Epigenetic clocks are predictors of biological age based on alterations in an individual's DNA methylation profile. Methylations – biochemical processes that modify the activity of a DNA segment without changing its sequence – occur naturally and regulate gene expression to control normal growth and development. With age, the methylation state of various genes may change. These changes are quantifiable and serve as a means to gauge epigenetic age, which often differs from chronological age.
The term "epigenetic clock" is also a collective designation referring to the natural biological mechanisms that drive DNA methylation. These innate mechanisms, which play critical roles in an organism's development and maintenance, leave a molecular "footprint" that reflects the biological life history of the organism. This overview focuses primarily on predictive epigenetic clocks, with a brief mention of the innate.
Epigenetics is a biological mechanism that regulates gene expression (how and when certain genes are turned on or off). Diet, lifestyle, and environmental exposures can drive epigenetic changes throughout an individual's lifespan to influence health and disease. For example, epigenetic processes are dysregulated in diseases such as cancer and Alzheimer's disease. Scientific evidence suggests that epigenetic changes can be passed from generation to generation.
Three biochemical processes are thought to drive epigenetic change: DNA methylation, histone modification, and non-coding RNA-associated gene silencing. DNA methylation has relevance for predicting biological age via epigenetic clocks.
DNA methylation occurs naturally when a methyl group – a chemical structure containing three hydrogen atoms and one carbon atom – attaches to one of DNA's four nucleotide bases (adenine [A], cytosine [C], guanine [G], or thymine [T]). A class of enzymes called methyltransferases facilitates the process of DNA methylation, while another class of enzymes, the ten-eleven transferases, or TET enzymes, reverses it. Methylation creates a biological record of the varied molecular processes that participate in an individual's development, maintenance, and decline. A vast array of factors promote methylation, including dietary intake, exercise, stress, smoking, and even social factors, such as maternal-infant interaction, among others.
The most common DNA methylation process involves the addition of a methyl group to one of the carbon atoms in the cytosine base, forming 5-methylcytosine. This addition alters the overall geometry of the DNA strand, ultimately influencing gene expression. The majority of 5-methylcytosine is found on areas of the DNA known as CpG islands – short stretches of DNA where the frequency of the cytosine-guanine (CG) sequence is higher than other regions. (The "p" in CpG simply reflects the presence of a phosphate group between the two nucleotides.) Methylation of a CpG island in the promoter region of a gene turns off, or "silences," the gene's expression. DNA methylation may promote age-related diseases such as cancer.
Methylation is a dynamic process that is not only reversible, but appears to be under circadian control in some tissues. It increases with age, and the rate of change over the lifespan varies among CpGs, averaging 3.2 percent organism-wide and ranging from 7 to 91 percent for certain individual genes.
A class of enzymes called methyltransferases facilitates the process of DNA methylation, while another class of enzymes, the ten-eleven transferases, or TET enzymes, reverses it. Evidence suggests both genetic and lifestyle factors influence the regulation of methyltransferases and TET enzymes, including transposons, genes, inflammation, cellular and environmental factors (such as diet), and others.   
Aging comprises the collective physiological, functional, and mental changes that accrue in a biological organism over time. It is the primary risk factor for many chronic diseases in humans, including cancer, Alzheimer's disease, and cardiovascular disease. Individuals age at different rates, however, a phenomenon observed across sexes, ethnic groups, and races. For example, women typically outlive men, despite having higher disease incidence. Similarly, Hispanics living in the United States exhibit what is commonly referred to as the "Hispanic paradox," a phenomenon in which their life expectancy is similar to whites, despite having lower income and education levels and reduced access to health care. Epigenetic clocks may provide a means of explaining these and other differential aging rates.
Aging in humans is measured according to three different standards: chronological age, epigenetic age, and biological (sometimes referred to as phenotypic) age.
An individual's chronological age simply reflects the number of months or years an individual has been alive. Although certain developmental milestones and characteristics correlate with chronological age, it is an unreliable measure of the aging process.
