Reading the Epigenetic Clock: A Comparative Analysis of DNA Methylation Markers for Age Estimation in Semen, Saliva, and Touch DNA

Abstract

Background. Predicting an individual's age from biological evidence is a significant advance in forensic intelligence. DNA methylation, a stable epigenetic mark, provides the molecular basis for epigenetic clocks, but their operational reliability requires validation across diverse sample types and populations, particularly for low-template touch DNA.

Objective. To develop and validate body-fluid-specific age prediction models from a curated five-CpG panel in semen, saliva and touch DNA, and to compare their accuracy in an Indonesian population.

Methods. Following approval from the Ethical Committee of CMHC Indonesia (No. 128/EC/CMHC/2023), 150 healthy Indonesian male volunteers aged 18–65 provided semen, saliva and high-yield standardized touch DNA. Methylation at five CpG sites (ELOVL2, FHL2, TRIM59, KCNQ1DN, C1orf132) was quantified by controlled pyrosequencing after bisulfite conversion with efficiency controls. Body-fluid-specific models were built by multiple linear regression and validated by 10-fold cross-validation.

Results. The semen and saliva models were highly accurate, with mean absolute deviations of 3.19 years (R²=0.94) and 3.55 years (R²=0.92). The touch DNA model was less precise but still informative (MAD 5.49 years, R²=0.85). All models satisfied the assumptions of linear regression, with variance inflation factors below 2.5, and 95% prediction intervals were narrowest for semen.

Conclusion. The panel is validated for age prediction in a Southeast Asian population. The semen and saliva models are accurate enough for consideration in casework, while the touch DNA model, interpreted cautiously, can generate investigative leads from trace evidence. The findings underline the importance of tissue-specific modeling and provide a methodological blueprint for responsible forensic age estimation.