DNA methylation clocks for dogs and humans.

DNA methylation profiles have been used to develop biomarkers of aging known as epigenetic clocks, which predict chronological age with remarkable accuracy and show promise for inferring health status as an indicator of biological age. Epigenetic clocks were first built to monitor human aging, but their underlying principles appear to be evolutionarily conserved, as they have now been successfully developed for many mammalian species. Here, we describe reliable and highly accurate epigenetic clocks shown to apply to 93 domestic dog breeds. The methylation profiles were generated using the mammalian methylation array, which utilizes DNA sequences that are conserved across all mammalian species. Canine epigenetic clocks were constructed to estimate age and also average time to death. We also present two highly accurate human­dog dual species epigenetic clocks (R = 0.97), which may facilitate the ready translation from canine to human use (or vice versa) of antiaging treatments being developed for longevity and preventive medicine. Finally, epigenome-wide association studies here reveal individual methylation sites that may underlie the inverse relationship between breed weight and lifespan. Overall, we describe robust biomarkers to measure aging and, potentially, health status in canines.

Early life adversity predicts an accelerated cellular aging phenotype through early timing of puberty.

BACKGROUND: The current study examined if early adversity was associated with accelerated biological aging, and if effects were mediated by the timing of puberty. METHODS: In early mid-life, 187 Black and 198 White (Mage = 39.4, s.d.age = 1.2) women reported on early abuse and age at first menstruation (menarche). Women provided saliva and blood to assess epigenetic aging, telomere length, and C-reactive protein. Using structural equation modeling, we created a latent variable of biological aging using epigenetic aging, telomere length, and C-reactive protein as indicators, and a latent variable of early abuse using indicators of abuse/threat events before age 13, physical abuse, and sexual abuse. We estimated the indirect effects of early abuse and of race on accelerated aging through age at menarche. Race was used as a proxy for adversity in the form of systemic racism. RESULTS: There was an indirect effect of early adversity on accelerated aging through age at menarche (b = 0.19, 95% CI 0.03-0.44), in that women who experienced more adversity were younger at menarche, which was associated with greater accelerated aging. There was also an indirect effect of race on accelerated aging through age at menarche (b = 0.25, 95% CI 0.04-0.52), in that Black women were younger at menarche, which led to greater accelerated aging. CONCLUSIONS: Early abuse and being Black in the USA may both induce a phenotype of accelerated aging. Early adversity may begin to accelerate aging during childhood, in the form of early pubertal timing.

Epigenetic Age Acceleration Reflects Long-Term Cardiovascular Health.

[Figure: see text].

Epigenetic Age and the Risk of Incident Atrial Fibrillation.

BACKGROUND: The most prominent risk factor for atrial fibrillation (AF) is chronological age; however, underlying mechanisms are unexplained. Algorithms using epigenetic modifications to the human genome effectively predict chronological age. Chronological and epigenetic predicted ages may diverge in a phenomenon referred to as epigenetic age acceleration (EAA), which may reflect accelerated biological aging. We sought to evaluate for associations between epigenetic age measures and incident AF. METHODS: Measures for 4 epigenetic clocks (Horvath, Hannum, DNA methylation [DNAm] PhenoAge, and DNAm GrimAge) and an epigenetic predictor of PAI-1 (plasminogen activator inhibitor-1) levels (ie, DNAm PAI-1) were determined for study participants from 3 population-based cohort studies. Cox models evaluated for associations with incident AF and results were combined via random-effects meta-analyses. Two-sample summary-level Mendelian randomization analyses evaluated for associations between genetic instruments of the EAA measures and AF. RESULTS: Among 5600 participants (mean age, 65.5 years; female, 60.1%; Black, 50.7%), there were 905 incident AF cases during a mean follow-up of 12.9 years. Unadjusted analyses revealed all 4 epigenetic clocks and the DNAm PAI-1 predictor were associated with statistically significant higher hazards of incident AF, though the magnitudes of their point estimates were smaller relative to the associations observed for chronological age. The pooled EAA estimates for each epigenetic measure, with the exception of Horvath EAA, were associated with incident AF in models adjusted for chronological age, race, sex, and smoking variables. After multivariable adjustment for additional known AF risk factors that could also potentially function as mediators, pooled EAA measures for 2 clocks remained statistically significant. Five-year increases in EAA measures for DNAm GrimAge and DNAm PhenoAge were associated with 19% (adjusted hazard ratio [HR], 1.19 [95% CI, 1.09-1.31]; P<0.01) and 15% (adjusted HR, 1.15 [95% CI, 1.05-1.25]; P<0.01) higher hazards of incident AF, respectively. Mendelian randomization analyses for the 5 EAA measures did not reveal statistically significant associations with AF. CONCLUSIONS: Our study identified adjusted associations between EAA measures and incident AF, suggesting that biological aging plays an important role independent of chronological age, though a potential underlying causal relationship remains unclear. These aging processes may be modifiable and not constrained by the immutable factor of time.

