Recent technological advances in epigenome profiling have led to an increasing number of studies investigating the role of the epigenome in obesity. There is also evidence that environmental exposures during early life can induce persistent alterations in the epigenome, which may lead to an increased risk of obesity later in life.
This paper provides a systematic review of studies investigating the association between obesity and either global, site-specific or genome-wide methylation of DNA. Studies on the impact of pre- and postnatal interventions on methylation and obesity are also reviewed. We discuss outstanding questions, and introduce EpiSCOPE, a multidisciplinary research program aimed at increasing the understanding of epigenetic changes in emergence of obesity.
An electronic search for relevant articles, published between September 2008 and September 2013 was performed. From the 319 articles identified, 46 studies were included and reviewed. The studies provided no consistent evidence for a relationship between global methylation and obesity. The studies did identify multiple obesity-associated differentially methylated sites, mainly in blood cells. Extensive, but small, alterations in methylation at specific sites were observed in weight loss intervention studies, and several associations between methylation marks at birth and later life obesity were found.
Overall, significant progress has been made in the field of epigenetics and obesity and the first potential epigenetic markers for obesity that could be detected at birth have been identified. Eventually this may help in predicting an individual’s obesity risk at a young age and opens possibilities for introducing targeted prevention strategies. It has also become clear that several epigenetic marks are modifiable, by changing the exposure in utero, but also by lifestyle changes in adult life, which implies that there is the potential for interventions to be introduced in postnatal life to modify unfavourable epigenomic profiles.
There is little contention that the rising incidence of obesity is a major public health issue worldwide.1 Obesity is a major risk factor for comorbidities, such as type 2 diabetes, cardiovascular disease and certain forms of cancer.2 Thus, the obesity epidemic threatens to reduce the length and quality of life of current and future generations, and it presents a significant challenge to future health-care budgets. There is a strong need for safe and effective strategies for obesity prevention and treatment. A multitude of campaigns have been launched by governments and health agencies, but for the most part with limited effects on reducing obesity rates in the medium to longer term.3 Part of the reason for this failure could be that these strategies are typically introduced after obesity is established, and it is becoming increasingly clear that at that stage obesity is difficult to reverse.4,5 The focus of anti-obesity campaigns should therefore be on prevention in order to achieve maximum long-term health gains. To improve prevention and treatment strategies a better understanding of factors contributing to the development of obesity is essential.
Epigenetics and human disease
Over the last decade, there has been increasing interest in the role of epigenetics in the development of complex conditions such as obesity. In contrast to genetic modifications, which lead to a change in the base sequence of DNA, epigenetic changes are typically reversible and refer to chemical modifications to DNA (or DNA-associated chromosomal proteins called histones) that occur in the absence of a change in the DNA sequence.6 Epigenetic marks are heritable through mitotic cell division and can alter the way the transcription of genes is controlled within a cell. This occurs through a number of processes, the best described being the addition of methyl groups to DNA (methylation) and posttranslational modifications to histone proteins, such as acetylation and methylation. Methylation of mammalian genomes occurs predominantly at cytosines adjacent to guanines (‘CpG’ sites). Epigenetic processes alter the accessibility of the transcriptional machinery to a particular gene, thereby determining whether or not the gene is active in a given cell at a given time. Importantly, although the DNA sequence of genes in an individual (the genome) is largely stable, the epigenome has the potential to be reversibly modified by exposure to a range of nutritional and environmental factors.6
The importance of epigenetic processes in human disease was first identified in the field of cancer in the 1980 s.7 Since then, there have been a plethora of studies that have described epigenetic changes in cancerous tissues, and in the blood of cancer patients, and alterations in the methylation level of specific genes have been proposed as novel biomarkers in cancer screening.8
More recently, the attention of the scientific community has turned to the potential role of epigenetic modifications in other disease states, including obesity. Initial studies were limited in sample size and number of CpG sites (CpGs) studied. With advances in technologies and the emergence of more affordable, high-throughput methylation screening methods, there has been an increase in large-scale studies and the first epigenome-wide association studies exploring the relationship between the environment, the epigenome and complex disease states. To date, DNA methylation, either at global, site-specific or genome-wide levels at single nucleotide resolution, is by far the most studied epigenetic mark. There have been few investigations of histone modifications in relation to obesity in humans, but some of the results to date do suggest an association between genome-wide histone modifications and the development of or susceptibility toward obesity.9
There is accumulating evidence that the propensity toward adult obesity has early developmental origins and follows an intergenerational cycle.10, 11, 12, 13, 14, 15 Epidemiological studies have shown that exposure to a suboptimal nutritional environment during development, as a result of either an excess or deficient maternal caloric or micronutrient intake, is associated with an increased risk of a range of chronic diseases, including obesity, type 2 diabetes and cardiovascular disease in later life.16, 17, 18, 19, 20 These findings have led to the developmental origins of health and disease hypothesis, which proposes that adult disease risk can be programmed by the perinatal environment.21
One consistent theme relating to this hypothesis is that transient environmental influences experienced early in life can cause permanent effects that emerge as increased disease risk much later in life. The mechanisms underpinning this nutritional ‘memory’ response are not clear but may include changes in the developmental trajectories of tissues, reprogramming of stem cells, changes in tissue structure, and the reprogramming of neural, endocrine and metabolic regulatory circuits. Epigenetic programming may be mechanistically involved in these processes or provide a readout of their occurrence. Moreover, once established, early life nutritionally induced epigenetic changes may lie dormant until their biological influence is triggered later in life.