Epigenetic age is based on an individual's DNA methylation profile. An individual's epigenetic age strongly correlates with their chronological age. However, some exceptions exist. For example, the epigenetic ages of semi-supercentenarians (people who live to be 105 to 109 years old) are markedly younger than their chronological ages.
An individual's biological age, sometimes referred to as phenotypic age, provides a measure of their physiological and functional state. It is a calculation of an individual's risk of disease and death compared to individuals of the same chronological age, based on biochemical measures of inflammation and metabolic and immune function.
Age acceleration is a phenomenon that occurs when an individual's epigenetic age exceeds their chronological age and may be the result of either intrinsic or extrinsic factors. Intrinsic factors are largely driven by internal physiological factors such as normal metabolism and genetics. Extrinsic factors are those associated with lifestyle and environmental exposures, such as diet, tobacco use, ultraviolet radiation, and mental illness. Markers of accelerated extrinsic aging have been observed in the blood of suicide completers, for example.
Several variations of DNA methylation-based epigenetic clocks have been identified. They are generally categorized according to the type and number of tissues used to formulate the calculation, as well as the type of age measured (e.g., epigenetic versus phenotypic). Each is named for the scientist who created the clock or for the clock's output. The most accurate and robust of these clocks are described here.
Created by Dr. Gregory Hannum, the Hannum clock is a single-tissue calculator of epigenetic age based on 71 CpGs present in DNA from human blood. Although highly accurate in regard to lifespan prediction, the Hannum clock is based on adult blood biomarkers, so it is inappropriate for use in children or against other tissue types.
One study used the Hannum clock to investigate associations between DNAm age (where "m" represents methylation) and chronological age in African Americans and whites and to identify links between race, poverty, sex, and epigenetic age acceleration. The study investigators analyzed methylation profiles present in DNA samples from 487 middle-aged African American and white men and women. The investigators found that African American men and women had more age-associated DNAm changes in genes implicated in age-related diseases and cellular pathways involved in growth and development than white men and women. Interestingly, African Americans also demonstrated slower extrinsic aging than whites. These findings may have relevance for age- and race-related health disparities.
Use of the Hannum clock also demonstrated that exposure to abuse, financial hardship, or neighborhood disadvantage that occurred around the age of 7.5 years alters methylation patterns, which may influence normal patterns of cellular aging.
"the Hannum clock demonstrated that exposure to abuse, financial hardship, or neighborhood disadvantage that occurred around the age of 7.5 years alters DNA methylation patterns, which may influence normal patterns of cellular aging" Click To Tweet
Another study investigated the effects of cigarette smoking on aging using the Hannum clock based on methylation data drawn from two large cohorts of adults living in the United States. The findings indicated that not only was smoking associated with accelerated biological aging, but even low levels of exposure elicited strong effects.
Multiple Horvath clocks exist, including the original Horvath clock, the GrimAge clock, and the DNAm PhenoAge clock.
The original Horvath epigenetic clock, created by Dr. Steven Horvath, predicts age based on methylation patterns and rates on 353 CpG islands in the DNA of 51 different tissue and cell types. This multi-tissue clock calculates epigenetic age by coupling a tissue's DNA methylation status on specific CpGs with a mathematical algorithm to provide an age estimate, referred to as DNAm age. The Horvath clock can identify the epigenetic age of a donor with 96 percent accuracy within approximately four years of actual age. Its accuracy extends across multiple tissue types and ages, including children.
The Horvath clock was used in a study that investigated links between lifestyle and aging by assessing measures of intrinsic versus extrinsic measures of epigenetic age acceleration. The study measured epigenetic aging in blood cell components, including plasmablasts, CD8+ T cells, CD4+ T cells, natural killer cells, monocytes, and granulocytes from more than 4,500 adults living in the United States and Italy. In blood cells, intrinsic epigenetic age acceleration is independent of age-related changes in blood cell composition such as those related to immunosenescence. Extrinsic epigenetic aging incorporates factors such as immune cell aging.
