Methylation profiling and validation of candidate tDMRs for identification of human blood, saliva, semen and vaginal fluid and its application in forensics.
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Identification of body fluids and tissues is an essential step in forensic investigation because it can be used as strong evidence in identifying suspects and victims. Currently in forensic investigations, catalytic, enzymatic and immunological techniques are used to identify body fluids, however, are limited due to lack of sensitivity and specificity. Hence, researchers are always on the lookout for novel methods that can be used to identify and analyse body fluids. Recently, DNA methylation-based markers have proven to be more sensitive and specific than conventional methods for body fluid identification. Genome-wide methylation studies have demonstrated that tissue specific differentially methylated regions (tDMRs) vary in methylation profiles in various cell types and tissues. The differences in methylation profiles of tDMRs can be targeted to be used as biomarkers to differentiate between body fluids and tissues. To date, only a few DNA methylation-based markers have been reported to identify body fluids. To enhance the specificity and robustness of DNA methylation-based identification, novel markers are required. Additionally, methylation-based markers require further interrogation, to evaluate the stability of their methylation profiles under simulated forensics conditions such as UV light, temperature, rain and microbes, which could cause DNA degradation and affect DNA recovery as well as the methylation status of body fluids. In a previous study, based on differential gene expression in blood, saliva, semen and vaginal fluid, gene body CpG islands were selected, in genes Zinc finger protein 282 (ZNF282), Protein tyrosine phosphatase, receptor S (PTPRS) and Hippocalcin like 1 (HPCAL1), that have potential tDMRs to differentiate between, blood, saliva, semen and vaginal fluid. It was proposed that differential gene expression could be possibly due to differences in methylation patterns. The present study was undertaken to establish the methylation status of potential tDMRs in target body fluids by using methylation specific PCR (MSP) and bisulfite sequencing (BS). In both MSP and BS, the methylation status of 3 genes ZNF282, PTPRS and HPCAL1 were analysed in 10 samples of each body fluid. With MSP analysis the ZNF282 and PTPRS1 tDMR displayed semen-specific hypomethylation while HPCAL1 tDMR showed saliva-specific hypomethylation. The PTPRS 2 tDMR did not differentiate between any body fluids due to presence of methylation and unmethylation for all body fluids. With quantitative analysis by BS the ZNF282 tDMR showed statistically significant difference in overall methylation status between semen and all other body fluids as well as at individual CpG sites (p < 0.05). Therefore, ZNF282 tDMR has the potential to be used to be a semen-specific hypomethylated marker. However, no statistically significant difference in methylation profiles was observed for PTPRS 1 and PTPRS 2 tDMR between body fluids or at individual CpG sites (p > 0.05). The BS study showed that the tDMR for the HPCAL1 gene displayed non-specific amplification therefore was not further analysed. Furthermore, a sensitivity and forensic simulation study was conducted to determine the stability of methylation profiles. To determine the lowest DNA concentration that can be evaluated with MSP, a sensitivity study was conducted using five-fold serial dilution (25, 20, 15, 10, 5, 1 ng) of blood DNA samples. Each DNA dilution was subjected to bisulfite modification, followed by amplification with ZNF282, PTPRS 1, PTPRS 2, and HPCAL1 primers. The results showed that the detection limits were 10 ng for ZNF282 tDMR, 5 ng for PTPRS 1, 15 ng for PTPRS 2, and 5 ng for HPCAL1 tDMR. Thus, it was concluded that a DNA concentration greater than 10 ng would yield successful results with MSP analyses. To evaluate whether environmental conditions has an effect on the stability of methylation profiles of the ZNF282 tDMR, five samples of each body fluid were subjected to five different forensic simulated conditions (dry at room temperature, wet in an exsiccator, outside on the ground, sprayed with alcohol and sprayed with bleach) for 50 days. Following the 50 days, vaginal fluid showed highest DNA recovery under all conditions while semen had least DNA quantity. Under outside on the ground condition, all body fluids except semen showed decrease in methylation level, however, significant decrease in methylation level was observed for saliva. A statistical significant difference was observed for saliva and semen (p < 0.05) in the outside on the ground condition. No differences in methylation level were observed for the ZNF282 tDMR under all conditions for vaginal fluid samples. Thus, ZNF282 tDMR is stable under environmental insults and can be used as reliable semen-specific hypomethylated marker. The analysis of tDMRs represents a unique, efficient and reliable technique that can be used to differentiate between human body fluids. In the future, identification and validation of new tDMRs based markers as well as determining methylation differences in other forensically relevant body fluids will be beneficial for forensics applications.