Evidence to support a role of epigenetics in developmental programming of disease has been predominately derived from animal studies that have demonstrated the impact of a suboptimal intrauterine nutritional environment on the epigenome and phenotype of the offspring.22, 23, 24, 25 There are relatively few human studies in this area, but one of the most significant studies was conducted in children who were born to women exposed to severe undernutrition during pregnancy as a result of the Dutch Hunger Winter during World War II, which reported a reduced methylation of the imprinted gene IGF2 in these individuals as adults.26,27 This has particular relevance given that these individuals have also been shown to be at increased risk of obesity or glucose intolerance, depending on the timing of the exposure to famine.28, 29, 30
In this review, we provide a systematic overview of the most recent findings in the research area of epigenetics and obesity, specifically focused on human studies. Studies investigating the association between either global methylation, site-specific methylation or genome-wide methylation of DNA and obesity, are summarised and discussed. In addition, the impact of interventions on DNA methylation profiles and obesity are summarised. Moreover, it discusses outstanding questions and introduces EpiSCOPE, a multidisciplinary research program with the goal of increasing the understanding of epigenetic changes in emergence of obesity.
Materials and methods
The PubMed database was searched for relevant studies published between 15 September 2008 and 15 September 2013, using the search terms ‘obesity OR body mass index OR overweight OR body fat OR adiposity OR adipose tissue’ AND ‘epigenetics OR methylation OR histone’. The first search was restricted to primary studies in humans, and an additional search was performed for articles that were not labelled as a human or animal study. The titles and abstracts, and in several cases the full texts, were scanned to determine their relevance to the scope of this review. Studies to be included either described an association between epigenetic marks and obesity in humans or reported an effect of a defined intervention on epigenetic marks and obesity in humans.
Studies identified by search strategy
From the 319 articles identified by the search strategy, 273 articles were excluded; 55 papers were reviews or commentaries, rather than original research articles, 62 studies were not conducted in humans, 71 did not assess any outcomes related to obesity, 18 did not assess an epigenetic outcome, 32 included only in vitro data and 35 were excluded for other reasons, (for example, methodology studies or studies that included individuals with hereditary diseases or cancer). Thus, a total of 46 articles were included in this review.
Summary of included studies
Of the 46 studies included, 15 studies assessed relationships between measures of obesity and global DNA methylation, 13 studies assessed relationships with DNA methylation in specific candidate genes, 5 studies used genome-wide approaches to assess differences in methylation between obese/lean individuals or the association with obesity measures, 8 studies assessed DNA methylation profiles in relation to weight loss interventions and 9 studies assessed relationships of DNA methylation at early life with either parental health measures or later life health outcomes. Some of the studies assessed multiple outcomes.
Global methylation and obesity
Global methylation refers to the overall level of methylcytosine in the genome, expressed as percentage of total cytosine (percentage 5-methylcytosine). A lowered global methylation level has been associated with chromosomal instability and increased mutation events and is considered as a hallmark of cancer,31 but less is known about global methylation in other disease states.
