Differentially methylated embryonal Fyn-associated substrate (EFS) gene as a blood-specific epigenetic marker and its potential application in forensic casework

- A novel blood-specific differentially methylated locus (EFS) was investigated.
- Most CpGs, especially CpG4, was found highly methylated in whole blood.
- EFS methylation has the potential to differentiate between whole and menstrual blood.
- The designed bisulfite pyrosequencing assay is both accurate and reproducible.
- The method's applicability to casework was demonstrated via aged and mixed stains.

DNA methylation patterns have the ability to reveal the activities of genes within a certain tissue at a particular time point. Tissue-specific DNA methylation patterns have been previously investigated for their applicability in the identification of forensically relevant body fluids, however there is still a lack in robust markers. While following a genome-wide scale investigation has a great potential to reveal useful tissue-specific changes, a gene-targeted approach can also lead to significant outcomes, especially in genomic locations not included in the genome-wide experiments. In this study, the potential of the candidate embryonal Fyn-associated substrate (EFS) gene for the positive identification of whole blood was investigated. For this purpose, the methylation profile of a selected genomic region containing a total of 10 CpG sites was analysed in 124 individuals via bisulfite pyrosequencing. Volunteers donated various forensically relevant tissues, including whole blood, saliva, seminal fluid, vaginal fluid and menstrual secretion. Whole blood showed the highest levels of DNA methylation (mean = 0.67), while semen samples were found to be very low methylated (mean = 0.06). The remaining tissues demonstrated partial mean methylation levels; more specifically, saliva − 0.43, vaginal fluid − 0.22 and menstrual blood − 0.22. One out of the 10 analysed CpG sites, CpG4, showed to be more robust, resulting in not only the highest methylation difference between blood and the rest of the tissues, but also the lowest inter-individual methylation difference. The proposed pyrosequencing assay was found to be accurate, linear and reproducible. Lastly, the method's applicability to forensic casework was assessed via the analysis of very old bloodstains stored up to 18 years, blood DNA samples stored long-term up to 9 years, mixed stains as well as other 'forensic-like' samples. In the majority of cases the expected methylation ratios were obtained indicating a stable DNA methylation pattern, however caution is necessary when analysing low quantity and/or quality samples due to potential stochastic effects. Future validation experiments can shed more light into the usefulness of EFS locus as a promising blood-specific epigenetic marker.