack of accurate and detailed data on PAE and incomplete information on contributing factors. The public health effectiveness of policies recommending complete abstinence from alcohol during pregnancy is challenged by the high frequency of unplanned pregnancies, where many women consumed some alcohol prior to pregnancy recognition. There is a need for research evidence emphasizing timing and dosage of PAE and its effects on child development.
Asking QUestions about Alcohol (AQUA) is a longitudinal cohort aiming to clarify the complex effects of low to moderate PAE using specifically developed and tested questions incorporating dose, pattern and timing of exposure. From 2011, 2146 pregnant women completed a questionnaire at 8-18 weeks of pregnancy. Further prenatal data collection took place via a questionnaire at 26-28 weeks and 35 weeks gestation. Extensive information was obtained on a large number of risk factors to assist in understanding the heterogeneous nature of PAE effects. 1571 women (73%) completed all three pregnancy questionnaires. A biobank of DNA from maternal and infant buccal cells, placental biopsies and cord blood mononuclear cells will be used to examine epigenetic state at birth as well as genetic factors in the mother and child. Participants will be followed up at 12 and 24 months after birth to assess child health and measure infant behavioural and sensory difficulties, as well as family environment and parenting styles. A subgroup of the cohort will have 3D facial photography of their child at 12 months and a comprehensive developmental assessment (Bayley Scales of Infant & Toddler Development, Bayley-III) at two years of age.
Using detailed, prospective methods of data collection, the AQUA study will comprehensively examine the effects of low to moderate alcohol consumption throughout pregnancy on child health and development, including the role of key mediators and confounders. These data will ultimately contribute to policy review and development, health professional education and information about alcohol consumption for pregnant women in the future.
Prenatal alcohol exposure; Fetal alcohol spectrum disorders; Cohort studies; Epidemiology; Pregnancy; Child health; Genetics; Epigenetics
Biomarkers are naturally-occurring characteristics by which a particular pathological process or disease can be identified or monitored. They can reflect past environmental exposures, predict disease onset or course, or determine a patient’s response to therapy. Epigenetic changes are such characteristics, with most epigenetic biomarkers discovered to date based on the epigenetic mark of DNA methylation. Many tissue types are suitable for the discovery of DNA methylation biomarkers including cell-based samples such as blood and tumor material and cell-free DNA samples such as plasma. DNA methylation biomarkers with diagnostic, prognostic and predictive power are already in clinical trials or in a clinical setting for cancer. Outside cancer, strong evidence that complex disease originates in early life is opening up exciting new avenues for the detection of DNA methylation biomarkers for adverse early life environment and for estimation of future disease risk. However, there are a number of limitations to overcome before such biomarkers reach the clinic. Nevertheless, DNA methylation biomarkers have great potential to contribute to personalized medicine throughout life. We review the current state of play for DNA methylation biomarkers, discuss the barriers that must be crossed on the way to implementation in a clinical setting, and predict their future use for human disease.
cancer; diabetes; obesity; smoking; stress; autism; schizophrenia; bipolar disorder; depression; environmental factors
Preterm birth confers a high risk of adverse long term health outcomes for survivors, yet the underlying molecular mechanisms are unclear. We hypothesized that effects of preterm birth can be mediated through measurable epigenomic changes throughout development. We therefore used a longitudinal birth cohort to measure the epigenetic mark of DNA methylation at birth and 18 years comparing survivors of extremely preterm birth with infants born at term.
Using 12 extreme preterm birth cases and 12 matched, term controls, we extracted DNA from archived neonatal blood spots and blood collected in a similar way at 18 years of age. DNA methylation was measured at 347,789 autosomal locations throughout the genome using Infinium HM450 arrays. Representative methylation differences were confirmed by Sequenom MassArray EpiTYPER.
At birth we found 1,555 sites with significant differences in methylation between term and preterm babies. At 18 years of age, these differences had largely resolved, suggesting that DNA methylation differences at birth are mainly driven by factors relating to gestational age, such as cell composition and/or maturity. Using matched longitudinal samples, we found evidence for an epigenetic legacy associated with preterm birth, identifying persistent methylation differences at ten genomic loci. Longitudinal comparisons of DNA methylation at birth and 18 years uncovered a significant overlap between sites that were differentially-methylated at birth and those that changed with age. However, we note that overlapping sites may either differ in the same (300/1,555) or opposite (431/1,555) direction during gestation and aging respectively.