A meta-analysis of genome-wide association studies of epigenetic age acceleration.

'Epigenetic age acceleration' is a valuable biomarker of ageing, predictive of morbidity and mortality, but for which the underlying biological mechanisms are not well established. Two commonly used measures, derived from DNA methylation, are Horvath-based (Horvath-EAA) and Hannum-based (Hannum-EAA) epigenetic age acceleration. We conducted genome-wide association studies of Horvath-EAA and Hannum-EAA in 13,493 unrelated individuals of European ancestry, to elucidate genetic determinants of differential epigenetic ageing. We identified ten independent SNPs associated with Horvath-EAA, five of which are novel. We also report 21 Horvath-EAA-associated genes including several involved in metabolism (NHLRC, TPMT) and immune system pathways (TRIM59, EDARADD). GWAS of Hannum-EAA identified one associated variant (rs1005277), and implicated 12 genes including several involved in innate immune system pathways (UBE2D3, MANBA, TRIM46), with metabolic functions (UBE2D3, MANBA), or linked to lifespan regulation (CISD2). Both measures had nominal inverse genetic correlations with father's age at death, a rough proxy for lifespan. Nominally significant genetic correlations between Hannum-EAA and lifestyle factors including smoking behaviours and education support the hypothesis that Hannum-based epigenetic ageing is sensitive to variations in environment, whereas Horvath-EAA is a more stable cellular ageing process. We identified novel SNPs and genes associated with epigenetic age acceleration, and highlighted differences in the genetic architecture of Horvath-based and Hannum-based epigenetic ageing measures. Understanding the biological mechanisms underlying individual differences in the rate of epigenetic ageing could help explain different trajectories of age-related decline.

Menopause accelerates biological aging.

Although epigenetic processes have been linked to aging and disease in other systems, it is not yet known whether they relate to reproductive aging. Recently, we developed a highly accurate epigenetic biomarker of age (known as the "epigenetic clock"), which is based on DNA methylation levels. Here we carry out an epigenetic clock analysis of blood, saliva, and buccal epithelium using data from four large studies: the Women's Health Initiative (n = 1,864); Invecchiare nel Chianti (n = 200); Parkinson's disease, Environment, and Genes (n = 256); and the United Kingdom Medical Research Council National Survey of Health and Development (n = 790). We find that increased epigenetic age acceleration in blood is significantly associated with earlier menopause (P = 0.00091), bilateral oophorectomy (P = 0.0018), and a longer time since menopause (P = 0.017). Conversely, epigenetic age acceleration in buccal epithelium and saliva do not relate to age at menopause; however, a higher epigenetic age in saliva is exhibited in women who undergo bilateral oophorectomy (P = 0.0079), while a lower epigenetic age in buccal epithelium was found for women who underwent menopausal hormone therapy (P = 0.00078). Using genetic data, we find evidence of coheritability between age at menopause and epigenetic age acceleration in blood. Using Mendelian randomization analysis, we find that two SNPs that are highly associated with age at menopause exhibit a significant association with epigenetic age acceleration. Overall, our Mendelian randomization approach and other lines of evidence suggest that menopause accelerates epigenetic aging of blood, but mechanistic studies will be needed to dissect cause-and-effect relationships further.

