Methylation of DNA is the best-known mode of epigenetic regulation (turning genes on and off). Methylation patterns are stable unless they are actively changed, and can persist over decades, even across generations.
Four years ago, biostatistician Steve Horvath of UCLA identified a set of 353 methylation sites that are best-correlated with human (chronological) age. These are sites where genes are turned on and off at particular stages of life. A computer analysis of a gene sample (from blood or skin or even urine) can determine a person’s age within about two years.
Two reasons the Horvath Clock is important. First, it is the best measure we have of a person’s biological age, so it provides an objective measure of whether our anti-aging interventions are working. Say you’re excited about a new drug and you want to know whether it really makes people younger. Before the Horvath clock, you had to give it to thousands of people and wait a long time to see if fewer of them were dying, compared to people who did not get the drug. The Horvath clock is a huge shortcut. You can give the drug to just a few people and measure their Horvath (methylation) age before and after. With just a few dozen people over a two-year period, you can get a very good idea whether your drug is working.
Second, there is evidence and theory to support the idea that the methylation sites that Horvath identified are not just markers of aging but causes of aging. That means that if we can figure out how to get inside the cell nucleus and re-configure the methylation patterns on the chromosomes, we should be able to address a root cause of aging. (Before we get too excited: “Gene therapy” has been around 20 years but is still in a developmental stage; “epigenetic therapy” is what we need, and it does not yet exist, but is technically feasible using genetically engineered viruses and CRISPR.)
In 2012-2013, three papers appeared proposing the idea that the deep cause of aging (in humans and many other higher animals) is an epigenetic program [Johnson, Mitteldorf, Rando]. Genes are turned on and off at various stages of life, producing growth, development and aging in seamless sequence. (A fourth paper by Blagosklonny proposed a similar idea, but focused on the role of a single transcription factor controlling gene expression (mTOR) and shied away from the conclusion that natural selection might have preferred aging affirmatively. Here’s an earlier presentiment by Blagosklonny.)
It’s a powerful hypothesis that proposes to resolve evolutionary and metabolic questions alike. It contains a seed of a prescription for anti-aging research—although epigenetics has proved to be so complicated that practical modification of the body’s gene expression schedule may require a lot more groundwork.
Unbeknownst to any of us working on these theoretical papers, Steve Horvath was already working on calibration and measurement of the epigenetic aging clock, and he published his basic result by the end of 2013.
One remarkable property of the Horvath clock is that it is more accurate than chronological age for predicting who will contract aging diseases and who will die. Even though the clock was derived with an algorithm that matched the output clock age as closely as possible to chronological age, the result proved to contain more information than chronological age. “In deriving the clock, chronological age was used as a proxy for biological age.” People whose “methylation age” is greater than their chronological age are likely to suffer health deterioration and to die sooner than people whose methylation age is less than their chronological age.
Horvath has openly shared his methodology and his computer program. Based on the Horvath clock, a California company began last year to offer a commercial test for methylation age. You can send a blood or urine sample to Zymo Research.
Candidate aging clocks
Horvath describes how he came up with the idea of a methylation clock by a process of elimination, beginning with four candidate clocks:
- Telomere length
- Gene expression profile
- Proteomic data
- DNA Methylation
- Telomere length – This had been measured easily and cheaply for more than a decade, but its correlation with chronological age (and with mortality) is not strong enough to be useful as a biological clock.
- Gene expression profile: Which genes are being transcribed into RNA at a given time? This can be measured by extracting RNA, and turns out to be highly tissue-specific. In other words, it varies according to which part of the body you’re looking at.
- Proteomic data: Genes, once transcribed, are translated into proteins. Some of these proteins stay in the cell while others circulate through the body. Gene CHIP technology measures levels of different proteins reliably and inexpensively.
- DNA Methylation: Easier to measure than (2) or (3). Methylation is only one of many mechanisms controlling gene expression, but it is one of the most persistent. Horvath found that a subset of DNA methylation sites seems to be characteristic of age no matter where in the body they are measured.
What is DNA methylation?
Adjacent to many genes is a promoter site, a location on the same chromosome which stores temporary information about whether the gene is turned on or off. Promoter sites contain the base sequence C-G-C-G-C-G-C repeated. This is called a CpG island (where the “p” just tells you that the C is linked to G on the same strand, rather than being linked across strands, in which C is paired with G.)
C stands for “Cytosine”, and the Cytosine molecule can be modified by adding an extra methyl group (CH3) to form 5-methyl Cytosine.