Repetitive elements, such as Alu and LINE1, comprise ~50% of the genome and the degree of methylation in these elements is often used as a surrogate to represent the overall methylation level of the genome. As a result of the relatively low cost and high-throughput, global methylation levels are reasonably easy to determine in large numbers of samples, which makes it ideal for screening purposes.
Details of the 15 studies that have examined global methylation in relation to obesity are summarised in Table 1. The majority of the studies used blood samples, but long interspersed nucleotide element 1 methylation in DNA from muscle, placenta and colon has also been studied. All of the studies used body mass index (BMI) or changes in BMI to classify obesity, and two studies also used percentage body fat.32,33 The majority of these investigations, including a large study combining four study populations, including up to 1254 individuals,34 did not find an association between obesity and global methylation.33,35, 36, 37, 38, 39, 40 Two studies that included only women,32,41 found reduced global methylation with increasing BMI, however, in one of these studies this only occurred in the presence of low concentrations of the methyl donor folate.32 In contrast, two other studies that included both men and women from two different populations (Samoa and China) reported a positive relationship between global methylation in peripheral blood leukocytes (PBLs) and BMI,42,43 and in a further study global methylation in placental tissues was higher in obese compared with lean women.40 Only one study examined global histone methylation in obesity, showing substantially decreased levels of histone H3 lysine 4 dimethylation in adipocytes of overweight individuals compared with lean, with increased levels of lysine 4 trimethylation observed in obese/diabetic individuals.9 Thus, while some studies do report significant associations between global methylation and obesity-related measures, the direction of change is not consistent, and both global hypomethylation as well as global hypermethylation have been related to obesity-related measures.32,33,41, 42, 43, 44, 45
There are a multitude of factors, including gender, ethnic background, age, exposure to chemicals, tobacco smoke, alcohol and diet,33,34,46, 47, 48, 49 that are known to affect global methylation levels, which could influence a potential association between global methylation and obesity. In several studies, corrections for at least a few of these confounding factors have been applied, but often not all confounding factors are known and taken into account.
Overall, the available global methylation studies in obesity do not provide consistent evidence for a relationship between global methylation and obesity. Compared with the situation in cancer, global methylation changes in obesity (if present) are likely to be more subtle and thus difficult to detect considering the influence of multiple other factors on this measure. Consequently, more specific methylation analyses, either for specific loci of interest in obesity or a genome-wide approach, are likely to provide a better picture of the association between obesity and DNA methylation. Larger sample sizes (>1000) may also be required to produce more robust results. In addition, the studies that have been conducted to date point to the potential for sex differences in the relationship between BMI and global methylation, which also require further investigation and suggest that it is important to examine associations separately in males and females.
Gene-specific DNA methylation and obesity
The epigenetic environment of individual genes provides a critical component contributing to their regulation and level of expression. As a result of the relative ease of analysis, gene-specific DNA methylation is the most extensively studied epigenetic mark in studies relating epigenetic changes to health outcomes, including obesity. Historically, elevated DNA methylation has been associated with repression of gene expression. However, with the advent of genome-wide methods of DNA methylation analysis, it is now recognised that the association of DNA methylation with gene expression is not as simple as previously thought, and appears to depend on where within the gene sequence the methylation occurs. In general, DNA methylation at gene promoters and enhancers is associated with gene silencing and higher methylation in the gene body with active gene expression, but even this is an oversimplification.50
The majority of studies examining the relationship between site-specific DNA methylation and obesity are cross-sectional; that is, both methylation levels and the phenotype are measured at the same time point. Hence, it cannot be established whether the association between a specific DNA methylation mark and obesity is a cause or a consequence of the obese phenotype.