We present evidence for widespread methylation differences between extreme preterm and term infants at birth that are largely resolved by 18 years of age. These results are consistent with methylation changes associated with blood cell development, cellular composition, immune induction and age at these time points. Finally, we identified ten probes significantly associated with preterm individuals and with greater than 5% methylation discordance at birth and 18 years that may reflect a long term epigenetic legacy of preterm birth.
Difficulties associated with long term culture of primary trophoblasts have proven to be a major hurdle in their functional characterization. In order to circumvent this issue, several model cell lines have been established over many years using a variety of different approaches. These include lines derived from gestational tumors, or transformation/immortalization of primary trophoblast cells. Due to their differing origins, gene expression profiles, and behavior in vitro, different model lines have been utilized to investigate specific aspects of trophoblast biology. However, generally speaking, the molecular mechanisms underlying functional differences remain unclear. In this study, we profiled genome-scale DNA methylation in primary first trimester trophoblast cells and seven commonly used trophoblast-derived cell lines in an attempt to identify functional pathways differentially regulated by epigenetic modification in these cells. We identified a general increase in DNA promoter methylation levels in four choriocarcinoma (CCA)-derived lines and transformed HTR-8/SVneo cells, including hypermethylation of several genes regularly seen in human cancers, while other differences in methylation were noted in genes linked to immune responsiveness, cell morphology, development and migration across the different cell populations. Interestingly, CCA-derived lines show an overall methylation profile more similar to unrelated solid cancers than to untransformed trophoblasts, highlighting the role of aberrant DNA methylation in CCA development and/or long term culturing. Comparison of DNA methylation and gene expression in CCA lines and cytotrophoblasts revealed a significant contribution of DNA methylation to overall expression profile, most likely underlying functional variation between cells of different origin. These data highlight the variability in epigenetic state between primary trophoblasts and cell models in pathways underpinning a wide range of cell functions, providing valuable candidate pathways for future functional investigation in different cell populations. This study also confirms the need for caution in the interpretation of data generated from manipulation of such pathways in vitro.
placenta; trophoblast; cell line; DNA methylation; epigenetics
The extent to which development- and age-associated epigenetic changes are influenced by genetic, environmental and stochastic factors remains to be discovered. Twins provide an ideal model with which to investigate these influences but previous cross-sectional twin studies provide contradictory evidence of within-pair epigenetic drift over time. Longitudinal twin studies can potentially address this discrepancy.
In a pilot, genome-scale study of DNA from buccal epithelium, a relatively homogeneous tissue, we show that one-third of the CpGs assayed show dynamic methylation between birth and 18 months. Although all classes of annotated genomic regions assessed show an increase in DNA methylation over time, probes located in intragenic regions, enhancers and low-density CpG promoters are significantly over-represented, while CpG islands and high-CpG density promoters are depleted among the most dynamic probes. Comparison of co-twins demonstrated that within-pair drift in DNA methylation in our cohort is specific to a subset of pairs, who show more differences at 18 months. The rest of the pairs show either minimal change in methylation discordance, or more similar, converging methylation profiles at 18 months. As with age-associated regions, sites that change in their level of within-pair discordance between birth and 18 months are enriched in genes involved in development, but the average magnitude of change is smaller than for longitudinal change.
Our findings suggest that DNA methylation in buccal epithelium is influenced by non-shared stochastic and environmental factors that could reflect a degree of epigenetic plasticity within an otherwise constrained developmental program.
The insulin-like growth factor 2 (IGF2) and H19 imprinted genes control growth and body composition. Adverse in-utero environments have been associated with obesity-related diseases and linked with altered DNA methylation at the IGF2/H19 locus. Postnatally, methylation at the IGF2/H19 imprinting control region (ICR) has been linked with cerebellum weight. We aimed to investigate whether decreased IGF2/H19 ICR methylation is associated with decreased birth and childhood anthropometry and increased contemporaneous adiposity.
