DNA methylation is known as a biomarker for age with applications in forensics. Here we describe the VISAGE (VISible Attributes through GEnomics) Consortium’s enhanced tool for epigenetic age estimation in somatic tissues. The tool is based on eight DNA methylation markers (44 CpGs), bisulfite multiplex PCR followed by sequencing on the MiSeq FGx platform, and three statistical prediction models for blood, buccal cells and bones. The model for blood is based on six CpGs from ELOVL2 , MIR29B2CHG , KLF14 , FHL2 , TRIM59 and PDE4C , and predicts age with a mean absolute error (MAE) of 3.2 years, while the model for buccal cells includes five CpGs from PDE4C , MIR29B2CHG , ELOVL2 , KLF14 and EDARADD and predicts age with MAE of 3.7 years, and the model for bones has six CpGs from ELOVL2, KLF14, PDE4C and ASPA and predicts age with MAE of 3.4 years. The VISAGE enhanced tool for age estimation in somatic tissues enables reliable collection of DNA methylation data from small amounts of DNA using a sensitive multiplex MPS assay that provides accurate estimation of age in blood, buccal swabs, and bones using the statistical model tailored to each tissue.

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DNA methylation analysis is becoming increasingly useful in biomedical research and forensic practice. The discovery of differentially methylated sites (DMSs) that continuously change over an individual's lifetime has led to breakthroughs in molecular age estimation. Although semen samples are often used in forensic DNA analysis, previous epigenetic age prediction studies mainly focused on somatic cell types. Here, Infinium MethylationEPIC BeadChip arrays were applied to semen-derived DNA samples, which identified numerous novel DMSs moderately correlated with age. Validation of the ten most age-correlated novel DMSs and three previously known sites in an independent set of semen-derived DNA samples using targeted bisulfite massively parallel sequencing, confirmed age-correlation for nine new and three previously known markers. Prediction modelling revealed the best model for semen, based on 6 CpGs from newly identified genes SH2B2, EXOC3, IFITM2, and GALR2 as well as the previously known FOLH1B gene, which predict age with a mean absolute error of 5.1 years in an independent test set. Further increases in the accuracy of age prediction from semen DNA will require technological progress to allow sensitive, simultaneous analysis of a much larger number of age correlated DMSs from the compromised DNA typical of forensic semen stains.

DNA methylation-based clocks provide the most accurate age estimates with practical implications for clinical and forensic genetics. However, the effects of external factors that may influence the estimates are poorly studied. Here, we evaluated the effect of alcohol consumption on epigenetic age prediction in a cohort of extreme alcohol abusers. Blood samples from deceased alcohol abusers and age- and sex-matched controls were analyzed using the VISAGE enhanced tool for age prediction from somatic tissues that enables examination of 44 CpGs within eight age markers. Significantly altered DNA methylation was recorded for alcohol abusers in MIR29B2CHG. This resulted in a mean predicted age of 1.4 years higher compared to the controls and this trend increased in older individuals. The association of alcohol abuse with epigenetic age acceleration, as determined by the prediction analysis performed based on MIR29B2CHG, was small but significant (β = 0.190; P-value = 0.007). However, the observed alteration in DNA methylation of MIR29B2CHG had a non-significant effect on age estimation with the VISAGE age prediction model. The mean absolute error in the alcohol-abusing cohort was 3.1 years, compared to 3.3 years in the control group. At the same time, upregulation of MIR29B2CHG expression may have a biological function, which merits further studies.