Genome-wide linkage and association analyses implicate FASN in predisposition to Uterine Leiomyomata.

Uterine leiomyomata (UL), the most prevalent pelvic tumors in women of reproductive age, pose a major public health problem given their high frequency, associated morbidities, and most common indication for hysterectomies. A genetic component to UL predisposition is supported by analyses of ethnic predisposition, twin studies, and familial aggregation. A genome-wide SNP linkage panel was genotyped and analyzed in 261 white UL-affected sister-pair families from the Finding Genes for Fibroids study. Two significant linkage regions were detected in 10p11 (LOD = 4.15) and 3p21 (LOD = 3.73), and five additional linkage regions were identified with LOD scores > 2.00 in 2q37, 5p13, 11p15, 12q14, and 17q25. Genome-wide association studies were performed in two independent cohorts of white women, and a meta-analysis was conducted. One SNP (rs4247357) was identified with a p value (p = 3.05 × 10(-8)) that reached genome-wide significance (odds ratio = 1.299). The candidate SNP is under a linkage peak and in a block of linkage disequilibrium in 17q25.3, which spans fatty acid synthase (FASN), coiled-coil-domain-containing 57 (CCDC57), and solute-carrier family 16, member 3 (SLC16A3). By tissue microarray immunohistochemistry, we found elevated (3-fold) FAS levels in UL-affected tissue compared to matched myometrial tissue. FAS transcripts and/or protein levels are upregulated in various neoplasms and implicated in tumor cell survival. FASN represents the initial UL risk allele identified in white women by a genome-wide, unbiased approach and opens a path to management and potential therapeutic intervention.

Fundamental equations linking methylation dynamics to maximum lifespan in mammals.

We describe a framework that addresses concern that the rate of change in any aging biomarker displays a trivial inverse relation with maximum lifespan. We apply this framework to methylation data from the Mammalian Methylation Consortium. We study the relationship of lifespan with the average rate of change in methylation (AROCM) from two datasets: one with 90 dog breeds and the other with 125 mammalian species. After examining 54 chromatin states, we conclude three key findings: First, a reciprocal relationship exists between the AROCM in bivalent promoter regions and maximum mammalian lifespan: AROCM ∝ 1/MaxLifespan. Second, the correlation between average methylation and age bears no relation to maximum lifespan, Cor(Methyl,Age) ⊥ MaxLifespan. Third, the rate of methylation change in young animals is related to that in old animals: Young animals' AROCM ∝ Old AROCM. These findings critically hinge on the chromatin context, as different results emerge in other chromatin contexts.

DNA methylation clocks for clawed frogs reveal evolutionary conservation of epigenetic aging.

To address how conserved DNA methylation-based epigenetic aging is in diverse branches of the tree of life, we generated DNA methylation data from African clawed frogs (Xenopus laevis) and Western clawed frogs (Xenopus tropicalis) and built multiple epigenetic clocks. Dual species clocks were developed that apply to both humans and frogs (human-clawed frog clocks), supporting that epigenetic aging processes are evolutionary conserved outside mammals. Highly conserved positively age-related CpGs are located in neural-developmental genes such as uncx, tfap2d as well as nr4a2 implicated in age-associated disease. We conclude that signatures of epigenetic aging are evolutionary conserved between frogs and mammals and that the associated genes relate to neural processes, altogether opening opportunities to employ Xenopus as a model organism to study aging.

Higher testosterone and testosterone/estradiol ratio in men are associated with decreased Pheno-/GrimAge and DNA-methylation based PAI1.

