DNA methylation (DNAm) has emerged as a powerful tool in forensic genetics, broadening traditional DNA profiling. In recent years, DNAm has enabled the inference of multiple traits from forensic samples, supporting a shift toward epigenomic profiling approaches that aim to extract comprehensive biological information from a single source. Applications such as age and sex estimation have proven particularly robust, with specific CpG sites showing strong and reproducible associations. Beyond these, DNAm also holds potential for inferring other characteristics, including aspects of health and disease susceptibility. Estimating the post-mortem interval (PMI) remains a key challenge, with epigenomic signatures offering potential to track time since death.

Objectives

Building on the potential of DNAm for epigenomic profiling, this study aims to evaluate its application in a post-mortem context. Specifically, this work investigates whether DNAm patterns can be used to simultaneously inform multiple traits, including age and sex, while also capturing temporal changes associated with decomposition, in the humid continental climate (Köppen classification Dfb) of southern Québec. By analyzing methylation profiles across post-mortem timepoints, the study assesses whether epigenomic signatures retain sufficient predictive power to support both biological inference and the estimation of time since death.

Material and methods

For this purpose, the Illumina EPICv2 methylation array was run to quantify total levels of DNAm from human cadavers. The process of decay of the human donors was monitored with pictures and soft-tissue samples were collected over a span of three years. The age at the time of death was estimated using multiple established epigenetic clocks, allowing comparison of their performance across post-mortem timepoints. Sex was established through a generalized linear model (glm) and additional analyses are currently underway to expand DNAm-based profiling of biological traits and to develop modeling approaches for PMI estimation.

Results and conclusions

A consistent decrease in DNAm levels was observed during the PMI. Epigenetic clocks consistently underestimated chronological age; however, their predictions followed similar trends across post-mortem timepoints, with comparable slopes. Sex modeling provided accurate predictions in all samples, even three years post-mortem. Preliminary results from ongoing analyses further support the potential of DNAm for multi-trait inference and PMI modeling within an epigenomic profiling framework. These findings highlight the potential of DNA methylation to extend beyond traditional applications in forensic genetics, as supported by a proof-of-concept study conducted in pigs as analogs for human decomposition in this work.

Authors

• Irina Badell (Trent University, Canada)
• Aaron B.A. Shafer (Trent University, Canada)
• Theresa Stotesbury (Ontario Tech University, Canada)
• Shari L. Forbes (University of Windsor, Canada)
• Lesley Hewett (Trent University, Canada)