Candidate gene studies
Multiple studies have used a hypothesis-driven, candidate gene approach (summarised in Table 2) where methylation sites in, or near, known candidate genes for obesity susceptibility have been the subject of investigation. In some cases, the choice of genes has been based on prior analysis of gene expression differences in the same subjects. The candidate gene methylation studies have focussed on a range of genes implicated in obesity, appetite control and/or metabolism, insulin signalling, immunity, growth, circadian clock regulation and imprinted genes, and assessed their relationship with a variety of obesity markers. Collectively, these studies have identified lower methylation of tumour necrosis factor alpha (TNFα) in PBL,51 pyruvate dehydrogenase kinase 4 (PDK4) in muscle52 and leptin (LEP) in whole blood (WB)53 and increased methylation of proopiomelanocortin (POMC) in WB,54 PPARγ coactivator 1 alpha (PGC1α)52 in muscle, and CLOCK and aryl hydrocarbon receptor nuclear translocator-like (BMAL1)55 genes in PBL in obese compared with lean individuals. Associations between BMI, adiposity, and waist circumference, with methylation in PDK4 in muscle,52 melanin-concentrating hormone receptor 1 (MCHR1) in WB,56 and the serotonin transporter (SLC6A4) gene,57 the androgen receptor (AR),58 11 b-hydroxysteroid dehydrogenase type 2 (HSD2),59 period circadian clock 2 (PER2)55 and glucocorticoid receptor (GR)59 in PBL have also been reported. The most consistently observed epigenetic association has been that of methylation at the IGF2/H19 imprinting region in blood cells with measures of adiposity.59,60 Collectively, these studies provide evidence that obesity is associated with altered epigenetic regulation of a number of metabolically important genes.
Genome-wide analyses and obesity
The recent development of genome-wide methods for quantifying site-specific DNA methylation has led to the initiation of studies that are not targeted to specific genes, but search for associations across a large number of genes and CpGs. Obesity-associated differentially methylated (DM) sites in peripheral blood cells were detected in four published genome-wide studies (Table 2).61, 62, 63, 64 Extensive, but small, alterations in methylation at specific sites have been observed. In one case, a signature of DM sites was used to predict obesity in a validation set,62 while in other studies specific DM CpGs were validated in a second cohort,63 or at a second time point.64
Overall, obesity-associated DM sites were enriched both in obesity candidate genes62 and in genes with a wide diversity of other functions, such as immune response,63 cell differentiation62 and regulation of transcription.61 DM sites were also identified in or near genes with no known function related to obesity or adipose tissue functioning.
Intervention studies in adults
It has long been assumed that DNA methylation profiles would remain stable throughout adult life; however, this view is now changing. Interventions such as exercise, diets and weight loss surgery have been shown to modulate methylation profiles in different tissue types (Table 3).52,65,66 Interestingly, methylation profiles of obese individuals became more similar to those of lean individuals following weight loss surgery.52 Although this was only demonstrated in a small study, it suggests that methylation profiles of obese individuals can be modified by reductions in body weight/fat mass. This conclusion may imply that some methylation marks are a consequence of the obese phenotype, rather than a programmed mark that predisposes people to become obese. These findings again highlight the importance of studies in which methylation marks are measured early in life before disease manifests, to define which acquired marks become permanent, and thus potential early markers for disease risk, and which ones are semipermanent and modifiable in later life.
In a separate group of studies, comparison of the methylation profiles of people who successfully lost weight during interventions and those who did not, has been used in order to determine whether there may be biomarkers that predict individual responsiveness to weight loss interventions (Table 3).55,66, 67, 68, 69 Methylation differences between these individuals were identified in genes involved in weight control, insulin secretion, inflammation and circadian rhythm. The association of such methylation differences with a propensity to lose weight may imply that methylation changes in those genes predisposes individuals to become and stay obese, even in situations of limited food intake. Adherence to the interventions was monitored in most studies through regular meetings with study dieticians, or attendance at group exercise or therapy sessions. However, it is notoriously difficult to accurately monitor compliance in nutritional intervention studies in humans, and it is therefore possible that failure to lose weight may also be a reflection of poor adherence of the participants to the intervention.
Prenatal and postnatal environment
The period of embryonic development has been recognised as a critical window in the establishment of the epigenome. There is compelling evidence that an adverse prenatal and early postnatal environment can increase obesity risk in later life27,70, 71, 72 and this has led to the search for nutritional interventions during pregnancy and lactation, which have the potential to mitigate or overcome this adverse programming.12 Diet and weight loss interventions in obese mothers may lead to a decreased risk of obesity in the offspring, possibly mediated through changes in insulin signalling, fat storage, energy expenditure or appetite control pathways. Epigenetic mechanisms are likely to have a role in this altered risk profile and the findings of obesity-associated methylation marks in genes involved in these processes also support this hypothesis. Human studies showing a direct relationship between specific prenatal (nutritional) exposures on methylation profiles of the offspring and subsequent risk of obesity in later life are scarce. However, there are a number of studies that have assessed differences in methylation of candidate genes in children in relation to maternal/paternal characteristics44,73, 74, 75 or have explored relationships between epigenetic markers in the cord blood at delivery and obesity/metabolic outcomes in childhood (Table 4). One such study compared methylation profiles of siblings born before and after maternal weight loss surgery, and reported differences between the siblings in obesity characteristics and in methylation profiles for genes involved in the regulation of glucose homeostasis and immune function,76,77 some of which translated into alterations in gene expression and insulin sensitivity. Although this was a small study, its findings suggest that significant weight loss, and presumably improved metabolic health profiles, in the mother are associated with a distinct epigenome and lower weight and waist circumference in the children.