Sex hormones are hypothesized to drive sex-specific health disparities. Here, we study the association between sex steroid hormones and DNA methylation-based (DNAm) biomarkers of age and mortality risk including Pheno Age Acceleration (AA), Grim AA, and DNAm-based estimators of Plasminogen Activator Inhibitor 1 (PAI1), and leptin concentrations. We pooled data from three population-based cohorts, the Framingham Heart Study Offspring Cohort, the Baltimore Longitudinal Study of Aging, and the InCHIANTI Study, including 1,062 postmenopausal women without hormone therapy and 1,612 men of European descent. Sex-stratified analyses using a linear mixed regression were performed, with a Benjamini-Hochberg (BH) adjustment for multiple testing. Sex Hormone Binding Globulin (SHBG) was associated with a decrease in DNAm PAI1 among men (per 1 standard deviation (SD): -478 pg/mL; 95%CI: -614 to -343; P:1e-11; BH-P: 1e-10), and women (-434 pg/mL; 95%CI: -589 to -279; P:1e-7; BH-P:2e-6). The testosterone/estradiol (TE) ratio was associated with a decrease in Pheno AA (-0.41 years; 95%CI: -0.70 to -0.12; P:0.01; BH-P: 0.04), and DNAm PAI1 (-351 pg/mL; 95%CI: -486 to -217; P:4e-7; BH-P:3e-6) among men. In men, testosterone was associated with a decrease in DNAm PAI1 (-481 pg/mL; 95%CI: -613 to -349; P:2e-12; BH-P:6e-11). SHBG was associated with lower DNAm PAI1 among men and women. Higher testosterone and testosterone/estradiol ratio were associated with lower DNAm PAI and a younger epigenetic age in men. A decrease in DNAm PAI1 is associated with lower mortality and morbidity risk indicating a potential protective effect of testosterone on lifespan and conceivably cardiovascular health via DNAm PAI1.

Causality-enriched epigenetic age uncouples damage and adaptation.

Machine learning models based on DNA methylation data can predict biological age but often lack causal insights. By harnessing large-scale genetic data through epigenome-wide Mendelian randomization, we identified CpG sites potentially causal for aging-related traits. Neither the existing epigenetic clocks nor age-related differential DNA methylation are enriched in these sites. These CpGs include sites that contribute to aging and protect against it, yet their combined contribution negatively affects age-related traits. We established a new framework to introduce causal information into epigenetic clocks, resulting in DamAge and AdaptAge-clocks that track detrimental and adaptive methylation changes, respectively. DamAge correlates with adverse outcomes, including mortality, while AdaptAge is associated with beneficial adaptations. These causality-enriched clocks exhibit sensitivity to short-term interventions. Our findings provide a detailed landscape of CpG sites with putative causal links to lifespan and healthspan, facilitating the development of aging biomarkers, assessing interventions, and studying reversibility of age-associated changes.

Effects of highly active antiretroviral therapy initiation on epigenomic DNA methylation in persons living with HIV.

Introduction: Highly active antiretroviral therapy (HAART) helps improve some measures of accelerated epigenetic aging in persons living with HIV (PLWH), but its overall impact on the epigenome is not fully understood. Methods: In this study, we analyzed the DNA methylation profiles of PLWH (n = 187) shortly before and approximately 2-3 years after they started HAART, as well as matched seronegative (SN) controls (n = 187), taken at two time intervals. Our aim was to identify specific CpGs and biologic pathways associated with HIV infection and initiation of HAART. Additionally, we attempted to identify epigenetic changes associated with HAART initiation that were independent of HIV-associated changes, using matched HIV seronegative (SN) controls (matched on age, hepatitis C status, and interval between visits) to identify CpGs that did not differ between PLWH and SN pre-HAART but were significantly associated with HAART initiation while being unrelated to HIV viral load. Epigenome-wide association studies (EWAS) on >850,000 CpG sites were performed using pre- and post-HAART samples from PLWH. The results were then annotated using the Genomic Regions Enrichment of Annotations Tool (GREAT). Results: When only pre- and post-HAART visits in PLWH were compared, gene ontologies related to immune function and diseases related to immune function were significant, though with less significance for PLWH with detectable HIV viral loads (>50 copies/mL) at the post-HAART visit. To specifically elucidate the effects of HAART separately from HIV-induced methylation changes, we performed EWAS of HAART while also controlling for HIV viral load, and found gene ontologies associated with transplant rejection, transplant-related diseases, and other immunologic signatures. Additionally, we performed a more focused analysis that examined CpGs reaching genome-wide significance (p < 1 × 10-7) from the viral load-controlled EWAS that did not differ between all PLWH and matched SN controls pre-HAART. These CpGs were found to be near genes that play a role in retroviral drug metabolism, diffuse large B cell lymphoma proliferation, and gastric cancer metastasis. Discussion: Overall, this study provides insight into potential biological functions associated with DNA methylation changes induced by HAART initiation in persons living with HIV.