DNA methylation in peripheral blood (n = 315) at 17 years old was measured at 12 cytosine-phosphate-guanine sites (CpGs), analysed as Sequenom MassARRAY EpiTYPER units within the IGF2/H19 ICR. Birth size, childhood head circumference (HC) at six time-points and anthropometry at age 17 years were measured. DNA methylation was investigated for its association with anthropometry using linear regression.
The principal component of IGF2/H19 ICR DNA methylation (representing mean methylation across all CpG units) positively correlated with skin fold thickness (at four CpG units) (P-values between 0.04 to 0.001) and subcutaneous adiposity (P = 0.023) at age 17, but not with weight, height, BMI, waist circumference or visceral adiposity. IGF2/H19 methylation did not associate with birth weight, length or HC, but CpG unit 13 to 14 methylation was negatively associated with HC between 1 and 10 years. β-coefficients of four out of five remaining CpG units also estimated lower methylation with increasing childhood HC.
As greater IGF2/H19 methylation was associated with greater subcutaneous fat measures, but not overall, visceral or central adiposity, we hypothesize that obesogenic pressures in youth result in excess fat being preferentially stored in peripheral fat depots via the IGF2/H19 domain. Secondly, as IGF2/H19 methylation was not associated with birth size but negatively with early childhood HC, we hypothesize that the HC may be a more sensitive marker of early life programming of the IGF axis and of fetal physiology than birth size. To verify this, investigations of the dynamics of IGF2/H19 methylation and expression from birth to adolescence are required.
Childhood; Fetal programming; DNA methylation; Insulin-like growth factor; Raine Study; Head circumference
Juvenile 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.
Methylation 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.
Our 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.
Epigenetics; Juvenile idiopathic arthritis; DNA methylation; Autoimmunity; Methylome; Methotrexate
Epigenome-wide association studies (EWAS) can be used to investigate links between early life environment, epigenetics and disease. However, such studies raise the question of which came first: the mark or the malady? A recent study has demonstrated that EWAS can be performed on neonatal 'Guthrie' heel-prick blood spots. As Guthrie cards are collected from all newborn infants and stored indefinitely in many countries, they represent an important timepoint to compare with later disease-associated epigenetic marks.
biomarkers; DNA; epigenetics; Guthrie cards; newborn screening
Like formalin fixed paraffin embedded (FFPE) tissues, archived bone marrow aspirate slides are an abundant and untapped resource of biospecimens that could enable retrospective molecular studies of disease. Historically, RNA obtained from slides is limited in utility because of their low quality and highly fragmented nature. MicroRNAs are small (≈22 nt) non-coding RNA that regulate gene expression, and are speculated to preserve well in FFPE tissue. Here we investigate the use of archived bone marrow aspirate slides for miRNA expression analysis in paediatric leukaemia. After determining the optimal method of miRNA extraction, we used TaqMan qRT-PCR to identify reference miRNA for normalisation of other miRNA species. We found hsa-miR-16 and hsa-miR-26b to be the most stably expressed between lymphoblastoid cell lines, primary bone marrow aspirates and archived samples. We found the average fold change in expression of hsa-miR-26b and two miRNA reportedly dysregulated in leukaemia (hsa-miR-128a, hsa-miR-223) was <0.5 between matching archived slide and bone marrow aspirates. Differential expression of hsa-miR-128a and hsa-miR-223 was observed between leukaemic and non-leukaemic bone marrow from archived slides or flash frozen bone marrow. The demonstration that archived bone marrow aspirate slides can be utilized for miRNA expression studies offers tremendous potential for future investigations into the role miRNA play in the development and long term outcome of hematologic, as well as non-hematologic, diseases.
The human placenta facilitates the exchange of nutrients, gas and waste between the fetal and maternal circulations. It also protects the fetus from the maternal immune response. Due to its role at the feto-maternal interface, the placenta is subject to many environmental exposures that can potentially alter its epigenetic profile. Previous studies have reported gene expression differences in placenta over gestation, as well as inter-individual variation in expression of some genes. However, the factors contributing to this variation in gene expression remain poorly understood.
