Genome-wide methylation profiling and a multiplex construction for the identification of body fluids using epigenetic markers
Introduction
Besides providing information on donor identity, body fluids found at the crime scenes play an important role in forensic investigations [1]. Determination of the type and origin of body fluids can provide important clues for crime scene reconstruction by linking donors and actual criminal acts. However, most currently used catalytic, enzymatic, and immunologic tests for forensic body fluid identification suffer from several limitations, such as low specificity, lack of sensitivity, sample destruction, instability of biomolecules being assayed or incompatibility with downstream individual identification [2].
In recent years, tissue-specific RNA expression and DNA methylation have been suggested as a promising new tool to overcome these limitations and to distinguish between different types of body fluids [3], [4], [5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17], [18]. Especially, recent approaches based on tissue-specific mRNA and miRNA expression have proven to be useful because of their high specificity, ability to be analyzed in multiplex, and unexpected long-term stability [3], [4], [5], [6], [7], [8], [9], [10], [11]. However, particular care and effort to inactivate ubiquitously present ribonucleases still prevents forensic experts from routinely integrating RNA analysis into a forensic casework scheme. On the other hand, the DNA methylation-based method uses the same biological source of DNA for personal identification profiling; thus, special training is not required for an assay. Moreover, body fluid-specific differential DNA methylation showed high specificity and multiplex analysis was also possible [13], [14], [15], [16], [17], [18]. Nevertheless, there is a need to identify more body fluid-specific DNA methylation markers. Many semen-specific markers have been identified, but markers specific to blood, saliva, vaginal fluid, menstrual blood, or skin have been reported only in a few publications and require more validation [12], [14], [18].
Meanwhile, rapid advances in epigenetics make genome-wide DNA methylation profiling accessible to many researchers. For example, Illumina’s HumanMethylation450 BeadChip array provides DNA methylation profiles at more than 450,000 CpG sites using only a small amount of DNA. Therefore, DNA methylation profiling for various types of body fluids using this technique would enable the identification of significant DNA methylation markers that are useful for forensic body fluid identification.
Therefore, we produced genome-wide DNA methylation profiles of blood, saliva, semen, vaginal fluid and menstrual blood using Illumina’s HumanMethylation450 BeadChip array and selected several body fluid-specific DNA methylation marker candidates. Then, we developed a multiplex methylation SNaPshot to analyze DNA methylation profiles at the selected body fluid-specific CpG sites. Body fluid specificity and forensic applicability of the multiplex system were also tested with various samples.
Section snippets
Samples
Blood, saliva, and semen from 84 males and blood, saliva, vaginal fluid, and menstrual blood from 16 female volunteers were collected using procedures approved by the Institutional Review Board of Severance Hospital, Yonsei University in Seoul, Korea. The 100 donors provided written informed consent after the goals, and procedures of the study were explained. Blood was collected in a syringe, and 200 μL aliquots were stored frozen. Saliva and freshly ejaculated semen were collected in a
HumanMethylation450 BeadChip array analysis
We analyzed HumanMethylation450 BeadChip array to obtain genome-wide DNA methylation profiles of 42 body fluid samples including 12 samples each from blood, saliva and semen, and 3 samples each from vaginal fluid and menstrual blood. To exclude the influence of age, analyses for blood, saliva and semen were performed with 36 samples from 18 male donors aged 20–59. Blood donors’ age was 24, 27, 28, 33, 37, 38, 41, 43, 48, 50, 57 and 59 years, saliva donors’ age was 20, 24, 27, 29, 32, 37, 38,
Conclusion
We analyzed the DNA methylation profile of 42 body fluid samples including venous blood, saliva, semen, vaginal fluid and menstrual blood using the Illumina’s Infinium Human Methylation450 BeadChip array and selected 64 body fluid-specific CpG marker candidates. Through further validation using targeted bisulfite sequencing, we confirmed that a subset of CpG markers showed body fluid-specific differential DNA methylation and could be useful biomarkers in forensic body fluid identification. The
Conflict of interest statement
The authors declare that they have no conflict of interest.
Acknowledgements
This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (NRF-2012R1A1A2007031) and a grant from the Ministry of Public Administration and Security through the National Forensic Service (Grant nos.1315000435 and 1315000544).
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