Cognitive rejuvenation in old rats by hippocampal OSKM gene therapy.

Several studies have indicated that interrupted epigenetic reprogramming using Yamanaka transcription factors (OSKM) can rejuvenate cells from old laboratory animals and humans. However, the potential of OSKM-induced rejuvenation in brain tissue has been less explored. Here, we aimed to restore cognitive performance in 25.3-month-old female Sprague-Dawley rats using OSKM gene therapy for 39 days. Their progress was then compared with the cognitive performance of untreated 3.5-month-old rats as well as old control rats treated with a placebo adenovector. The Barnes maze test, used to assess cognitive performance, demonstrated enhanced cognitive abilities in old rats treated with OSKM compared to old control animals. In the treated old rats, there was a noticeable trend towards improved spatial memory relative to the old controls. Further, OSKM gene expression did not lead to any pathological alterations within the 39 days. Analysis of DNA methylation following OSKM treatment yielded three insights. First, epigenetic clocks for rats suggested a marginally significant epigenetic rejuvenation. Second, chromatin state analysis revealed that OSKM treatment rejuvenated the methylome of the hippocampus. Third, an epigenome-wide association analysis indicated that OSKM expression in the hippocampus of old rats partially reversed the age-related increase in methylation. In summary, the administration of Yamanaka genes via viral vectors rejuvenates the functional capabilities and the epigenetic landscape of the rat hippocampus.

Epigenetic predictors of species maximum life span and other life-history traits in mammals.

By analyzing 15,000 samples from 348 mammalian species, we derive DNA methylation (DNAm) predictors of maximum life span (R = 0.89), gestation time (R = 0.96), and age at sexual maturity (R = 0.85). Our maximum life-span predictor indicates a potential innate longevity advantage for females over males in 17 mammalian species including humans. The DNAm maximum life-span predictions are not affected by caloric restriction or partial reprogramming. Genetic disruptions in the somatotropic axis such as growth hormone receptors have an impact on DNAm maximum life span only in select tissues. Cancer mortality rates show no correlation with our epigenetic estimates of life-history traits. The DNAm maximum life-span predictor does not detect variation in life span between individuals of the same species, such as between the breeds of dogs. Maximum life span is determined in part by an epigenetic signature that is an intrinsic species property and is distinct from the signatures that relate to individual mortality risk.

Higher testosterone and testosterone/estradiol ratio in men are associated with better epigenetic estimators of mortality risk.

