What are biomarkers of aging and why are they needed?

Epigenetic clock and other biomarkers of aging

Yuri Deigin, Geektimes

We all know someone who is "perfectly preserved" for his age, and someone who is "old beyond his years."

Actually, we need biomarkers of aging in order to be able to objectively say: yes, you are 50, but your health is at the level of a 35-year-old. But you, a young man, should take a closer look at your health – your biological age is 10 years older than the chronological one, and this is fraught with a 48% increase in the risk of death. 

What does aging and the likelihood of death have to do with it? And despite the fact that in humans, as in most mammals, aging is accompanied by an exponential risk of mortality:

1

And if at the age of 30 you have an annual chance of dying at 1 chance in a thousand, then by 80 it increases 100 times. It is this age-related increase in the probability of dying that gerontologists call aging. And no, not "all living things" are getting old. There are species that, on the contrary, "get younger" with age – their probability of death decreases, and fertility increases:

So, the goal of the fight against aging is to learn how to roll back the biological age to the level of today's healthy 25-year-old person, and fix it there. And the task of the biomarker of aging is to separate the biological age from the chronological (passport) one. That is, to reliably show exactly where you are on this curve:

Therefore, a good biomarker should be highly correlated with mortality so that biological age can be determined with its help. For example, if a biomarker shows that your current annual probability of death is 1/1000, then you are biologically 30, and if 1/100, then 60. Regardless of what your passport says. Because we die not by passport, but by health.

And, of course, it is important for us to see the reverse dynamics of aging biomarkers: they "improved" the body with some proven anti-aging therapy (for example, mice with the same fasting or rapamycin) and saw a decrease in biological age.

What is epigenetics?

Epigenetics is a "superstructure" over genetics (epi- = over), a mechanism for controlling genes. Rather, there are several such mechanisms: methylation of the genes themselves, acetylation or methylation of histones on which these genes are "wound", and many other things that fall under the definition of epigenetic control.

Why do genes need to be controlled at all? Firstly, because the DNA of an organism is the same in all types of cells, and different sets of genes must be active in a brain cell and a skin cell. And also because different genes are responsible for different stages of the development of the organism – the caterpillar and the butterfly have a very different activity profile of these very genes. As with us, in fact: some genes are active in the womb, others in childhood, and still others in old age. 

And, as it turns out, with age, the on/off profile of various genes changes in almost the same way for all people. And what is even more interesting, it changes in a similar way in mice and other animals. That is, the epigenetic aging of a mouse is similar to the epigenetic aging of a human, only accelerated by 40 times:

We observed tissue-specific age-related changes in DNA methylation [in mice], the direction vector of which coincided with the observed changes in humans. These results further support the view that changes in DNA methylation are related to chronological age and indicate that these processes are similar in different tissues, as well as between mammalian species.

At the same time, mice and I even have common "gears" in these aging clocks – the same genes that make up them:

Differentially methylated regions in mice have a high similarity in nucleotide sequence with humans, and the nature of their methylation is also largely similar between the two species.

What is an epigenetic clock?

In fact, the "methylation clock" is just a set of on/off parameters that best correlates with age. With what age exactly – chronological or biological?

Initially, both with one and the other – after all, in animals in the wild, both chronological and biological are almost identical. They don't drink or smoke, and they don't eat at McDonald's. Therefore, initially, the methylation clocks are set (calibrated) according to the chronological age of each species, and only then various ways to speed them up or slow them down are tested to check whether those effects that prolong life at the same time slow down these clocks, and those effects that shorten life accelerate these clocks?

And still yes! In smokers, diabetics, AIDS patients or people with Down syndrome (who age much faster), the biological age really turned out to be higher than their chronological age. And in mice receiving various rejuvenation therapy, the biological age also decreased.

But more on that later. For now, I will only mention that a high correlation between the methylation clock and age has been established for a number of animals besides humans: rotifers, mice, chimpanzees, and even whales:

And what is so special about this very watch?

The special thing about methylation hours is that they are highly correlated with mortality. For example, in this large-scale study by Horvath on thirteen thousand people, it was found that for every year ahead of the chronological age methylation clock (that is, if you are 45 and the clock shows 46), there is a 2% to 4% increase in the risk of mortality.

Moreover, this observation worked both ways and had a cumulative effect: in those whose biological clock was 10 years ahead of age, the risk of death increased by as much as 48% (1.04 ¹⁰ =1.48), and those who were 5 years "younger" than their age were 18% less at risk of death.