Additional studies have explored the association of DNA methylation at birth with adiposity in later life (Table 4).78, 79, 80, 81 Methylation variation in the promoter of the retinoid X receptor alpha gene (RXRα) in umbilical cord tissue was found to explain up to 26% of the variation in childhood adiposity.79 RXRα is a nuclear receptor with a known role in adipogenesis;82 it forms a heterodimer with the transcription factor PPARγ to activate transcription of genes involved in adipocyte differentiation, glucose metabolism, inflammation and energy homeostasis. DNA methylation appears to be also important in the regulation of PPARγ,83 and variation in DNA methylation of PGC1A, interacting with PPARγ, has been associated with weight loss, obesity, and risk for type 2 diabetes mellitus.52,84,85 Variation in methylation within tumour-associated calcium signal transducer 2 (TACST2) at birth was also found to correlate with fat mass in later life, however, further analysis including single nuclear polymorphism data of this gene showed that reverse causation or confounding was likely to account for the observed correlation.81 IGF2 is another example of a gene showing loci-specific variation in methylation at birth, and also at childhood, that is associated with growth characteristics and obesity in later life.59,60,78 The epigenetic regulation of IGF2 has been of particular interest given its role in control of fetal growth and development. Differences in the degree of methylation near IGF2 has often been linked to exposure to a suboptimal environment in utero.26,27,59,86 Contradictory findings, however, come from recent mice studies that showed no effect of maternal nutrition on IGF2 DMRs (DM regions).87
The finding of an association between variation in matrix metallopeptidase 9 (MMP9) methylation levels at birth and childhood adiposity80 is also of interest, given the critical role that metalloproteinases have in extra cellular matrix remodelling during adipose tissue formation, and coupled with the fact that altered MMP9 plasma levels and gene expression has previously been found in obese individuals.88 Moreover, variation in methylation near MMP9, and another metalloproteinase called PM20D1, was associated with BMI in a genome-wide study at two time points 11 years apart.64 These findings show that these marks are most likely established at an early age and may be associated with adiposity at different stages in later life, which suggests that these methylation changes could be potentially useful to predict obesity risk from an early age.
Measurement of DNA methylation
The advent of genome-wide, array-based methods for determining site-specific DNA methylation is opening a new window for identifying phenotype DM or regions. An overview of different methods of DNA methylation analysis and discussion of their respective advantages and disadvantages, including those used in the reviewed studies, can be found in Supplementary Table 1. Although a number of technologies have been used to date, the Illumina Infinium Methylation450 bead Chip is emerging as the most widely used platform. Early experience suggests that the technology provides reproducible quantitative data and its widespread use will facilitate cross-study comparisons. As assessment is made of only a selected set of CpGs and regions (<5% of total number of CpGs), it will be important that other technologies that explore the genome more widely continue to be used.
Notably, both from candidate gene and genome-wide approaches, the methylation differences associated with obesity or interventions reported to date have generally been small. In array-based approaches, as the number of individuals is low compared with the high number of sites measured, often only a few DM sites remain significant after strict multiple testing corrections. The presence of co-ordinate variation at CpGs within a localised region can provide more confidence that differences seen are real and methods looking at variable methylated regions, as opposed to single sites, are increasingly being used in analysis of methylation data to overcome the problems associated with strict multiple testing corrections.89 It has been proposed that particular regions of the genome show high levels of variance in methylation levels and that this may provide potential for gene expression levels to stably respond to environmental conditions.90 This adaptability would confer a significant fitness advantage and has probably been subject to strong selection during the longer time scales of evolution, that is, the genetic blueprint has likely been altered by evolution and natural selection to enhance epigenetically mediated adaptability traits. In support of this, a significant overlap in variably methylated regions and DM regions has been observed.62 Given the modest levels of change in DNA methylation, it is expected that the effect size on phenotype of individual DM sites is generally likely to be small. However, it is noteworthy that methylation at the RXRA locus at birth could statistically account for 26% of variation in adiposity of children at 9 years.79 Similar to genetic variations, it will probably not be one single DM site but more likely combinations of multiple (in)dependent DM sites, possibly in several genes, which can explain variations in phenotype and that will need to be used in combination to develop predictive signatures.