The cell has molecular workers that are deployed to go around specifically adding methyl groups in some parts of the DNA or removing them in others. The bottom line is that methylated Cytosine is a sign that says “don’t transcribe the adjacent gene.” When the methyl groups are removed, it is a signal that the gene are to be transcribed once more.
Enzymes called methyl transferases are deployed to precise regions of the genome to turn genes on and off. Methylation can be transient. There is evidence for circadian cycles of methylation. Or it can be quite long-lasting. Methylation patterns can persist for decades, and are copied when cells replicate, so that methylation patterns can be passed to offspring as part of one’s epigenetic legacy. Inherited methylation sites are the exception however; most of the genome is programmed fresh with age-zero, pluripotent methylation patterns when egg and sperm cells are generated.
How the methylation clock works
Using a standard statistical algorithm, Horvath identified 353 CpG sites that were most strongly correlated with chronological age, no matter where in the body he looked. The same algorithm provided 353 numbers to be multiplied by methylation levels at each site, then added up to produce a number. The number is not directly a measure of age, but in the last step a table is used (an empirically-derived curve) to associate the number with an age.
This is the raw output of the function before it is transformed into an age. Notice that methylation changes very rapidly during the first 5 years of life, gradually slowing during the growth phase and straightening out to constant slope after about age 18.
Even though the Horvath clock was designed to be independent of what part of the body DNA was drawn from, some variations appear. Most noticeable is female breast tissue, which ages faster than the rest of the body, and brain tissue, which ages more slowly. Blood and bone tissue tend to age a little faster. (Sperm and egg cells are “age zero” no matter the age of the person from whom the germ cells were drawn. Placentas from women of all ages are age zero.) Similarly, induced stem cells (using the 4 Yamanaka factors) have zero age. In contrast, a similar treatment can change one differentiated cell type into another, for example, turning a skin cell into a neuron. This does not affect epigentic age.
Liver cells tend to be older than the rest of the body in people who are overweight, and younger than the rest of the body in people who are underweight. Other tissues don’t seem to show this relationship. For example, fat cells do not have older methylation ages in people who are obese. And, perhaps surprisingly, weight loss does not reverse the accelerated methylation age of the liver (at least, not within the 9-month time frame of the one study looking at this).
Studies have been done correlating methylation age with various diseases and, of course, mortality. Corrections are made for every kind of environmental factor, including smoking, obesity, exercise, workplace hazards, etc, called collectively the “extrinsic factors”. The result is that methylation age rises with extrinsic factors, and independently methylation age is also correlated with intrinsic (genetic) factors that affect lifespan. Horvath estimates that genetics controls 40% of the variation in methylation age (as it differs from chronological age).
Men are slightly older than women in methylation age. This is already evident by age 2. Delayed menopause is associated with lower epigenetic age. Cognitive function correlates inversely with methylation age of the brain.
Speaking before Horvath at the same conference, Jim Watson claims there are many supplements and medications that can slow the Horvath clock. The one he focuses on is metformin, which, he says, has epigenetic effects via an entirely different pathway from lowering blood sugar (the purpose for which it has been prescribed to tens of millions of diabetics).
Here’s a curious clue: There is a tiny number of children who never develop or grow, and continue to look like babies through age 20 and perhaps beyond. These children have normal methylation age. Whatever it is that blocks their growth, it is not the methylation changes in their DNA. Does this mean that there are other epigenetic controls, more powerful than methylation, that control growth and development? Or does it mean that children with this syndrome have normal epigenetic development, but something downstream from gene expression is blocking their growth? Conversely, Hutchinson-Gilford progeria is caused by a defect in the LMNA gene which causes children to age and die before they even grow up. Hutchinson-Gilford children have normal methylation ages by the Horvath clock.
Radiation, like smoking and exposure to environmental oxidation, tends to age the body faster. This is independent of methylation age—which is unaffected by radiation. Neither smoking nor radiation exposure affect epigenetic age. HIV also accelerates aging, and HIV does affect methylation age.
Methylation age and telomere age are both correlated with chronological age, and they both predict mortality and morbidity independent of chronological age. But the two measures are not correlated with each other. In other words, the information contained in the methylation clock and in measures of telomere length complement one another to offer a better predictor of future aging decline than either of them separately.
Diet has a weak effect on methylation age. Very high carbohydrate, very low protein diets are noticeably terrible. Beyond this, there seem to be two sweet spots: one for the Ornish-style protein-restricted diet and one for the Zone/Atkins style diet. Weak evidence to be sure, but suggestive that they both work.