Introduction: Sex hormones are hypothesized to drive sex-specific health disparities. Here, we study the association between sex steroid hormones and DNA methylation-based (DNAm) biomarkers of age and mortality risk including Pheno Age Acceleration (AA), Grim AA, and DNAm-based estimators of Plasminogen Activator Inhibitor 1 (PAI1), and leptin concentrations. Methods: We pooled data from three population-based cohorts, the Framingham Heart Study Offspring Cohort (FHS), the Baltimore Longitudinal Study of Aging (BLSA), and the InCHIANTI Study, including 1,062 postmenopausal women without hormone therapy and 1,612 men of European descent. Sex hormone concentrations were standardized with mean 0 and standard deviation of 1, for each study and sex separately. Sex-stratified analyses using a linear mixed regression were performed, with a Benjamini-Hochberg (BH) adjustment for multiple testing. Sensitivity analysis was performed excluding the previously used training-set for the development of Pheno and Grim age. Results: Sex Hormone Binding Globulin (SHBG) is associated with a decrease in DNAm PAI1 among men (per 1 standard deviation (SD): -478 pg/mL; 95%CI: -614 to -343; P:1e-11; BH-P: 1e-10), and women (-434 pg/mL; 95%CI: -589 to -279; P:1e-7; BH-P:2e-6). The testosterone/estradiol (TE) ratio was associated with a decrease in Pheno AA (-0.41 years; 95%CI: -0.70 to -0.12; P:0.01; BH-P: 0.04), and DNAm PAI1 (-351 pg/mL; 95%CI: -486 to -217; P:4e-7; BH-P:3e-6) among men. In men, 1 SD increase in total testosterone was associated with a decrease in DNAm PAI1 (-481 pg/mL; 95%CI: -613 to -349; P:2e-12; BH-P:6e-11). Conclusion: SHBG was associated with lower DNAm PAI1 among men and women. Higher testosterone and testosterone/estradiol ratio were associated with lower DNAm PAI and a younger epigenetic age in men. A decrease in DNAm PAI1 is associated with lower mortality and morbidity risk indicating a potential protective effect of testosterone on lifespan and conceivably cardiovascular health via DNAm PAI1.

DNA methylation-based biomarkers for ageing long-lived cetaceans.

Epigenetic approaches for estimating the age of living organisms are revolutionizing studies of long-lived species. Molecular biomarkers that allow age estimates from small tissue biopsies promise to enhance studies of long-lived whales, addressing a fundamental and challenging parameter in wildlife management. DNA methylation (DNAm) can affect gene expression, and strong correlations between DNAm patterns and age have been documented in humans and nonhuman vertebrates and used to construct "epigenetic clocks". We present several epigenetic clocks for skin samples from two of the longest-lived cetaceans, killer whales and bowhead whales. Applying the mammalian methylation array to genomic DNA from skin samples we validate four different clocks with median errors of 2.3-3.7 years. These epigenetic clocks demonstrate the validity of using cytosine methylation data to estimate the age of long-lived cetaceans and have broad applications supporting the conservation and management of long-lived cetaceans using genomic DNA from remote tissue biopsies.

Multi-omic rejuvenation and life span extension on exposure to youthful circulation.

Heterochronic parabiosis (HPB) is known for its functional rejuvenation effects across several mouse tissues. However, its impact on biological age and long-term health is unknown. Here we performed extended (3-month) HPB, followed by a 2-month detachment period of anastomosed pairs. Old detached mice exhibited improved physiological parameters and lived longer than control isochronic mice. HPB drastically reduced the epigenetic age of blood and liver based on several clock models using two independent platforms. Remarkably, this rejuvenation effect persisted even after 2 months of detachment. Transcriptomic and epigenomic profiles of anastomosed mice showed an intermediate phenotype between old and young, suggesting a global multi-omic rejuvenation effect. In addition, old HPB mice showed gene expression changes opposite to aging but akin to several life span-extending interventions. Altogether, we reveal that long-term HPB results in lasting epigenetic and transcriptome remodeling, culminating in the extension of life span and health span.

Bloom syndrome patients and mice display accelerated epigenetic aging.

Bloom syndrome (BSyn) is an autosomal recessive disorder caused by variants in the BLM gene, which is involved in genome stability. Patients with BSyn present with poor growth, sun sensitivity, mild immunodeficiency, diabetes, and increased risk of cancer, most commonly leukemias. Interestingly, patients with BSyn do not have other signs of premature aging such as early, progressive hair loss and cataracts. We set out to determine epigenetic age in BSyn, which can be a better predictor of health and disease over chronological age. Our results show for the first time that patients with BSyn have evidence of accelerated epigenetic aging across several measures in blood lymphocytes, as compared to carriers. Additionally, homozygous Blm mice exhibit accelerated methylation age in multiple tissues, including brain, blood, kidney, heart, and skin, according to the brain methylation clock. Overall, we find that Bloom syndrome is associated with accelerated epigenetic aging effects in multiple tissues and more generally a strong effect on CpG methylation levels.

