Epigenetic age prediction

Advanced age is the main common risk factor for cancer, cardiovascular disease and neurodegeneration. Yet, more is known about the molecular basis of any of these groups of diseases than the changes that accompany ageing itself. Progress in molecular ageing research was slow because the tools predicting whether someone aged slowly or fast (biological age) were unreliable. To understand ageing as a risk factor for disease and to develop interventions, the molecular ageing field needed a quantitative measure; a clock for biological age. Over the past decade, a number of age predictors utilising DNA methylation have been developed, referred to as epigenetic clocks. While they appear to estimate biological age, it remains unclear whether the methylation changes used to train the clocks are a reflection of other underlying cellular or molecular processes, or whether methylation itself is involved in the ageing process. The precise aspects of ageing that the epigenetic clocks capture remain hidden and seem to vary between predictors. Nonetheless, the use of epigenetic clocks has opened the door towards studying biological ageing quantitatively, and new clocks and applications, such as forensics, appear frequently. In this review, we will discuss the range of epigenetic clocks available, their strengths and weaknesses, and their applicability to various scientific queries.     

Intersection clock reveals a rejuvenation event during human embryogenesis

Recent research revealed a rejuvenation event during early development of mice. Here, by examining epigenetic age dynamics of human embryogenesis, we tested whether a similar event exists in humans. For this purpose, we developed an epigenetic clock method, the intersection clock, that utilizes bisulfite sequencing in a way that maximizes the use of informative CpG sites with no missing clock CpG sites in test samples and applied it to human embryo development data. We observed no changes in the predicted epigenetic age between cleavage stage and blastocyst stage embryos; however, a significant decrease was observed between blastocysts and cells representing the epiblast. Additionally, by applying the intersection clock to datasets spanning pre and postimplantation, we found no significant change in the epigenetic age during preimplantation stages; however, the epigenetic age of postimplantation samples was lower compared to the preimplantation stages. We further investigated the epigenetic age of primed (representing early postimplantation) and naïve (representing preimplantation) pluripotent stem cells and observed that in all cases the epigenetic age of primed cells was significantly lower than that of naïve cells. Together, our data suggest that human embryos are rejuvenated during early embryogenesis. Hence, the rejuvenation event is conserved between the mouse and human, and it occurs around the gastrulation stage in both species. Beyond this advance, the intersection clock opens the way for other epigenetic age studies based on human bisulfite sequencing datasets as opposed to methylation arrays.     

DNA methylation and healthy human aging

The process of aging results in a host of changes at the cellular and molecular levels, which include senescence, telomere shortening, and changes in gene expression. Epigenetic patterns also change over the lifespan, suggesting that epigenetic changes may constitute an important component of the aging process. The epigenetic mark that has been most highly studied is DNA methylation, the presence of methyl groups at CpG dinucleotides. These dinucleotides are often located near gene promoters and associate with gene expression levels. Early studies indicated that global levels of DNA methylation increase over the first few years of life and then decrease beginning in late adulthood. Recently, with the advent of microarray and next‐generation sequencing technologies, increases in variability of DNA methylation with age have been observed, and a number of site‐specific patterns have been identified. It has also been shown that certain CpG sites are highly associated with age, to the extent that prediction models using a small number of these sites can accurately predict the chronological age of the donor. Together, these observations point to the existence of two phenomena that both contribute to age‐related DNA methylation changes: epigenetic drift and the epigenetic clock. In this review, we focus on healthy human aging throughout the lifetime and discuss the dynamics of DNA methylation as well as how interactions between the genome, environment, and the epigenome influence aging rates. We also discuss the impact of determining ‘epigenetic age’ for human health and outline some important caveats to existing and future studies.     