In this study, we performed a genome-wide DNA methylation analysis of gene promoters in placenta tissue from three pregnancy trimesters. We identified large-scale differences in DNA methylation levels between first, second and third trimesters, with an overall progressive increase in average methylation from first to third trimester. The most differentially methylated genes included many immune regulators, reflecting the change in placental immuno-modulation as pregnancy progresses. We also detected increased inter-individual variation in the third trimester relative to first and second, supporting an accumulation of environmentally induced (or stochastic) changes in DNA methylation pattern. These highly variable genes were enriched for those involved in amino acid and other metabolic pathways, potentially reflecting the adaptation of the human placenta to different environments.
The identification of cellular pathways subject to drift in response to environmental influences provide a basis for future studies examining the role of specific environmental factors on DNA methylation pattern and placenta-associated adverse pregnancy outcomes.
Hyperplastic Polyposis Syndrome (HPS) is a condition associated with multiple serrated polyps, and an increased risk of colorectal cancer (CRC). At least half of CRCs arising in HPS show a CpG island methylator phenotype (CIMP), potentially linked to aberrant DNA methyltransferase (DNMT) activity. CIMP is associated with methylation of tumor suppressor genes including regulators of DNA mismatch repair (such as MLH1, MGMT), and negative regulators of Wnt signaling (such as WIF1). In this study, we investigated the potential for interaction of genetic and epigenetic variation in DNMT genes, in the aetiology of HPS.
We utilized high resolution melting (HRM) analysis to screen 45 cases with HPS for novel sequence variants in DNMT1, DNMT3A, DNMT3B, and DNMT3L. 21 polyps from 13 patients were screened for BRAF and KRAS mutations, with assessment of promoter methylation in the DNMT1, DNMT3A, DNMT3B, DNMT3L MLH1, MGMT, and WIF1 gene promoters.
No pathologic germline mutations were observed in any DNA-methyltransferase gene. However, the T allele of rs62106244 (intron 10 of DNMT1 gene) was over-represented in cases with HPS (p<0.01) compared with population controls. The DNMT1, DNMT3A and DNMT3B promoters were unmethylated in all instances. Interestingly, the DNMT3L promoter showed low levels of methylation in polyps and normal colonic mucosa relative to matched disease free cells with methylation level negatively correlated to expression level in normal colonic tissue. DNMT3L promoter hypomethylation was more often found in polyps harbouring KRAS mutations (p = 0.0053). BRAF mutations were common (11 out of 21 polyps), whilst KRAS mutations were identified in 4 of 21 polyps.
Genetic or epigenetic alterations in DNMT genes do not appear to be associated with HPS, but further investigation of genetic variation at rs62106244 is justified given the high frequency of the minor allele in this case series.
Epigenetic modification can mediate environmental influences on gene expression and can modulate the disease risk associated with genetic variation. Epigenetic analysis therefore holds substantial promise for identifying mechanisms through which genetic and environmental factors jointly contribute to disease risk. The spatial and temporal variance in epigenetic profile is of particular relevance for developmental epidemiology and the study of aging, including the variable age at onset for many common diseases. This review serves as a general introduction to the topic by describing epigenetic mechanisms, with a focus on DNA methylation; genetic and environmental factors that influence DNA methylation; epigenetic influences on development, aging, and disease; and current methodology for measuring epigenetic profile. Methodological considerations for epidemiologic studies that seek to include epigenetic analysis are also discussed.
DNA methylation; environment; epigenesis, genetic; folic acid
The genome of extraembryonic tissue, such as the placenta, is hypomethylated relative to that in somatic tissues. However, the origin and role of this hypomethylation remains unclear. The DNA methyltransferases DNMT1, -3A, and -3B are the primary mediators of the establishment and maintenance of DNA methylation in mammals. In this study, we investigated promoter methylation-mediated epigenetic down-regulation of DNMT genes as a potential regulator of global methylation levels in placental tissue. Although DNMT3A and -3B promoters lack methylation in all somatic and extraembryonic tissues tested, we found specific hypermethylation of the maintenance DNA methyltransferase (DNMT1) gene and found hypomethylation of the DNMT3L gene in full term and first trimester placental tissues. Bisulfite DNA sequencing revealed monoallelic methylation of DNMT1, with no evidence of imprinting (parent of origin effect). In vitro reporter experiments confirmed that DNMT1 promoter methylation attenuates transcriptional activity in trophoblast cells. However, global hypomethylation in the absence of DNMT1 down-regulation is apparent in non-primate placentas and in vitro derived human cytotrophoblast stem cells, suggesting that DNMT1 down-regulation is not an absolute requirement for genomic hypomethylation in all instances. These data represent the first demonstration of methylation-mediated regulation of the DNMT1 gene in any system and demonstrate that the unique epigenome of the human placenta includes down-regulation of DNMT1 with concomitant hypomethylation of the DNMT3L gene. This strongly implicates epigenetic regulation of the DNMT gene family in the establishment of the unique epigenetic profile of extraembryonic tissue in humans.
