Forensic DNA Phenotyping (FDP) has emerged as a promising tool for inferring investigative leads from biological traces when conventional DNA profiling fails. Among FDP, chronological age estimation represents a key parameter to narrow down potential individuals in forensic investigations. In recent years, DNA methylation at specific CpG sites has proven to be one of the most reliable biomarkers for age prediction, leading to the development of so-called epigenetic clocks.

This study aims to validate an epigenetic age prediction model based on the VISAGE Consortium approach in an Italian population, within the framework of the project “Legal, ethical and social challenges of the Forensic DNA Phenotyping in Italy (LetFor)”. The LetFor project, funded by the Italian Ministry of University and Research, focused on two main goals. The first was to design and validate genetic and epigenetic tools for FDP in the Italian population through the use of massive parallel sequencing (MPS), addressing the current lack of comprehensive Italian genetic data. The second aim was to explore public awareness and perceptions of FDP in Italy, as well as to assess its potential advantages and challenges from ethical, social, and regulatory viewpoints.

With regard to the first objective, a total of 36 peripheral blood samples were collected from Italian volunteers aged between 18 and 67 years. DNA was extracted, quantified, and subjected to bisulfite conversion to distinguish methylated from unmethylated cytosines. MPS was performed using the Ion GeneStudio™ S5 platform, targeting forty-four CpG sites included in the VISAGE epigenetic clock. Sequencing data were processed through dedicated bioinformatic pipelines to obtain methylation levels (β values) for each CpG site. Quality control measures were applied, including bisulfite conversion efficiency assessment and minimum coverage thresholds.

For model development, samples were divided into a training set and a test set and the relationship between methylation levels and chronological age was evaluated. 

The results confirmed a significant correlation between DNA methylation levels at selected CpG sites and chronological age in the studied population. The model demonstrated good predictive performance, with a MAE (mean absolute error) of 3.18 years, consistent with previously reported studies and supporting its applicability in a forensic context. However, variability in prediction accuracy was observed, potentially due to biological factors and technical limitations such as sample size and sequencing variability.

Overall, this study provides further evidence supporting the robustness of epigenetic clocks for age estimation in forensic genetics and highlights the importance of population-specific validation.

Authors

• Chiara Turchi (Department of Biomedical Sciences and Public Health, Section of Legal Medicine, Marche Polytechnic University, Italy)
• Federica Alessandrini (Department of Biomedical Sciences and Public Health, Section of Legal Medicine, Marche Polytechnic University, Italy)
• Aleksandra Pisarek-Pacek (Institute of Zoology and Biomedical Research, Jagiellonian University, Poland)
• Rosaria Gesuita (Center of Epidemiology, Biostatistics and Medical Information Technology, Department of Biomedical Sciences and Public Health, Marche Polytechnic University, Italy)
• Edlira Skrami (Center of Epidemiology, Biostatistics and Medical Information Technology, Department of Biomedical Sciences and Public Health, Marche Polytechnic University, Italy)
• Wojciech Branicki (Institute of Zoology and Biomedical Research, Jagiellonian University, Poland)
• Ewelina Pośpiech (Department of Genomics and Forensic Genetics, Pomeranian Medical University in Szczecin, Poland)
• Carla Bini (Department of Medical and Surgical Sciences, Section of Legal Medicine, University of Bologna, Italy)
• Susi Pelotti (Department of Medical and Surgical Sciences, Section of Legal Medicine, University of Bologna, Italy)
• Mauro Pesaresi (Department of Biomedical Sciences and Public Health, Section of Legal Medicine, Marche Polytechnic University, Italy)