Whereas factors associated with slowed intrinsic aging included higher poultry intake, factors associated with slowed extrinsic aging included higher fish intake, higher carotenoid levels (a marker of fruit and vegetable intake), higher education, moderate alcohol consumption, and higher physical activity. Factors associated with accelerated intrinsic aging included higher BMI, but metabolic syndrome (which is associated with higher BMI) was linked with both intrinsic and extrinsic accelerated epigenetic aging.
In a study in which the Horvath clock was used to assess epigenetic age acceleration in people with nonalcoholic steatohepatitis, or NASH, a type of fatty liver disease characterized by liver inflammation and damage, the clock accurately predicted the chronological age of all the study subjects. Furthermore, the investigators found that NASH was associated with accelerated aging and closely correlated with hepatic collagen content, a measure of liver fibrosis.
Preliminary findings from an interventional trial indicate that vitamin D status influences epigenetic age. The study involved 51 overweight or obese African Americans between the ages of 13 and 45 years who had suboptimal vitamin D levels. Participants took a supplement providing approximately 600 IU, 2000 IU, or 4000 IU of vitamin D or a placebo daily for 16 weeks. Whereas taking 4000 IU of vitamin D per day was associated with a decrease in Horvath epigenetic aging of 1.85 years, taking 2000 IU per day was associated with a decrease in Hannum epigenetic aging of 1.90 years. These findings suggest that other diet and lifestyle factors may be able to slow epigenetic aging, as well.
"Whereas taking 4000 IU of vitamin D per day was associated with a decrease in Horvath epigenetic aging of 1.85 years, taking 2000 IU per day was associated with a decrease in Hannum epigenetic aging of 1.90 years" Click To Tweet
The recently-identified GrimAge (named creatively for the Grim Reaper) predicts lifespan and healthspan in units of years and tests whether potential lifestyle interventions may slow or reverse biological aging. A composite measure based on seven DNAm surrogates and a DNAm-based estimator of smoking pack-years, it is 18 percent more accurate than calendar age and 14 percent more accurate than other epigenetic clocks. Using data from more than 2,300 adults, GrimAge accurately predicted time-to-death, time-to-coronary heart disease, time-to-cancer, and age-at-menopause and closely aligned with computed tomography data for fatty liver disease and excess visceral fat. Information on how to calculate DNAm age using Horvath's method is freely available.
Dr. Morgan Levine and Dr. Steve Horvath created a multi-tissue clock that calculates an individual's phenotypic age, called DNAm PhenoAge. This clock, sometimes referred to as the "Levine clock," is distinct from other clocks in that it predicts time to death based on DNA methylation at 513 CpG islands as well as biochemical markers of age-related disease, including albumin, creatinine, glucose, C-reactive protein, alkaline phosphatase, and several blood components. Several physiological responses are associated with accelerated phenotypic aging, including increased activation of proinflammatory pathways and decreased DNA repair activities.
The DNAm PhenoAge predicts mortality risk among people of the same chronological age. It was used in a study to estimate the 10-year mortality risk (converted into units of years) in a cohort of people living in the United States (NHANES data), based on nine clinical biomarkers of aging that are highly predictive of cardiovascular disease and coronary heart disease. The findings demonstrated that each one-year increase in DNAm PhenoAge was associated with a 9 percent increase in all-cause mortality, a 10 percent increase in CVD-related mortality, a 7 percent increase in cancer-related mortality, a 20 percent increase in diabetes-related mortality, and a 9 percent increase in chronic lower respiratory disease mortality, even after adjusting for chronological age.
Further analysis using data from five large, independent cohorts revealed that a one-year increase in DNAm PhenoAge was associated with a 4.5 percent increase in all-cause mortality risk (independent of chronological age). Accelerated PhenoAge was linked with higher inflammatory biomarkers (such as C-reactive protein), higher insulin, higher glucose, higher triglycerides, and lower HDL. The DNA methylation PhenoAge acceleration was also linked to known age-related changes in blood cells, such as decreased populations of CD4 and CD8 T-cells and increased granulocytes.