Development Differentiation/Tissue; DNA/Methylation; DNA/Methyltransferase; Epigenetics; Gene Transcription; Extraembryonic Tissue; Placenta; Trophoblast
Plasma concentrations of biologically active vitamin D
(1,25-(OH)2D) are tightly controlled via feedback regulation of
renal 1α-hydroxylase (CYP27B1; positive) and 24-hydroxylase
(CYP24A1; catabolic) enzymes. In pregnancy, this regulation is
uncoupled, and 1,25-(OH)2D levels are significantly elevated,
suggesting a role in pregnancy progression. Epigenetic regulation of
CYP27B1 and CYP24A1 has previously been described in cell
and animal models, and despite emerging evidence for a critical role of
epigenetics in placentation generally, little is known about the regulation of
enzymes modulating vitamin D homeostasis at the fetomaternal interface. In
this study, we investigated the methylation status of genes regulating vitamin
D bioavailability and activity in the placenta. No methylation of the
VDR (vitamin D receptor) and CYP27B1 genes was found in any
placental tissues. In contrast, the CYP24A1 gene is methylated in
human placenta, purified cytotrophoblasts, and primary and cultured chorionic
villus sampling tissue. No methylation was detected in any somatic human
tissue tested. Methylation was also evident in marmoset and mouse placental
tissue. All three genes were hypermethylated in choriocarcinoma cell lines,
highlighting the role of vitamin D deregulation in this cancer. Gene
expression analysis confirmed a reduced capacity for CYP24A1
induction with promoter methylation in primary cells and in vitro
reporter analysis demonstrated that promoter methylation directly
down-regulates basal promoter activity and abolishes vitamin D-mediated
feedback activation. This study strongly suggests that epigenetic decoupling
of vitamin D feedback catabolism plays an important role in maximizing active
vitamin D bioavailability at the fetomaternal interface.
DNA methylation is a hallmark of transcriptional silencing, yet transcription has been reported at the centromere. To address this apparent paradox, we employed a fully sequence-defined ectopic human centromere (or neocentromere) to investigate the relationship between DNA methylation and transcription. We used sodium bisulfite PCR and sequencing to determine the methylation status of 2,041 CpG dinucleotides distributed across a 6.76-Mbp chromosomal region containing a neocentromere. These CpG dinucleotides were associated with conventional and nonconventional CpG islands. We found an overall hypermethylation of the neocentric DNA at nonconventional CpG islands that we designated as CpG islets and CpG orphans. The observed hypermethylation was consistent with the presence of a presumed transcriptionally silent chromatin state at the neocentromere. Within this neocentric chromatin, specific sites of active transcription and the centromeric chromatin boundary are defined by DNA hypomethylation. Our data demonstrate, for the first time to our knowledge, a correlation between DNA methylation and centromere formation in mammals, and that transcription and “chromatin-boundary activity” are permissible at the centromere through the selective hypomethylation of pockets of sequences without compromising the overall silent chromatin state and function of the centromere.
The centromere is a chromosomal structure that is vital for the correct partitioning of chromosomes during cell division. Recent studies in a number of different species have shown that transcription is permissible within the centromere, but the mode of transcription regulation at the centromere remains unclear. DNA methylation is a well-characterized mechanism for the genomic regulation of transcription. Here, the authors investigate the relationship between DNA methylation and transcription activity at a functional human centromere. They demonstrate a high level of DNA methylation across the centromere but identify pockets of DNA sequences within the methylated domain that are non-methylated. These pockets correspond to sites of transcription and/or boundaries that separate major centromeric chromatin sub-domains. This study shows the complexity of the centromere as it uses DNA methylation to both maintain a tight chromatin structure and to allow transcription to occur.