The vast majority of epigenetic studies published to date focused only on DNA methylation, thereby ignoring other epigenetic information, such as histone modifications and non-coding RNAs. Histone modifications can influence DNA methylation patterns and vice versa, and there is evidence that histone modifications have key roles in adipogenesis.91 Thus, these are likely to have an important role in the development of obesity and should be taken into account in epigenetic studies. Integration of epigenome and transcriptome data will also be crucial to obtain a more complete picture of the interaction between epigenetic modifications and regulation of gene expression.
DNA methylation in different tissue types
DNA from peripheral blood cells is the most frequently used source of DNA for epigenetic studies. As blood is easily accessible, it is often the only biological material that is routinely sampled in large-scale studies. A potential issue with the use of blood is that it consists of a mixture of different cell types with different methylation profiles.92,93 It has been shown that although methylation of some CpGs is dependent on variation in blood cell types,58,94 global methylation and methylation at most sites appear to be unaffected.95, 96, 97 However, if one wants to minimise potential effects of cellular heterogeneity on methylation profiles, a correction based on the numbers of each respective cell type in the sample may need to be applied.98,99
Another important consideration when using blood in epigenetic studies is that the blood cell methylation profile in blood may not necessarily report the epigenetic state in other tissues. As the haematopoietic system is established very early in development, it has been suggested that methylation changes induced around conception and in early embryo development may be reflected in all germ layers and thus detectable in blood and most tissue types.64 Later in development, however, environmentally induced epigenetic changes may be more tissue specific and may not be detectable in blood. A recent study comparing methylomes across 30 human tissues and cell types showed that there is a large ‘common methylation profile’ across tissues and only a small fraction (~20%) of CpGs show dynamic regulation during development.100 Efforts to provide a ‘Blue Print of Human Epigenomes’, as undertaken by consortia like the International Human Epigenome Consortium, will be of considerable value to get a better insight in tissue-specific epigenetic signatures and their role in disease development.
To increase our understanding of the role of epigenetics in obesity, adipose tissue is of high interest. Adipose tissue is not only the main tissue for energy storage, but also has important endocrine functions, which are often disrupted in obesity. Adipose tissue contains functionally different cellular subtypes and different depots have distinct characteristics depending on their anatomical location. Metabolic disturbances are often linked to increased fat deposition in visceral depots, whereas storage in subcutaneous depots is considered less problematic from a metabolic stand-point. Recently, there has been renewed interest in understanding the role of brown fat in humans, in particular in relation to human obesity, as a consequence of its fat-burning properties.101 As DNA methylation is of major importance in defining cellular identity and differentiation of adipocytes, the study of DNA methylation profiles in different adipose tissue depots under different metabolic conditions could provide information about how epigenetic regulation of adipose tissue is involved in the development of obesity and associated comorbidities, and how this could potentially be manipulated. Studies have already shown that DNA methylation in adipose tissue can change after exercise intervention and display differences between high and low responders to weight loss interventions,65,68,69 which indicates that epigenetic regulation in this tissue is likely to be of importance and should be further investigated.
For diagnostic purposes in a clinical setting, particularly when screening young children, epigenetic marks should be detectable in easily accessible samples, such as peripheral blood or possibly buccal cells. Blood and buccal cells originate from different germ layers and the degree of similarity in methylation between both tissues varies across the sites that have been examined so far. An extensive comparison of genome-wide methylation profiles in both tissues has not yet been done and would be of significant interest to the field.
For screening at birth, DNA isolated from the umbilical cord may also be of interest, especially in light of the findings that variation in methylation in this tissue is highly associated with body fat mass in later life.79 Cord blood and newborn blood spotted on filter paper (Guthrie cards) are other relatively accessible materials. Guthrie cards are routinely collected worldwide for screening of genetic diseases in newborns and a number of studies have demonstrated that DNA extracted from these spots can be successfully used for methylome profiling.102,103 In countries where these samples are routinely stored for extended periods of time, they provide a unique resource for researchers to determine whether specific methylation marks detected at older ages were already present in the individuals at birth.