Another study showed a high correlation of methylation hours and the risk of lung cancer in smokers. Moreover, both the risk and the correlation reliability coefficient increased with age:

We also showed that the IEAA's ability to predict lung cancer is highest among people aged 70 and older. An increase in the IEAA by one unit was associated with a 2.5-fold increase in lung cancer among a subgroup of people aged 70+, while for the entire cohort aged 50+, it increased the risk by only 50%.

In this study, only 10 methylation sites were identified (for comparison, there were 353 such sites in the "Horvath clock" mentioned above), abnormal methylation of at least six of which increased the risk of mortality at times – both from any causes, and from cancer or cardiovascular diseases:

And in this study, the Japanese showed generally damning facts for the mitochondrial theory of aging: defects in mitochondrial respiration are caused not by the accumulation of breakdowns, but by epigenetic (programmed) changes. And with the epigenetic rollback of such old cells with the help of Yamanaki factors, all defects of mitochondrial respiration disappear:

We reprogrammed human fibroblast lines by generating iPSCs and showed that reprogramming fibroblasts obtained from elderly people restores age-related mitochondrial respiration defects. Therefore, these age-related phenotypes found in elderly fibroblasts are regulated reversibly and are similar to differentiation phenotypes, since both are controlled by epigenetic regulation rather than mutations in nuclear or mtDNA. Given that human aging can be considered as a consequence of a programmed phenomenon, it is possible that epigenetic regulation also controls human aging.

The Japanese are talking business. And the Japanese presented the greatest gift to humanity in the form of those Yamanaki factors. After all, they not only reset the epigenetic clock (both in humans and mice), but also significantly prolong the life of animals:

So how to get younger?

The main point of biomarkers of aging is to use them to look for the most effective ways to combat it. Therefore, immediately after the methylation clock proved to be such a potential biomarker, scientists rushed to investigate them to see if they reflected the effectiveness of various anti-aging effects. And indeed, such a relationship is beginning to manifest itself. Here is a summary of the latest study by the Croat mentioned a couple of times, who is one of the leading experts in this direction (blue arrows reduce the methylation hours, and red ones increase):

Much of the above is expected. A couple of surprises for me were that both moderate alcohol consumption and "good" cholesterol reduce methylation hours. Well, there will be a reason to drink to the health of the Croat.

A much more targeted study in mice to assess the effects on the clock of rapamycin methylation, calorie restriction and life-prolonging genetic mutations was published not so long ago. And it also confirmed that all these interventions have a rejuvenating effect on the methylation clock:

We formulated a model of epigenetic aging in mice and used it to find evidence that known life extension interventions slow down the epigenetic clock in the liver of mice.

By the way, the same researchers released a parallel article where they tried to identify which regions of the genome are susceptible to age-related changes in methylation. After analyzing 42 million (!) methylation sites, the authors concluded that the main objects of age-related changes are promoters and enhancers of highly expressed genes.

Which for me confirms the hypothesis of programmed aging – it seems that the key age-related change is a change in the expression profile of just a few key gene regulators at the top of the hierarchy of homeostasis control, and this cascade entails all other age-related changes.

Vadim Gladyshev and his colleagues describe very similar findings indicating several key genes in their latest work:

The significance of the various methylation sites was unevenly distributed in the methylation hours. The sites formed several different clusters associated with the Hsf4, Kcns2, Map10, Tns2, Wnt3a and Zscan2 genes. We found that 17 of the 18 CpG sites common to subsets of methylation clocks 1 and 2 were also present among 90 CpG sites of mDNAm clocks. Most of these 17 CpG sites were located inside the introns Ciita, Cd200r4, Rasgef1c, Wnt3a and Zscan2, and some were grouped.

Slowing down the methylation clock with various aging-slowing interventions was also shown in this beautiful work:

It is important to note that we have found that biological interventions affect the methylation clock of mice, and therefore we assume that the clock predictions reflect not only chronological, but also biological age.

I really liked this graph of age-related changes in the methylation level of 329 sites in different tissues:

It shows how some genes are activated with age, others – on the contrary, and others remain unchanged.

By the way, the results of a study on twins confirm the correlation of methylation hours with mortality. The higher the biological age of one of the twins, the higher the probability that he will die first:

This hypothesis was confirmed by a classical survival analysis showing an increase in the risk of mortality by 35% (4-77%) for each 5-year-old exceeding the age of methylation hours over chronological age. In addition, the paired analysis of twins revealed more than double the risk of mortality for a twin with a large age by the methylation clock, as well as the dose dependence of the probability of death, which increased 3.2 (1.05-10.1) times for every 5 years of the age difference by the methylation clock between twins, thereby demonstrating a stronger association of the methylation clock with the probability of death in older people, taking into account family factors. In conclusion, our results confirm that the methylation clock can be considered a biomarker of aging.

