The last imprint: Differential methylation assay for forensic identification of brain tissue and death determination.

Background: The ability to test samples for the presence of specific tissues is useful for numerous forensic applications. More specifically, the identification of vital organ remains, such as the brain, in a crime scene or battlefield, can assist in determining the death of a missing person. In many cases, tissue samples are of insufficient quality or quantity for the application of histological methods, leaving forensic labs mostly restricted to immunological and catalytic assays designed to identify blood, semen, and saliva. Recent studies have suggested expression profiling-based methods for tissue and bodily fluid identification. Objective: We present a methylation-based assay for the detection of brain tissue in forensic samples. Methods: Genome-wide methylation data from 12 human tissues were analyzed to identify CpG sites uniquely methylated in brain tissue. Four candidate regions were selected based on high inter-tissue specificity and low intra-tissue variability. Targeted assays were developed using bisulfite conversion, polymerase chain reaction amplification, and next-generation sequencing, and validated based on reference tissues, mixtures, and environmentally degraded DNA samples. Results: Four regions displayed consistent brain-specific methylation with >94% single-read accuracy and complete sample-level discrimination at ≥5% brain DNA. The assay retained diagnostic performance in mixed and degraded samples, demonstrating robustness under typical forensic conditions. Conclusion: This study presents a sensitive and specific methylation-based assay for brain tissue identification. The approach enables reliable detection in degraded or composite materials and supports future integration of epigenetic biomarkers into forensic workflows for organ-source attribution.