Stability and inter-individual variation in DNA methylation
When considering methylation marks as biomarkers for disease risk, it is essential that they show substantial variation between individuals, and are relatively stable over time. For the design and interpretation of epigenetic studies, it is also important to know the actual scale and extent of inter-individual variation in DNA methylation.
Variability in DNA methylation across healthy individuals is a combination of genetic influence, environmental influence and stochastic events.64,104 A number of studies have investigated the stability and inter-individual variation in DNA methylation, mainly in blood, comparing changes in DNA methylation profiles over short time spans (days) to longer periods (years). The period from birth to childhood is considered a dynamic period for DNA methylation; Wang et al.105 showed that methylation status of ∼5% of the measured 27 000 CpGs substantially changed from birth to 2 years of age and in another study ~8% of the sites on the Illumina arrays showed age-associated methylation in childhood.106 Multiple studies have demonstrated that some methylation marks show considerable variation over time, while others are highly stable.64,96,107,108 It has been suggested that the stable epigenetic marks represent those determined by genetics, whereas the variable marks are more likely to be influenced by the environment.64 A comparison of methylation profiles between populations of different ethnic backgrounds showed that about two-thirds of population-specific CpGs are associated with genetic background,104 whereas one-third may be influenced by other factors.
The stability and inter-individual variation in methylation is also dependent on its genomic location. Low levels of inter-individual variation have been found at commonly unmethylated regions such as CpG islands, and higher variation is measured in regions adjacent to the CpG islands, such as the CpG shores.105,106,109,110 DM regions also show enrichment of single-nucleotide polymorphisms (SNPs), which may help explain why several DMRs were found within or near obesity candidate genes. DNA methylation at sites that associate with SNPs may be a mechanism through which some SNPs affect gene function.111,112 In this regard, the presence of SNPs that are associated with cis-acting regional changes in DNA methylation status are of particular interest.113
Study design and analysis
Robustly designed, well-conducted and adequately powered studies are crucial for the identification of early epigenetic biomarkers (whether causative or not) for obesity risk and in establishing the effects of the in utero environment.
In most current studies, methylation levels and the phenotype are measured at the same time point, which makes it impossible to define whether specific DNA methylation marks are a cause or consequence of obesity. However, although they may not be contributing to the aetiology of obesity, they may still be useful for predicting obesity risk. The association between methylation and obesity may be indirect; there is most likely a third factor (nutritional or environmental) involved that independently affects both methylation and obesity.
Where no longitudinal data are available, statistical approaches have been developed in epidemiological studies to infer causality and these are beginning to be applied to epigenomic data. For example, Liu et al.98 inferred that methylation at specific sites mediated genetic risk for rheumatoid arthritis. The Mendelian randomisation strategy, as proposed by Relton and Davey Smith114 makes use of SNP data as a proxy for DNA methylation and the modifiable exposure of interest, and may be a useful approach to gain a better understanding of the direction of causality. However, this approach also has its limitations, and suitable SNP data are not always available.
Although an effect of early nutritional exposures on later life disease risk in humans has been demonstrated, evidence showing involvement of epigenetic processes in linking early nutritional exposure to later obesity risk in humans is scarce. To successfully address this knowledge gap, large randomised controlled trials and prospective studies will be needed. It may not be ethical to conduct a randomised trial in all situations (for example, restricting breastfeeding or randomly assigning children to be breast or formula fed). However, in the case of nutritional interventions applied during pregnancy, adequately powered randomised controlled trials are the only way to clearly dissect out the impact of the specific nutritional intervention from those of other environmental/demographic variables. In addition, in longitudinal studies, extensive data on parental phenotype and lifestyle, as well as parental blood or buccal cell samples, should ideally be collected before birth, and the phenotype and epigenome of the offspring should be followed prospectively from birth throughout their life course. As DNA methylation profiles at birth have not been affected by postnatal environment or disease state in later life, such studies will provide better support for a link between early epigenetic marks and disease risk than cross-sectional studies. Another advantage of longitudinal studies is that genotype is constant and its direct effects on methylation profiles can be measured and removed. In addition, these studies may provide information on the dynamics of methylation changes during life time, this may help in identifying at what point during development an intervention would be most effective. Animal studies will also remain essential, particularly to give more mechanistic insight and answer outstanding questions related to the underlying biology that are impossible to address in humans.
