Enhancing forensic casework interpretation through DNA methylation-based analysis: A case study of pooled blood...

In forensic casework, the meticulous investigation of unidentified biological material is critical for developing decisive investigative leads. Integrated genetic and epigenetic profiling allows for the reconstruction of incident scenarios and the assessment of criminal involvement probability. Specifically, DNA methylation-based analyses facilitate the determination of cellular composition, donor age, and smoking status, progressively delineating probable incident circumstances and converging on donor identification. We report a complex case involving a large volume of pooled blood discovered on an apartment staircase half-landing with an unknown donor identity. The investigation was refined using multiple genetic and epigenetic assays, including STR typing, Y-haplotyping, SNaPshot-based body fluid identification, and array-based DNA methylation profiling. Two samples collected at the scene-one presumed blood and one unknown-were confirmed to originate from the same donor: a Korean male belonging to Y-haplogroup O3a2. SNaPshot analysis indicated the presumed blood sample was a blood-saliva mixture, while the unknown sample remained unassigned to any specific fluid type. Array-based cell/tissue deconvolution and unsupervised clustering analysis further revealed that the presumed blood sample was predominantly composed of whole blood with a minor contribution from upper gastrointestinal tissue. Integrating the results of body fluid identification with cell-proportion deconvolution supported the interpretation of hematemesis (vomiting blood), thereby favoring a medical scenario over criminal involvement. The unknown sample exhibited methylation patterns distinct from all candidate tissue groups, possibly reflecting sample quality limitations or tissue mixture. Chronological epigenetic clock analyses estimated the donor's age to be middle-aged (approximately 50-60 years), while smoking status prediction classified the donor as a current smoker. Despite suboptimal sample quality and the complete absence of contextual information, this comprehensive analysis provided detailed donor profiling and incident reconstruction. This case exemplifies the power of integrated genetic and epigenetic methodologies-particularly tissue-of-origin profiling-to generate investigative leads, facilitate the inference of incident circumstances, and provide substantive contributions to forensic investigations.