DNA methylation clock

Saket Choudhary

saketc@iitb.ac.in

Computational Multi-omics of Ageing

DH 603

Lecture 02 || Friday, 7^th March 2025

History of ageing

History of ageing: Gilgamesh

• 4000 year old Epic of Gligamesh → from Mesopotamia
• The titular hero, king of the city of Uruk, befriends a wild warrior Enkidu
• Enkidu unfortunately dies motivating Gilgamesh to conquer death
• Gilgamesh consults Utnapishtim who offers him two routes to immortality:
• 1: Escape death if you stay awake for a week straigh
• But he falls asleep and fails
• 2: Consume a herb found underwater
• Gilgamesh successfully obtains it and plans to test it by first feeding it to the older people of Uruk
• But a snake slithers away with the herb

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Source

History of ageing: China

• First emperor of unified China
• Having won many battles, conquered territories, he wanted to win the elixir of life
• Sent missionaries to find the elixir with a punishment of execution if they fail to do so
• Would ultimately die before constructing his mausoleum possibly due to mercury poisoning from one of the potions he took to prolong his life

Qin Shin Huang

The story of culturing immortality

Alexis Carrel

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• Alexis Carrel was a famous (Nobel laureate) french surgeon famous for pioneering techniques to connect severed blood vessels
• With the goal of keeping tissues alive, he began a series of experiments at Rockefeller Institute to keep a culture of cells alive from a tissue outside the human body, indefinitely
• Took cells from heart of chicken embryo, kept steadily supplying them with nutrients → the culture could be maintained for years → big breakthrough
• 1921 issue of Scientific American reads, “Perhaps the day is not far away when most of us may reasonably anticipate a hundred years of life. And if a hundred, why not a thousand?”

But Carrel’s finding could not be replicated in other labs for years!

The Hayflick Limit

Leonard Hayflick

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• Overturned the “central dogma” that all vertebrate cells grown in culture are immortal
• Hayflick and Moorhead performed an experiment using human fibroblasts which showed that fibroblasts doubled a finite number of times, after which the cells stopped dividing and entered what Hayflick termed the phase III phenomenon
• Simple experiment: Mix equal numbers of normal human male fibroblasts that had divided many times with female fibroblasts that had divided only a few time.
• When the male ‘control’ culture stopped dividing, the mixed culture was examined and only female cells were found → old cells ‘remembered’ they were old, even when surrounded by young cells

A brief timeline

Source

Is human lifespan limited?

Supercentenarians database

https://www.supercentenarians.org/en/

Is there a limit to human lifespan?

• A hard limit can exist only if a programmed process leading to death exists
• The human lifespan can be modeled using statistical distribution using variations on the generalized extreme value distribution (GEV) → statistical model of the probability distribution of the oldest age a person can survive to
• After fitting the statistical model, if the shape parameter is negative (the orange curve), the density function has support (nonzero probability) from –∞ to ω → there is no chance for anyone to live past age ω (a hard limit to lifespan)
• When the shape parameter is 0 (the blue curve), the density function has support over the interval (–∞,+∞); survival to any age is possible, although trending very close to zero for higher ages (a soft limit to lifespan)

Methylation clocks

DNA-sequences can undergo modifications

Fi

Moor et al. 2012

• A family of enzymes (DNA methyltransferase, DNMT) catalyze the transfer of methyl group to the 5th carbon of cytosine to form 5-methylcytosine (5mC)
• Primarily occurs in the CpG (C followed by a G connected by a phospodiester bond
• DNMT3a and DNMT3b are de-novo methyl transferase → transfer methyl group to naked DNA
• DNMT1 is a maintenance methyltransferase

5mC

Methylation happens primarily at CpG sites

Fi

• CpG sites (C followed by G) occur with high frequency in the genome
• Cytosines in CpG dinucleotides can be methylated to form 5-methylcytosines → Mammals have 70-80% CpGs methylated
• Methylated CpG can change the expression of the nearby gene

Source

Variation of methylation status across contexts

Fi

Breiling et al. 2011

• Methylation quantified as a percentage of ‘reads’ originating from a loci that are methylated (details later)
• Mammalian ‘methylomes’ have pervasive methylation (80% of CpG sites are methylated in humans)
• Organism like bees have sporadic methylation
• Organisms like drosophila have almost no methylation → methylation is not a requirement for development or fate specification
• Absence of conservation of methylation across organisms hints that it is not a

Methylation arrays capture CpG sites across genome

Fi

• Methylation arrays capture 27k (Illumina 27k array) 450k (Illumina 450k array) or 850k (“EPIC”) CpG sites across the genome spanning ~20k genes
• Bisuflite treatment converts unmethylated C to U
• Bead contains sequences that hybridize to sequences across the genome (containing the CpG dicnuleotide)
• Beta values = Methylated C (Methylated C + Unmethylated U)

Constant offset

Methylation clock - the science

Steve Horvath

Some definitions

Chronological age Age of an organism as measured by physical time, for example, calendar years from birth.

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Biological age A correlative measure of chronological age that is more informative than chronological age of the current and future health status of an individual. An example of a directly quantifiable biological age is mortality risk.

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Chronological age is captured by CpG methylation

7.5k samples from 27k or 450k arrays→ 353 informative CpGs

Horvath 2013

Works across species

Horvath 2013

Ever expanding table of clocks

Elastic Net

• First term encourages sparse solution to coefficients
• Second term encourages highly correlated features to be averaged

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Linear regression

Under constraints on beta

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Questions?