Human and animal data suggest that not only maternal health and diet may be of importance for the offspring; there are also indications that the paternal contribution to methylation of imprinted genes and phenotype should be taken into account.73
Cohorts, including multiple generations, siblings or ideally monozygotic twins discordant for obesity, would be valuable in unravelling the impact of genetics and environment on the epigenome and phenotype. These types of studies are expensive and time consuming, but some large-scale initiatives such as ARIES, MuTHER, EpiTwin, PETS and KORA are underway.115, 116, 117, 118, 119
EpiSCOPE (Epigenome Study Consortium for Obesity primed in the Perinatal Environment) is a recently initiated multi-institutional Australian research program that aims to add valuable new information to our understanding of the role of epigenetics in the aetiology of obesity. The research will centre around three areas: (1) the epigenome of human adipocytes; EpiSCOPE will develop high-resolution epigenome and transcriptome maps of visceral and subcutaneous adipocytes of healthy, lean individuals for comparison with obese and obese/diabetic subjects. Cross-tissue comparisons will be performed to establish the translatability of epigenetic patterns in adipocytes to blood. (2) Early epigenetic marks for obesity and the effect of in utero fish oil exposure; this study is utilising blood samples collected both at birth and during childhood from children from the large randomised controlled DOMInO trial120 that provides the opportunity to investigate both the relationship of epigenetic marks to childhood metabolic outcomes and the effect of in utero fish oil exposure on the development of childhood obesity and to determine whether epigenetic changes are involved in mediating this. (3) The effect of periconceptional obesity, without the presence of obesity during pregnancy, on the epigenome of the offspring. A sheep model was chosen because of the similarities in the prenatal development of many organ systems, including adipose tissue, in sheep and humans. The model will be used to investigate the molecular mechanisms in specific organs involved in risk of obesity and that contribute to the transmission of a vulnerability to obesity in the offspring of obese mothers.
Using this three-pronged approach, EpiSCOPE aims to improve our understanding of the role of epigenetics in determining long-term metabolic outcomes and to provide an evidence base for the development of novel therapeutic and diagnostic targets to lower the burden and risk of a dominant chronic and to date intractable disease.
Overall, significant progress has been made in the field of epigenetics and obesity, but there is still much to be learned before we fully understand the role of the epigenome in development of complex diseases such as obesity. Epigenetics is a rapidly evolving area of research and the first steps are already being made in identifying potential biomarkers for obesity that could be detected at birth. Eventually this may help in predicting an individual’s obesity risk at a young age, before the phenotype develops, and opens possibilities for introducing targeted strategies to prevent the condition. It is also now clear that several epigenetic marks are modifiable, not only by changing the exposure in utero, but also by lifestyle changes in adult life, which implies that there is the potential for interventions to be introduced in postnatal life to modify or rescue unfavourable epigenomic profiles.
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Funding for EpiSCOPE is received from the Science and Industry Endowment Fund (Australia), grant RP03-064. BSM and JLM are each supported by a Career Development Fellowship from the National Health and Medical Research Council of Australia. We thank Natalie Luscombe-Marsh and Nathan O’Callaghan for their critical reading of the manuscript.
The authors declare no conflict of interest.
M Buckley, CSIRO Computational Informatics, North Ryde, NSW, Australia; SJ Clark, Epigenetics Group, Cancer Research Program, Garvan Institute of Medical Research, Darlinghurst, NSW, Australia; IC McMillen, School of Pharmacy and Medical Sciences, Sansom Institute for Health Research, University of South Australia, Adelaide, SA, Australia; M Noakes, CSIRO Animal, Food and Health Sciences, Adelaide, SA, Australia; K Samaras, Diabetes and Obesity Program, Garvan Institute of Medical Research and Department of Endocrinology, St Vincent's Hospital, Darlinghurst, NSW, Australia; RL Tellam, CSIRO Animal, Food and Health Sciences, St Lucia, QLD, Australia.
Supplementary Information accompanies this paper on International Journal of Obesity website
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van Dijk, S., Molloy, P., Varinli, H. et al. Epigenetics and human obesity. Int J Obes 39, 85–97 (2015). https://doi.org/10.1038/ijo.2014.34
- DNA methylation
- adipose tissue
- developmental programming
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