Alcohol consumption and epigenetic age acceleration in young adults.

Alcohol is a widely consumed substance in the United States, however its effect on aging remains understudied. In this study of young adults, we examined whether cumulative alcohol consumption, i.e., alcohol years of beer, liquor, wine, and total alcohol, and recent binge drinking, were associated with four measures of age-related epigenetic changes via blood DNA methylation. A random subset of study participants in the Coronary Artery Risk Development in Young Adults Study underwent DNA methylation profiling using the Illumina MethylationEPIC Beadchip. Participants with alcohol consumption and methylation data at examination years 15 (n = 1,030) and 20 (n = 945) were included. Liquor and total alcohol consumption were associated with a 0.31-year (P = 0.002) and a 0.12-year (P = 0.013) greater GrimAge acceleration (GAA) per additional five alcohol years, while beer and wine consumption observed marginal (P = 0.075) and no associations (P = 0.359) with GAA, respectively. Any recent binge drinking and the number of days of binge drinking were associated with a 1.38-year (P < 0.001) and a 0.15-year (P < 0.001) higher GAA, respectively. We observed statistical interactions between cumulative beer (P < 0.001) and total alcohol (P = 0.004) consumption with chronological age, with younger participants exhibiting a higher average in GAA compared to older participants. No associations were observed with the other measures of epigenetic aging. These results suggest cumulative liquor and total alcohol consumption and recent binge drinking may alter age-related epigenetic changes as captured by GAA. With the increasing aging population and widespread consumption of alcohol, these findings may have potential implications for lifestyle modification to promote healthy aging.

Refining epigenetic prediction of chronological and biological age.

BACKGROUND: Epigenetic clocks can track both chronological age (cAge) and biological age (bAge). The latter is typically defined by physiological biomarkers and risk of adverse health outcomes, including all-cause mortality. As cohort sample sizes increase, estimates of cAge and bAge become more precise. Here, we aim to develop accurate epigenetic predictors of cAge and bAge, whilst improving our understanding of their epigenomic architecture. METHODS: First, we perform large-scale (N = 18,413) epigenome-wide association studies (EWAS) of chronological age and all-cause mortality. Next, to create a cAge predictor, we use methylation data from 24,674 participants from the Generation Scotland study, the Lothian Birth Cohorts (LBC) of 1921 and 1936, and 8 other cohorts with publicly available data. In addition, we train a predictor of time to all-cause mortality as a proxy for bAge using the Generation Scotland cohort (1214 observed deaths). For this purpose, we use epigenetic surrogates (EpiScores) for 109 plasma proteins and the 8 component parts of GrimAge, one of the current best epigenetic predictors of survival. We test this bAge predictor in four external cohorts (LBC1921, LBC1936, the Framingham Heart Study and the Women's Health Initiative study). RESULTS: Through the inclusion of linear and non-linear age-CpG associations from the EWAS, feature pre-selection in advance of elastic net regression, and a leave-one-cohort-out (LOCO) cross-validation framework, we obtain cAge prediction with a median absolute error equal to 2.3 years. Our bAge predictor was found to slightly outperform GrimAge in terms of the strength of its association to survival (HRGrimAge = 1.47 [1.40, 1.54] with p = 1.08 × 10-52, and HRbAge = 1.52 [1.44, 1.59] with p = 2.20 × 10-60). Finally, we introduce MethylBrowsR, an online tool to visualise epigenome-wide CpG-age associations. CONCLUSIONS: The integration of multiple large datasets, EpiScores, non-linear DNAm effects, and new approaches to feature selection has facilitated improvements to the blood-based epigenetic prediction of biological and chronological age.