Forensic Epigenetic Approaches in Human Biological Samples from Medico-Legal Investigations: A Systematic Review of Public Health

Abstract

Forensic epigenetics has emerged as a promising extension of conventional forensic genetics, enabling the inference of physiologically and socially relevant characteristics from biological evidence beyond individual identification. In particular, DNA methylation–based approaches have demonstrated utility in estimating age, sex, and lifestyle-related traits, with growing relevance for medico-legal investigations and public health intelligence. This systematic review synthesizes current evidence on forensic epigenetic methodologies applied to human biological samples, with a focus on their applications in age, sex, and lifestyle estimation.

A systematic literature search was conducted across PubMed/MEDLINE, Scopus, Web of Science, Embase, and Google Scholar using predefined search strategies. Study selection was guided by the Population–Exposure–Outcome (PEO) framework, while methodological quality was appraised using the Critical Appraisal Skills Programme (CASP) checklist. Owing to methodological heterogeneity among included studies, a narrative synthesis approach was employed. Five studies met the inclusion criteria. DNA methylation–based age estimation emerged as the most robust and consistently validated application, particularly using age-associated CpG markers such as ELOVL2, although performance varied by tissue type. Epigenetic approaches to sex estimation and biological differentiation demonstrated potential, particularly in resolving complex forensic scenarios such as monozygotic twin discrimination but remain less mature than age prediction models. Additionally, lifestyle and environmental exposures were shown to modulate epigenetic signatures, underscoring their dynamic nature. Tissue specificity significantly influenced analytical accuracy, with blood and semen yielding more stable and reliable signals compared to touch DNA.

DNA methylation–based forensic epigenetic approaches represent a valuable tool for age estimation and supplementary biological inference in medico-legal contexts. Despite ongoing methodological and interpretative challenges, these approaches hold substantial promise for advancing forensic investigations and informing population-level public health applications.