A clockwork fish: Age prediction using DNA methylation‐based biomarkers in the European seabass

Age‐related changes in DNA methylation do occur. Taking advantage of this, mammalian and avian epigenetic clocks have been constructed to predict age. In fish, studies on age‐related DNA methylation changes are scarce and no epigenetic clocks have been constructed. However, in fisheries and population dynamics studies there is a need for accurate estimation of age, something that is often impossible for some economically important species with the currently available methods. Here, we used the European sea bass, a marine fish the age of which can be determined with accuracy, to construct a piscine epigenetic clock, the first one in a cold‐blooded vertebrate. We used targeted bisulfite sequencing to amplify 48 CpGs from four genes in muscle samples and applied penalized regressions to predict age. We thus developed an age predictor in fish that is highly accurate (0.824) and precise (2.149 years). In juvenile fish, accelerated growth due to elevated temperatures had no effect on age prediction, indicating that the clock is able to predict the chronological age independently of environmentally‐driven perturbations. An epigenetic clock developed using muscle samples accurately predicted age in samples of testis but not ovaries, possibly reflecting the reproductive biology of fish. In conclusion, we report the development of the first piscine epigenetic clock, paving the way for similar studies in other species. Piscine epigenetic clocks should be of great utility for fisheries management and conservation purposes, where age determination is of crucial importance.     

Physical activity is associated with slower epigenetic ageing—Findings from the Rhineland study

Epigenetic ageing, i.e., age-associated changes in DNA methylation patterns, is a sensitive marker of biological ageing, a major determinant of morbidity and functional decline. We examined the association of physical activity with epigenetic ageing and the role of immune function and cardiovascular risk factors in mediating this relation. Moreover, we aimed to identify novel molecular processes underlying the association between physical activity and epigenetic ageing. We analysed cross-sectional data from 3567 eligible participants (mean age: 55.5 years, range: 30–94 years, 54.8% women) of the Rhineland Study, a community-based cohort study in Bonn, Germany. Physical activity components (metabolic equivalent (MET)-Hours, step counts, sedentary, light-intensity and moderate-to-vigorous intensity activities) were recorded with accelerometers. DNA methylation was measured with the Illumina HumanMethylationEPIC BeadChip. Epigenetic age acceleration (Hannum's age, Horvath's age, PhenoAge and GrimAge) was calculated based on published algorithms. The relation between physical activity and epigenetic ageing was examined with multivariable regression, while structural equation modeling was used for mediation analysis. Moreover, we conducted an epigenome-wide association study of physical activity across 850,000 CpG sites. After adjustment for age, sex, season, education, smoking, cell proportions and batch effects, physical activity (step counts, MET-Hours and %time spend in moderate-to-vigorous activities) was non-linearly associated with slower epigenetic ageing, in part through its beneficial effects on immune function and cardiovascular health. Additionally, we identified 12 and 7 CpGs associated with MET-Hours and %time spent in moderate-to-vigorous activities, respectively (p < 1 × 10⁻⁵). Our findings suggest that regular physical activity slows epigenetic ageing by counteracting immunosenescence and lowering cardiovascular risk.     

Development of DNA methylation-based epigenetic age predictors in loblolly pine (Pinus taeda)

Biological ageing is connected to life history variation across ecological scales and informs a basic understanding of age-related declines in organismal function. Altered DNA methylation dynamics are a conserved aspect of biological ageing and have recently been modelled to predict chronological age among vertebrate species. In addition to their utility in estimating individual age, differences between chronological and predicted ages arise due to acceleration or deceleration of epigenetic ageing, and these discrepancies are linked to disease risk and multiple life history traits. Although evidence suggests that patterns of DNA methylation can describe ageing in plants, predictions with epigenetic clocks have yet to be performed. Here, we resolve the DNA methylome across CpG, CHG, and CHH-methylation contexts in the loblolly pine tree (Pinus taeda) and construct epigenetic clocks capable of predicting ages in this species within 6% of its maximum lifespan. Although patterns of CHH-methylation showed little association with age, both CpG and CHG-methylation contexts were strongly associated with ageing, largely becoming hypomethylated with age. Among age-associated loci were those in close proximity to malate dehydrogenase, NADH dehydrogenase, and 18S and 26S ribosomal RNA genes. This study reports one of the first epigenetic clocks in plants and demonstrates the universality of age-associated DNA methylation dynamics which can inform conservation and management practices, as well as our ecological and evolutionary understanding of biological ageing in plants.     

MOABS: model based analysis of bisulfite sequencing data

Background

Human aging is associated with DNA methylation changes at specific sites in the genome. These epigenetic modifications may be used to track donor age for forensic analysis or to estimate biological age.