“The epigenetic clock is broken in cancer tissue.” [ref]
Building on the original clock
The original clock was optimized to track chronological age, and yet it fortuitously provided more information than chronological age. In a second iteration, Horvath set out explicitly to track biological age. He used historic blood samples from the 1990s, and paired them with hospital records and death certificates to search for methylation sites that correlate best with aging-related health outcomes. The result was the phenotypic clock, DNAm phenoAge. This uses 513 methylation sites to predict
- all-cause mortality
- cardiovascular mortality
- lung disease
- (loss of) physical strength
- (loss of) cognitive ability
On the drawing board: An epigenetic clock specialized to work well with skin and blood cells, (which are the most accessible). (Enough skin cells can be scraped painlessly from the inside of your mouth (buccal epithelial cells) to do a DNAm test.)
Connection to Parabiosis and Plasma Transfusions
Several groups have begun to experiment with transfusions of blood plasma from a young donor as a possible path to rejuvenation. Horvath reports an encouraging finding: Sometimes older people contract a form of leukemia that requires a blood and marrow transfusion (including the stem cells that give rise to new blood) from a donor. The finding is that after this treatment, the blood of the patient continues to show the methylation age of the donor, not the patient.
Epigenetic Aging and Telomere Aging Bound to a See-Saw Relationship
(This was the most exciting new result for me personally, because it relates to an idea I have held dear for more than a decade.)
Methylation age is older or younger than chronological age in different people, generally by about +2 years. 40% of the variation is due to genetics. Some common genetic variants can make the clock run faster or slower. The most prominent genetic variants link telomere aging to methylation aging. The faster your epigenetic clock runs, the longer your telomeres. The slower your epigenetic clock runs, the shorter your telomeres. [preprint]
There’s a word for this in the genetic theory of aging. It’s called Antagonistic Pleiotropy. Back in 1957, George Williams theorized that the genes causing aging ought to have simultaneous beneficial and detrimental effects. That would explain why natural selection has permitted aging to occur, despite the fact that it cuts off fitness. Williams said: Nature had no choice but to accept the genes that cause aging because there was no other way to get the benefits of these same genes (which he surmised ought to enhance fertility).
My theory of Antagonistic Pleiotropy is that it is not a situation of “forced choice”; rather, aging is important for the health of the community, and mother nature has been faced with the dilemma: how to keep aging in place despite efficient natural selection against it on the individual level. Aging is so important to the community that evolution has been motivated to find ways to keep it in place, despite the short-term temptation for natural selection to favor those with longer lives (thus greater opportunities to leave offspring). In my hypothesis, evolution invented pleiotropy to address this problem. The telomerase-epigenetic clock connection is an example. There is no physically necessary connection between telomerase and epigenetic aging, but the two have evolved a see-saw link so that it is more difficult to mutate aging away.
This also relates to my coverage last fall of the telomerase-cancer connection. At the time, I was scratching my head, why should genetic variants that lengthen telomeres be associated with higher rates of some cancers? Here is a clue: The same genetic variants that lengthen telomeres also accelerate the epigenetic aging program. The specific example of a cancer that is most closely tied to higher telomerase levels is melanoma, which is a cancer that is less sensitive to age than other cancers. People tend to get melanoma earlier in life than other skin cancers. Therefore, I predict that other pleiotropic links will be found between these genetic variants that promote longer telomeres and other mechanisms linked specifically to melanoma.
The Bottom Line
All these data in a field so new is a tribute to Horvath’s industriousness and to the promise and fruitfulness of a new methodology.
The data so far suggest that methylation programming is a big part of the driver of aging, but not the whole story. Smoking affects life expectancy, but it doesn’t affect methylation age. Weight loss benefits life expectancy, but it is invisible to methylation age. Most curious are those children who fail to develop, or age prematurely, even though their methylation age is progressing on schedule.
What does it mean that radiation ages the body without advancing the methylation clock? Perhaps that accumulation of damage is part of the phenotype of aging, though I remain hopeful that the body remains capable of undoing that damage even late in life, if it is re-programmed to want to do so. What does it mean that AIDS advances the aging clock? Perhaps that the immune system is a central signaling mechanism in the aging process.
So, it’s “methylation plus”. Plus what? Not just methylation plus damage”; though we can certainly shorten our lifespan with radiation or smoking, we can’t increase our lifespan by avoiding toxins. “Methylation plus other epigenetic programs”—this would be my first guess. “Methylation plus mitochondrial state” would be a close second. Methylation is all in the nucleus, and the cytoplasm of the cell seems to store independent information, and can even re-program the state of the nucleus, as suggested by parabiosis experiments. There is also evidence for“Methylation plus telomere shortening”.