Epigenetic clock

The strong correlation between aging and DNA methylation levels has been known since the late 1960s. New epigenetic clocks based on other markers continue being developed. An aging clock based on nucleosome positioning derived from cell-free DNA was introduced in 2024. In 2025, age-related changes in histone marks have been leveraged to build a new class of epigenetic clocks histone modifications. These new predictors show promise as an alternative to clocks that use cytosine methylation. New age estimation tools are being developed continuously, which also facilitate the prognosis of certain diseases. == Most robustly age associated loci ==

ELOVL2 Elongation Of Very Long Chain Fatty Acids-Like 2 is a gene that codes for a transmembrane protein that plays a role in the synthesis of VLCFAs. The inhibition of its expression has been associated with increased aging of the retina in mice while its upregulation resulted in a slower aging of the retina. Methylation sites in the promoter region of this gene have consistently been part of the top most age correlated in different studies. The methylation in those sites increases with age which reduce its expression. FHL2 Four-and-a-Half LIM domain protein 2 is a gene implicated in signal transduction. Increase in its expression has been associated with obesity. The methylation in its promoter is also strongly correlated with age in numerous studies. In this case the methylation, which increases with age, is associated with an increase in FHL2 expression but surprisingly also with a decreased expression in some tissues. ==Relationship to a cause of biological aging==

It is not yet known what exactly is measured by DNA methylation age. Horvath hypothesized that DNA methylation age measures the cumulative effect of an epigenetic maintenance system but details are unknown. The fact that DNA methylation age of blood predicts all-cause mortality in later life Rather, the epigenetic clock captures an emergent property of the epigenome. On the other hand, the aging clock based on nucleosome positioning is linked to a mechanistic effect of increasing of average genomic distances between nucleosomes with aging. extended this theory, proposing an epigenetic clock theory of aging with the following tenets: • Biological aging results as an unintended consequence of both developmental programs and maintenance program, the molecular footprints of which give rise to DNA methylation age estimators. • The precise mechanisms linking the innate molecular processes (underlying DNAm age) to the decline in tissue function probably relate to both intracellular changes (leading to a loss of cellular identity) and subtle changes in cell composition, for example, fully functioning somatic stem cells. • At the molecular level, DNAm age is a proximal readout of a collection of innate aging processes that conspire with other, independent root causes of aging to the detriment of tissue function. ==Motivation for biological clocks==

In general, biological aging clocks and biomarkers of aging are expected to find many uses in biological research since age is a fundamental characteristic of most organisms. Accurate measures of biological age (biological aging clocks) could be useful for • testing the validity of various theories of biological aging, • diagnosing various age related diseases and for defining cancer subtypes, • predicting/prognosticating the onset of various diseases, • serving as surrogate markers for evaluating therapeutic interventions including rejuvenation approaches, • studying developmental biology and cell differentiation, • forensic applications, for example to estimate the age of a suspect based on blood left on a crime scene. Overall, biological clocks are expected to be useful for studying what causes aging and what can be done against it. However, they can only capture the effects of interventions that affect the rate of future aging, i.e. the slope of the Gompertz curve by which mortality increases with age, and not that of interventions that act at one moment in time, e.g. to lower mortality across all ages, i.e. the intercept of the Gompertz curve. ==Properties of Horvath's clock==

The clock is defined as an age estimation method based on 353 epigenetic markers on the DNA. The 353 markers measure DNA methylation of CpG dinucleotides. Estimated age ("predicted age" in mathematical usage), also referred to as 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 (which are used as human analogs for biological testing purposes). Organismal growth (and concomitant cell division) leads to a high ticking rate of the epigenetic clock that slows down to a constant ticking rate (linear dependence) after adulthood (age 20). Salient features of Horvath's epigenetic clock include its applicability to a broad spectrum of tissues and cell types. Since it allows one to contrast the ages of different tissues from the same subject, it can be used to identify tissues that show evidence of accelerated age due to disease. Genetic estimators in the Horvath clock The Horvath clock, specifically the IEAA variant, is associated with several ageing-related genes:14 • TRIM59: of the tripartite motif family, strongly associated with chronological age and whose expression has been observed in multiple cancers • SMC4: inhibits cellular senescence, an established hallmark of ageing • KPNA4: member of the importin family, nuclear transport receptors. Dysfunction of nuclear transport has been proposed as a marker of ageing • CD46: encodes a regulator of T-cell function and the complement system, a key component of the innate immune system where it promotes inflammation • ATP8B4: encodes for a lipid transporter protein and contains variants that have been reported in association with Alzheimer's disease • CXXC4: encodes Idax, an inhibitor of Wnt signalling Statistical approach The basic approach is to form a weighted average of the 353 clock CpGs, which is then transformed to DNAm age using a calibration function. The calibration function reveals that the epigenetic clock has a high ticking rate until adulthood, after which it slows to a constant ticking rate. Using the training data sets, Horvath used a penalized regression model (Elastic net regularization) to regress a calibrated version of chronological age on 21,369 CpG probes that were present both on the Illumina 450K and 27K platform and had fewer than 10 missing values. DNAm age is defined as estimated ("predicted") age. The elastic net predictor automatically selected 353 CpGs. 193 of the 353 CpGs correlate positively with age while the remaining 160 CpGs correlate negatively with age. R software and a freely available web-based tool can be found at the following webpage. The correlation between chronological age and telomere length is r = −0.51 in women and r = −0.55 in men. The correlation between chronological age and expression levels of p16INK4a in T cells is r = 0.56. ==Applications of epigenetic clocks==