Results

We perform a comprehensive analysis of methylation profiles to narrow down 102 age-related CpG sites in blood. We demonstrate that most of these age-associated methylation changes are reversed in induced pluripotent stem cells (iPSCs). Methylation levels at three age-related CpGs - located in the genes ITGA2B, ASPA and PDE4C - were subsequently analyzed by bisulfite pyrosequencing of 151 blood samples. This epigenetic aging signature facilitates age predictions with a mean absolute deviation from chronological age of less than 5 years. This precision is higher than age predictions based on telomere length. Variation of age predictions correlates moderately with clinical and lifestyle parameters supporting the notion that age-associated methylation changes are associated more with biological age than with chronological age. Furthermore, patients with acquired aplastic anemia or dyskeratosis congenita - two diseases associated with progressive bone marrow failure and severe telomere attrition - are predicted to be prematurely aged.

Conclusions

Our epigenetic aging signature provides a simple biomarker to estimate the state of aging in blood. Age-associated DNA methylation changes are counteracted in iPSCs. On the other hand, over-estimation of chronological age in bone marrow failure syndromes is indicative for exhaustion of the hematopoietic cell pool. Thus, epigenetic changes upon aging seem to reflect biological aging of blood.     

Epigenome-wide scans identify differentially methylated regions for age and age-related phenotypes in a healthy ageing population

Age-related changes in DNA methylation have been implicated in cellular senescence and longevity, yet the causes and functional consequences of these variants remain unclear. To elucidate the role of age-related epigenetic changes in healthy ageing and potential longevity, we tested for association between whole-blood DNA methylation patterns in 172 female twins aged 32 to 80 with age and age-related phenotypes. Twin-based DNA methylation levels at 26,690 CpG-sites showed evidence for mean genome-wide heritability of 18%, which was supported by the identification of 1,537 CpG-sites with methylation QTLs in cis at FDR 5%. We performed genome-wide analyses to discover differentially methylated regions (DMRs) for sixteen age-related phenotypes (ap-DMRs) and chronological age (a-DMRs). Epigenome-wide association scans (EWAS) identified age-related phenotype DMRs (ap-DMRs) associated with LDL (STAT5A), lung function (WT1), and maternal longevity (ARL4A, TBX20). In contrast, EWAS for chronological age identified hundreds of predominantly hyper-methylated age DMRs (490 a-DMRs at FDR 5%), of which only one (TBX20) was also associated with an age-related phenotype. Therefore, the majority of age-related changes in DNA methylation are not associated with phenotypic measures of healthy ageing in later life. We replicated a large proportion of a-DMRs in a sample of 44 younger adult MZ twins aged 20 to 61, suggesting that a-DMRs may initiate at an earlier age. We next explored potential genetic and environmental mechanisms underlying a-DMRs and ap-DMRs. Genome-wide overlap across cis-meQTLs, genotype-phenotype associations, and EWAS ap-DMRs identified CpG-sites that had cis-meQTLs with evidence for genotype-phenotype association, where the CpG-site was also an ap-DMR for the same phenotype. Monozygotic twin methylation difference analyses identified one potential environmentally-mediated ap-DMR associated with total cholesterol and LDL (CSMD1). Our results suggest that in a small set of genes DNA methylation may be a candidate mechanism of mediating not only environmental, but also genetic effects on age-related phenotypes.     

DNA methylation age of human tissues and cell types

Background

It is not yet known whether DNA methylation levels can be used to accurately predict age across a broad spectrum of human tissues and cell types, nor whether the resulting age prediction is a biologically meaningful measure.

Results

I developed a multi-tissue predictor of age that allows one to estimate the DNA methylation age of most tissues and cell types. The predictor, which is freely available, was developed using 8,000 samples from 82 Illumina DNA methylation array datasets, encompassing 51 healthy tissues and cell types. I found that DNA methylation age has the following properties: first, it is close to zero for embryonic and induced pluripotent stem cells; second, it correlates with cell passage number; third, it gives rise to a highly heritable measure of age acceleration; and, fourth, it is applicable to chimpanzee tissues. Analysis of 6,000 cancer samples from 32 datasets showed that all of the considered 20 cancer types exhibit significant age acceleration, with an average of 36 years. Low age-acceleration of cancer tissue is associated with a high number of somatic mutations and TP53 mutations, while mutations in steroid receptors greatly accelerate DNA methylation age in breast cancer. Finally, I characterize the 353 CpG sites that together form an aging clock in terms of chromatin states and tissue variance.