Summarizing this part, I can't help but say that I believe that in order to become radically younger, you need to learn how to roll back the epigenetic clock directly. While we are just beginning to understand how to do it: thanks to the results of the Belmonte group, we have learned to gently knock on this clock with a "hammer" in the form of Yamanaki factors. But ideally I would like to pick up a key to our watch.

What other biomarkers of aging are there?

Among other biomarkers of aging, I want to mention locomotor activity, on the basis of which the Russian company Gero recently released a cool application that determines biological age by the pattern of motor activity from your FITBIT.

Also, a good predictor of mortality is the thickness of the arterial intima-media complex. Everything is complicated about the IGF-1 (1, 2, 3, 4, 5, but 6, 7, 8), so I will deal with it in a separate post. Well, the grandfather of all biomarkers is frailty index, over the unambiguous translation of which gerontologists are still struggling: "senility index" sounds insulting, and "fragility index" or "vulnerability index" does not fully convey the original meaning. 

By the way, not so long ago I saw the news about a fresh article that boldly claimed that the new version of this index developed by its authors reflects biological age even better than the "methylation clock". At least, her title was very ambitious: “The frailty index outperforms DNA methylation age and its derivatives as an indicator of biological age.”

But after reading it, I realized that the boldness of the authors' statements is unfounded. Not only is their hypothesis, to put it mildly, doubtful, but their article itself refutes this hypothesis. To begin with, their new and improved index is a simple questionnaire of 34 items. Which at the same time assigns an equal role to infarction and cystitis:

And loud statements are not supported by their own data. Here is a table of the results of their analysis:

From it, we see that regression by methylation hours (model No. ²) has a higher R2 than regression by their senility index (model No. 3) – and almost 2 times (0.22 vs. 0.13). At the same time, the usual chronological age (model No. 1) has R ² almost 4 times higher than their index, and 2 times higher than the methylation clock. I'm not even talking about the fact that the R ² of all their models is extremely low.

How do the authors justify their loud statements that their FI is better suited to the role of a biological clock than DNAmAge? But what is the lower p-value in model No. 7, and even limited in age only to octogenarians:

In all Cox regression models for the entire study cohort, which includes age categories from 60 to 103, chronological age was the best predictor of mortality (Fig. 3a). When Cox regression was limited to only 80-year-olds, FI34 was a better predictor of mortality than chronological age (P=0.035 vs. P=0.054, respectively, Figure 3b). This indicates that FI34 is the best indicator of biological age in later years, when the accumulation of health deficits accelerates in the oldest.

That is, a lower p-value for them means better predictive power. A typical misconception of those who do not understand statistics well:

Misconception #13: Statistical significance is a property of the phenomenon being studied, and thus statistical tests reveal significance.No!
This misinterpretation is promoted when researchers claim that they have found or have not found "evidence" of a statistically significant effect. The effect under study either exists or does not exist. "Statistical significance" is a dichotomous description of the P value (which is below the selected cutoff) and, therefore, is a property of the statistical test result, but it is not a property of the effect or the population being studied.

Moreover, for their key conclusion, the authors took as a basis model No. 7, where both parameters under consideration (FI and DNAmAge) are presented simultaneously, and even together with the chronological age with which they both correlate (that is, most likely, they are not independent, violating one of the regression conditions). Moreover, they narrowed the sample only to 80-year–olds - that is, to a very narrow segment of that parameter (chronological age), which best explains the variation of the entire data array (since model No. 1 R ² is several times higher than other models), and then they happily reported that the age at such a narrow the age segment has a low p-value.

The fact that the model has their decrepitude index R ² is almost 2 times lower than the DNAmAge model, I have already mentioned (0.13 vs. 0.22). And the fact that adding additional parameters to the model to the already existing chronological age is meaningless, can be seen by the fact that R ² of such models (0.48–0.50) is almost identical the original (0.48 of model No. 1).

Well, in conclusion, it is worth noting that the value of the very concept of the senility index proposed by the authors is close to zero. The biomarker is valuable because it can change in both directions, because aging therapy is designed to reduce biological age. And the proposed senility index is mainly based on historical parameters (whether you had a stroke/heart attack/etc.). Therefore, even if aging therapy rejuvenates your heart, brain and kidneys, it will practically not affect the historical senility index. And on the methylation clock or locomotor – quite.

So I congratulate us all on the new biomarkers of aging, and wish us happiness, health and very long life!

Portal "Eternal youth" http://vechnayamolodost.ru  27.06.2017