Reproducibility and validation of a targeted and flexible epigenetic clock for forensic applications

DNA methylation variants have been widely used as biomarkers of ageing and several mathematical models have been developed to estimate the biological age. More recently, DNA technology has triggered efforts toward the simplification of the array-based epigenetic clocks and targeted approaches, based on the assessment of a small number of CpG sites have been developed. Among the markers included in these clocks, ELOVL2, FHL2, KLF14, C1orf132/MIR29B2C, and TRIM59 resulted to be the most strongly validated markers. We tested the reproducibility and validation of a previously developed targeted epigenetic clock purposely optimized for the measurement of chronological age in blood samples. The clock includes DNAm biomarkers strongly correlated with chronological age whose DNA methylation levels were measured by using a multiplex methylation SNaPshot assay. We found that epigenetic age, calculated using the developed clock, was highly correlated with age (r = 0.97) in a total of 201 blood samples covering a full spectrum of human ages. For 74 of these, methylation profiles of the whole genome were obtained through the Infinium Methylation EPIC v2.0 Kit which also allowed to estimate the most frequently used clocks of Horvath. These results show the potential of our efficient and affordable test for simultaneously measuring DNA methylation levels at multiple target CpG sites to assess chronological age. We observed a strong correlation between the prediction models for the analyzed CpG sites measured using the SNaPshot method and those obtained with the Illumina EPIC array, especially with the Horvath2 clock, which was specifically developed for DNA from skin and blood cells.