Conclusions

I propose that DNA methylation age measures the cumulative effect of an epigenetic maintenance system. This novel epigenetic clock can be used to address a host of questions in developmental biology, cancer and aging research.     

Aberrant DNA methylation profiles in the premature aging disorders Hutchinson-Gilford Progeria and Werner syndrome

DNA methylation gradiently changes with age and is likely to be involved in aging-related processes with subsequent phenotype changes and increased susceptibility to certain diseases. The Hutchinson-Gilford Progeria (HGP) and Werner Syndrome (WS) are two premature aging diseases showing features of common natural aging early in life. Mutations in the LMNA and WRN genes were associated to disease onset; however, for a subset of patients the underlying causative mechanisms remain elusive. We aimed to evaluate the role of epigenetic alteration on premature aging diseases by performing comprehensive DNA methylation profiling of HGP and WS patients. We observed profound changes in the DNA methylation landscapes of WRN and LMNA mutant patients, which were narrowed down to a set of aging related genes and processes. Although of low overall variance, non-mutant patients revealed differential DNA methylation at distinct loci. Hence, we propose DNA methylation to have an impact on premature aging diseases.     

New findings showing how DNA methylation influences diseases

In 1975, Holliday and Pugh as well as Riggs independently hypothesized that DNA methylation in eukaryotes could act as a hereditary regulation mechanism that influences gene expression and cell differentiation. Interest in the study of epigenetic processes has been inspired by their reversibility as well as their potentially preventable or treatable consequences. Recently, we have begun to understand that the features of DNA methylation are not the same for all cells.Major differences have been found between differentiated cells and stem cells.Methylation influences various pathologies, and it is very important to improve the understanding of the pathogenic mechanisms. Epigenetic modifications may take place throughout life and have been related to cancer, brain aging, memory disturbances, changes in synaptic plasticity, and neurodegenerative diseases,such as Parkinson's disease and Huntington's disease. DNA methylation also has a very important role in tumor biology. Many oncogenes are activated by mutations in carcinogenesis. However, many genes with tumor-suppressor functions are "silenced" by the methylation of CpG sites in some of their regions.Moreover, the role of epigenetic alterations has been demonstrated in neurological diseases. In neuronal precursors, many genes associated with development and differentiation are silenced by CpG methylation. In addition,recent studies show that DNA methylation can also influence diseases that do not appear to be related to the environment, such as IgA nephropathy, thus affecting,the expression of some genes involved in the T-cell receptor signaling. In conclusion, DNA methylation provides a whole series of fundamental information for the cell to regulate gene expression, including how and when the genes are read, and it does not depend on the DNA sequence.     

DNA Methylation and Epigenotypes

The science of epigenetics is the study of all those mechanisms that control the unfolding of the genetic program for development and determine the phenotypes of differentiated cells. The pattern of gene expression in each of these cells is called the epigenotype. The best known and most thoroughly studied epigenetic mechanism is DNA methylation, which provides a basis both for the switching of gene activities, and the maintenance of stable phenotypes. The human epigenome project is the determination of the pattern of DNA methylation in multiple cell types. Some methylation sites, such as those in repeated genetic elements, are likely to be the same in all cell types, but genes with specialized functions will have distinct patterns of DNA methylation. Another project for the future is the study of the reprogramming of the genome in gametogenesis and early development. Much is already known about the de novo methylation of tumor suppressor genes in cancer cells, but the significance of epigenetic defects during ageing and in some familial diseases remains to be determined.     

