[10][11] Horvath spent over 4 years collecting publicly available Illumina DNA methylation data and identifying suitable statistical methods.
[13] The age estimator was developed using 8,000 samples from 82 Illumina DNA methylation array datasets, encompassing 51 healthy tissues and cell types.
The major innovation of Horvath's epigenetic clock lies in its wide applicability: the same set of 353 CpGs and the same prediction algorithm is used irrespective of the DNA source within the organism, i.e. it does not require any adjustments or offsets.
Shortly afterwards, a derivation of Horvath's clock, the IEAA (Intrinsic Epigenetic Age Acceleration), an estimator based on the cellular composition of the blood, was developed.
Specifically, pan-mammalian epigenetic clocks determine the age of tissues from all mammalian species by analyzing cytosine methylation in DNA regions that are highly conserved.
[14] More recently in 2025, age-related changes in histone marks have been leveraged to build a new class of epigenetic clocks that do not rely on DNA methylation.
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.
[17] Methylation sites in the promoter region of this gene have consistently been part of the top most age correlated in different studies.
Horvath hypothesized that DNA methylation age measures the cumulative effect of an epigenetic maintenance system but details are unknown.
[10] The fact that DNA methylation age of blood predicts all-cause mortality in later life even after adjusting for known risk factors[27][28] is compatible with a variety of causal relationships, e.g. a common cause for both.
[36] Salient features of Horvath's epigenetic clock include its applicability to a broad spectrum of tissues and cell types.
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.
[43] Genome-wide association studies (GWAS) of epigenetic age acceleration in postmortem brain samples have identified several SNPs at a genomewide significance level.
[44][45] GWAS of age acceleration in blood have identified several genome-wide significant genetic loci including the telomerase reverse transcriptase gene (TERT) locus.
[46] Genetic variants associated with longer leukocyte telomere length in TERT gene paradoxically confer higher epigenetic age acceleration in blood.
[47][48][49] 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[48] and the risks associated with metabolic syndrome.
However, studies suggest that high levels of alcohol consumption are associated with accelerated aging of certain epigenetic clocks.
[49] The epigenetic clock was used to study the relationship between high body mass index (BMI) and the DNA methylation ages of human blood, liver, muscle and adipose tissue.
[47] The same large study found that various biomarkers of metabolic syndrome (glucose-, insulin-, triglyceride levels, C-reactive protein, waist-to-hip ratio) were associated with epigenetic age acceleration in blood.
[47] Conversely, high levels of HDL cholesterol were associated with a lower epigenetic aging rate of blood.
[47] Other research suggests very strong associations between higher body mass index, waist-to-hip ratio, and waist circumference and accelerated epigenetic clocks, with evidence that physical activity may lessen these effects.
[51] 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.
[10] Colorectal cancer samples with a BRAF (V600E) mutation or promoter hypermethylation of the mismatch repair gene MLH1 are associated with an increased age acceleration.
According to the epigenetic clock, trisomy 21 significantly increases the age of blood and brain tissue (on average by 6.6 years).
[55] Epigenetic age acceleration of the human prefrontal cortex was found to be correlated with several neuropathological measurements that play a role in Alzheimer's disease[43] Further, it was found to be associated with a decline in global cognitive functioning, and memory functioning among individuals with Alzheimer's disease.
[56] This finding might explain why the cerebellum exhibits fewer neuropathological hallmarks of age related dementias compared to other brain regions.
[58] These results are consistent with an independent study that also found an age advancement of 5 years in blood of HIV patients and a strong effect of the HLA locus.
[59] A large-scale study suggests that the blood of Parkinson's disease subjects, in particular, their granulocyte ratio, exhibits (relatively weak) accelerated aging effects.
[60] Children with a very rare disorder known as syndrome X maintain the façade of persistent toddler-like features while aging from birth to adulthood.
[62] Second, surgical menopause (due to bilateral oophorectomy) is associated with epigenetic age acceleration in blood and saliva.