Identifying human remains in mass disasters or unsolved cold cases presents a significant challenge for forensic practitioners. While traditional STR profiling remains the gold standard, its success depends on the availability of direct reference samples or close relatives. As part of the ForMAT project, we explore the use of DNA methylation (DNAm) to facilitate Disaster Victim Identification (DVI). Epigenetic markers can aid in distinguishing between tissue types and enable subsequent age estimation. Importantly, in addition to bone samples, which can be challenging to analyze, cartilage may serve as a more practical alternative for human identification. This study employs a multi-phase approach to select and validate CpG sites that are informative for both tissue type and age. First, we conducted a comprehensive review of the scientific literature to identify candidate markers. To this end, we analyzed publicly available data from the Gene Expression Omnibus (GEO), using three datasets for bone tissue (GSE36002, GSE138307, GSE64490) and three datasets for cartilage (GSE43269, GSE63695, GSE63106), with aggregated sample sizes of N = 84 and N = 68, respectively. The top markers, exhibiting the strongest effect sizes, including those located in ELOVL2, PDE4C, KLF14, ASPA, TRIM59, PI4KB, FAM59B, PRMT8 and SCGN for tissue differentiation and age prediction, were identified through meta-analyses of GEO data. In the final stage, to refine and validate these markers, we generated novel genome-wide DNA methylation data using the Illumina Infinium MethylationEPIC v2.0 BeadChip for N=96 bone samples (48 femur and 48 skull) and N=40 rib cartilage samples, all collected on site during autopsy procedures. The final set of markers, derived from both publicly available and newly generated data, represents a compact panel that balances predictive accuracy with panel size. These markers are an important part of the DNAm DVI kit that is being developed as part of the ForMAT project.

The study received funding from the European Union’s Horizon Europe Programme under Grant agreement no. 101225631. Views and opinions expressed are however those of the author(s) only and do not necessarily reflect those of the European Union or REA.

Authors

• Kamila Marszałek (Department of Genomics and Forensic Genetics, Pomeranian Medical University in Szczecin, Poland)
• Aleksandra Pisarek-Pacek (Institute of Zoology and Biomedical Research, Jagiellonian University, Poland)
• Balakrishnan Subramanian (Institute of Zoology and Biomedical Research, Jagiellonian University, Poland)
• Michał Kowalski (Department of Genomics and Forensic Genetics, Pomeranian Medical University in Szczecin, Poland)
• Kamila Stanisz (Department of Genomics and Forensic Genetics, Pomeranian Medical University in Szczecin, Poland)
• Maria Stępień (Department of Genomics and Forensic Genetics, Pomeranian Medical University in Szczecin, Poland)
• Miguel Boullón-Cassau (Forensic Genetics Unit, Institute of Forensic Sciences, University of Santiago de Compostela, Spain)
• Mafalda Silva (Forensic Genetics Unit, Institute of Forensic Sciences, University of Santiago de Compostela, Spain)
• Adrián Ambroa-Conde (Forensic Genetics Unit, Institute of Forensic Sciences, University of Santiago de Compostela, Spain)
• Andrzej Ossowski (Department of Genomics and Forensic Genetics, Pomeranian Medical University in Szczecin, Poland)
• Antonia Heidegger (Institute of Legal Medicine, Medical University of Innsbruck, Austria)
• Walther Parson (Institute of Legal Medicine, Medical University of Innsbruck, Austria)
• María Victoria Lareu (Forensic Genetics Unit, Institute of Forensic Sciences, University of Santiago de Compostela, Spain)
• Ana Freire-Aradas (Forensic Genetics Unit, Institute of Forensic Sciences, University of Santiago de Compostela, Spain)
• 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)