Many chronological aging clocks can be found throughout the epigenome: Implications for quantifying biological aging

Epigenetic alterations are a hallmark of aging and age‐related diseases. Computational models using DNA methylation data can create “epigenetic clocks” which are proposed to reflect “biological” aging. Thus, it is important to understand the relationship between predictive clock sites and aging biology. To do this, we examined over 450,000 methylation sites from 9,699 samples. We found ~20% of the measured genomic cytosines can be used to make many different epigenetic clocks whose age prediction performance surpasses that of telomere length. Of these predictive sites, the average methylation change over a lifetime was small (~1.5%) and these sites were under‐represented in canonical regions of epigenetic regulation. There was only a weak association between “accelerated” epigenetic aging and disease. We also compare tissue‐specific and pan‐tissue clock performance. This is critical to applying clocks both to new sample sets in basic research, as well as understanding if clinically available tissues will be feasible samples to evaluate “epigenetic aging” in unavailable tissues (e.g., brain). Despite the reproducible and accurate age predictions from DNA methylation data, these findings suggest they may have limited utility as currently designed in understanding the molecular biology of aging and may not be suitable as surrogate endpoints in studies of anti‐aging interventions. Purpose‐built clocks for specific tissues age ranges or phenotypes may perform better for their specific purpose. However, if purpose‐built clocks are necessary for meaningful predictions, then the utility of clocks and their application in the field needs to be considered in that context.     

Keynote Symposium

DNA Methylation, 5meC, is an epigenetic modification that acts as an important regulator of genomic stability and gene expressivity. Genome-wide changes in methylation have been associated with lineage-specific changes in gene expression profiles during development and in some cell-based pathologies, including oncogenesis. Cost-effective and rapid platforms for the detection of changes in the global levels of methylation are of value for the investigation of the processes that regulate methylation. Flow cytometry allows rapid and quantitative analysis of epitopes within a large number of cells. We have recently optimised the conditions required for valid detection of 5meC by immunofluorescence microscopy. These studies showed that immunological detection of 5meC requires the sequential denaturation of chromatin by a brief period of acidification followed by a partial tryptic digestion step. We have assessed the reliability of flow cytometry for the detection of changes in 5meC when coupled with this optimised epitope retrieval strategy. This study provides support for the use of high throughput screening of 5meC by flow cytometry for the analysis of the epigenetic regulation of important cell transitions.     

Peripheral Nerve Injury Is Associated with Chronic,Reversible Changes in Global DNA Methylation in the Mouse Prefrontal Cortex

Changes in brain structure and cortical function are associated with many chronic pain conditions including low back pain and fibromyalgia. The magnitude of these changes correlates with the duration and/or the intensity of chronic pain. Most studies report changes in common areas involved in pain modulation, including the prefrontal cortex (PFC), and pain-related pathological changes in the PFC can be reversed with effective treatment. While the mechanisms underlying these changes are unknown, they must be dynamically regulated. Epigenetic modulation of gene expression in response to experience and environment is reversible and dynamic. Epigenetic modulation by DNA methylation is associated with abnormal behavior and pathological gene expression in the central nervous system. DNA methylation might also be involved in mediating the pathologies associated with chronic pain in the brain. We therefore tested a) whether alterations in DNA methylation are found in the brain long after chronic neuropathic pain is induced in the periphery using the spared nerve injury modal and b) whether these injury-associated changes are reversible by interventions that reverse the pathologies associated with chronic pain. Six months following peripheral nerve injury, abnormal sensory thresholds and increased anxiety were accompanied by decreased global methylation in the PFC and the amygdala but not in the visual cortex or the thalamus. Environmental enrichment attenuated nerve injury-induced hypersensitivity and reversed the changes in global PFC methylation. Furthermore, global PFC methylation correlated with mechanical and thermal sensitivity in neuropathic mice. In summary, induction of chronic pain by peripheral nerve injury is associated with epigenetic changes in the brain. These changes are detected long after the original injury, at a long distance from the site of injury and are reversible with environmental manipulation. Changes in brain structure and cortical function that are associated with chronic pain conditions may therefore be mediated by epigenetic mechanisms.     

Genome wide analysis reveals association of a FTO gene variant with epigenetic changes

Variants of the FTO gene show strong association with obesity, but the mechanisms behind this association remain unclear. We determined the genome wide DNA methylation profile in blood from 47 female preadolescents. We identified sites associated with the genes KARS, TERF2IP, DEXI, MSI1, STON1 and BCAS3 that had a significant differential methylation level in the carriers of the FTO risk allele (rs9939609). In addition, we identified 20 differentially methylated sites associated with obesity. Our findings suggest that the effect of the FTO obesity risk allele may be mediated through epigenetic changes. Further, these sites might prove to be valuable biomarkers for the understanding of obesity and its comorbidites.