The findings also revealed that people who aged the top 5 percent of the fastest agers in the cohorts had a 62 percent higher risk of premature death than people with an average PhenoAge and a 158 percent higher risk of premature death than the slowest agers. The DNAm PhenoAge clock predicted that the median life expectancy at age 50 was approximately 81 years for the fastest agers, 83.5 years for average agers, and 86 years for the slowest agers.
The DNAm PhenoAge epigenetic clock was also employed as a means to assess breast cancer risk. The study gauged the DNA methylation age of more than 1,500 women with breast cancer and determined that for every 5-year acceleration in a woman's epigenetic aging, her risk of developing breast cancer increased by 15 percent.
One study used the DNAm PhenoAge epigenetic clock to investigate the effects of heavy, chronic alcohol intake on epigenetic age acceleration using clinical biomarkers such as liver function enzymes. The study, which estimated DNA methylation age in 331 people with alcohol use disorder, found that the disorder accelerated aging by an average of 2.2 years. A genome-wide meta-analysis of accelerated epigenetic aging among the study subjects indicated that the presence of a single nucleotide polymorphism in the APOL2 gene (a member of the apolipoprotein-L family) further accelerated aging.
Exposure to air pollutants is associated with poor health outcomes and increased risk of disease. A study using the Levine clock to gauge epigenetic age of more than 2,700 white women living in the United States who were exposed to particulate air pollutants found that the women's epigenetic aging was accelerated by as much as six years.
"A study using the Levine clock to gauge epigenetic age of more than 2,700 white women living in the United States who were exposed to particulate air pollutants found that the women's epigenetic aging was accelerated by as much as six years" Click To Tweet
In general, the native epigenetic clock's ticking rate across multiple types of tissue from a single individual is fairly consistent. However, the cerebellum tends to age more slowly, while female breast tissue tends to age more quickly. Interestingly, in vitro evidence suggests that the epigenetic age of adult cells can be reset. Dr. Shinya Yamanaka discovered a group of proteins that can reprogram differentiated (mature) cells into pluripotent stem cells. These proteins, now called Yamanaka factors, are highly expressed in embryonic stem cells in mice and humans. Their short-term expression can ameliorate cellular and physiological hallmarks of aging and prolong lifespan partly by resetting the innate epigenetic clock.
The role of Yamanaka factors was demonstrated in vivo as well, in a mouse model of premature aging in which short-term induction of Yamanaka factors improved markers of aging, including those associated with tumor suppression, mitochondrial dysfunction, and oxidative stress. The same study showed that in older, normal mice, short-term induction of Yamanaka factors mitigated the deleterious effects of pancreatic or muscle injury, which could have implications for age-related metabolic dysfunction or strength losses, respectively.
Some individuals may be genetically predisposed to a slower overall clock rate. For example, one study analyzed the DNA methylation levels of peripheral blood mononuclear cells (a type of white blood cell) from semi-supercentenarians and their offspring. The investigators found that the average epigenetic age of the semi-supercentenarians was nearly nine years younger than their chronological age. The epigenetic age of their offspring was approximately five years younger than that of their age-matched controls.
"the average epigenetic age of the semi-supercentenarians was nearly nine years younger than their chronological age. The epigenetic age of their offspring was approximately five years younger than that of their age-matched controls" Click To Tweet
Lifestyle factors and exposures can influence the native ticking rate, as well. For example, an obesogenic diet can increase methylation and the clock's subsequent ticking rate. And, as described above, smoking cigarettes and exposure to particulate air pollutants increases the epigenetic aging rate. Some interventions have been identified that may slow the aging rate, however. These measures have been studied for their longevity-enhancing effects and include caloric restriction and administration of rapamycin, an immunosuppressant drug.
Epigenetic clocks predict biological age based on molecular markers on an individual's DNA. Several variants of clocks have been identified, and they differ based on the type and number of tissues in which the markers are measured, as well as the final output. In general, the epigenetic aging rate across multiple types of tissue from a single individual is fairly consistent, but some exceptions do exist. The use of epigenetic clocks may have widespread applications in health and society, including forensic science and early prevention and treatment of disease to promote healthy aging. In the future, rather than asking whether a person's biomarkers look better, soon clinical trials may ask whether the person is simply aging better.