By contrasting DNA methylation age (estimated age) with chronological age, one can define measures of age acceleration. Age acceleration can be defined as the difference between DNA methylation age and chronological age. Alternatively, it can be defined as the residual that results from regressing DNAm age on chronological age. The latter measure is attractive because it does not correlate with chronological age. A positive/negative value of epigenetic age acceleration suggests that the underlying tissue ages faster/slower than expected. Genetic studies of epigenetic age acceleration The broad sense heritability (defined via Falconer's formula) of age acceleration of blood from older subjects is around 40% but it appears to be much higher in newborns. Genetic variants associated with longer leukocyte telomere length in TERT gene paradoxically confer higher epigenetic age acceleration in blood. Cross sectional studies of extrinsic epigenetic aging rates in blood show reduced epigenetic aging correlates with higher education, eating a high plant diet with lean meats, moderate alcohol consumption, and physical activity Breast cancer In a study of three epigenetic clocks and breast cancer risk, DNAm age was found to be accelerated in blood samples of cancer-free women, years before diagnosis. Cancer tissue Cancer tissues show both positive and negative age acceleration effects. For most tumor types, no significant relationship can be observed between age acceleration and tumor morphology (grade/stage). Trisomy 21 (Down syndrome) Down syndrome entails an increased risk of many chronic diseases that are typically associated with older age. The clinical manifestations of accelerated aging suggest that trisomy 21 increases the biological age of tissues, but molecular evidence for this hypothesis has been sparse. According to the epigenetic clock, trisomy 21 significantly increases the age of blood and brain tissue (on average by 6.6 years). Centenarians age slowly The offspring of semi-supercentenarians (subjects who reached an age of 105–109 years) have a lower epigenetic age than age-matched controls (age difference = 5.1 years in blood) and centenarians are younger (8.6 years) than expected based on their chronological age. A progressing infection with Simian Immunodeficiency Virus in rhesus macaque (SIVmac) - a non-human primate model of AIDS - causes epigenetic age acceleration in PBMCs and internal tissues. Parkinson's disease A large-scale study suggests that the blood of Parkinson's disease subjects, in particular, their granulocyte ratio, exhibits (relatively weak) accelerated aging effects. Developmental disorder: syndrome X Children with a very rare disorder known as syndrome X maintain the façade of persistent toddler-like features while aging from birth to adulthood. Since the physical development of these children is dramatically delayed, these children appear to be a toddler or at best a preschooler. According to an epigenetic clock analysis, blood tissue from syndrome X cases is not younger than expected. These results highlight the independence of cellular senescence from epigenetic aging. Consistent with this, telomerase-immortalised cells continued to age (according to the epigenetic clock) without having been treated with any senescence inducers or DNA-damaging agents, re-affirming the independence of the process of epigenetic ageing from telomeres, cellular senescence, and the DNA damage response pathway. Although the uncoupling of senescence from cellular aging appears at first sight to be inconsistent with the fact that senescent cells contribute to the physical manifestation of organism ageing, as demonstrated by Baker et al., where removal of senescent cells slowed down aging. The epigenetic clock analysis of senescence, however, suggests that cellular senescence is a state that cells are forced into as a result of external pressures such as DNA damage, ectopic oncogene expression and exhaustive proliferation of cells to replenish those eliminated by external/environmental factors. The epigenetic clock method applies to all examined racial/ethnic groups in the sense that DNAm age is highly correlated with chronological age. But ethnicity can be associated with epigenetic age acceleration. Progeria Adult progeria also known as Werner syndrome is associated with epigenetic age acceleration in blood. ==Biological mechanism behind the epigenetic clock==

Possible explanation 1: Epigenomic maintenance system Horvath hypothesized that his clock arises from a methylation footprint left by an epigenomic maintenance system. and about 10,000 oxidative damages per day (see DNA damage (naturally occurring)). During repair of double-strand breaks many epigenetic alterations are introduced, and in a percentage of cases epigenetic alterations remain after repair is completed, including increased methylation of CpG island promoters. Similar, but usually transient epigenetic alterations were recently found during repair of oxidative damages caused by H2O2, and it was suggested that occasionally these epigenetic alterations may also remain after repair. These accumulated epigenetic alterations may contribute to the epigenetic clock. Accumulation of epigenetic alterations may parallel the accumulation of un-repaired DNA damages that are proposed to cause aging (see DNA damage theory of aging). In line with stochastic DNA damage accumulation, age-related alterations in DNA methylation have been observed to predominantly undergo stochastic changes as individuals age. This accumulation of stochastic variation has demonstrated sufficient capacity to build aging clocks, further supporting the notion that epigenetic changes may be driven by the gradual accrual of unprogrammed stochastic damage. == References ==