Identification of tissue specific differential methylation in human body fluids and its potential application in forensics.
In forensic investigations, human biological traces have great potential to serve as strong evidence in placing a suspect at the scene of a crime. In cases where activities leading to the deposition of an individual’s biological materials are disputed, the identification of body fluids and tissues may be crucial. Current catalytic, enzymatic and immunological techniques used to identify body fluids present numerous limitations such as lack of sensitivity and specificity. Hence, forensic scientists are constantly seeking exciting, novel methods to identify and analyse body fluids and aid in the reconstruction of crime scenes. Recently, DNA methylation-based markers have emerged as a reliable tool for identification of body fluids. Genome-wide methylation analysis using high throughput DNA technologies has discovered numerous differentially methylated regions (tDMRs) that differ in levels of methylation in various cell types and tissues. tDMRs may be unmethylated in particular body fluids/tissues yet display varying degrees of methylation in others, hence providing distinguishing characteristics between tissues. tDMRs can be targeted to develop markers for body fluid identification. To date, only a few DNA methylation-based markers have been reported to identify body fluids, most of which have yet to be validated. To enhance the specificity and robustness of DNA methylation-based identification, novel markers are required. Furthermore, as DNA methylation levels have been found to differ between ethnic groups of human populations, it is worthwhile to test previously documented tDMR-based markers on different ethnic groups to determine if there are significant methylation differences. The present study developed new potential tDMRs-based markers to differentiate between saliva, semen, blood and vaginal fluid; and tested the methylation status of previously documented tDMRs for saliva on the diverse South African population. To identify new tDMRs, 1833 differentially expressed (over-expressed) genes, proposed to be regulated by DNA methylation, were identified in four body fluids; namely saliva, blood, semen and vaginal fluid. CpG dinucleotide methylation information from non-target tissues was mapped to genes and heavily methylated CpG islands (CGIs) were targeted for primer design. Sixty-three CGI sequences were selected and analysed for specificity in the human genome, and a total of four CGIs were targeted to design PCR primers. The primers were tested on saliva, blood, semen and vaginal fluid by methylation-specific restriction enzyme (MSRE)–PCR. The study has identified two potential body fluid-specific tDMRs: a tDMR of the HPCAL1 gene was identified as a potential blood-specific hypomethylation marker, and a tDMR of the PTPRS gene is a potential vaginal fluid-specific hypermethylation marker. To our knowledge, this is the first study where these genes have been targeted to identify tDMRs and develop markers for body fluid identification. To determine if DNA methylation levels of previously documented tDMRs differ between four ethnic groups of South Africa, saliva samples were collected from 80 healthy individuals, male and female, belonging to four different ethnic groups of SA; Blacks, Indians, Whites and Coloureds. A multiplex MSRE-PCR assay was used to determine the methylation levels of four tDMRs in the USP49, DACT1, L81528 and PFN3 genes. The methylation levels of all selected tDMRs were highest in the Coloured ethnic group, while the lowest methylation levels were evident in the Black ethnic group. Promising results were found for two tDMRs; DACT1 and L81528, as both these markers displayed significant variations between the Coloured and Black ethnic groups. Significant differences in DNA methylation levels could assist forensic analysts in future, not only to accurately identify saliva but also to narrow down the search of sample donors, link them to the crime or exonerate them with confidence. The analysis of tDMRs represents a novel, efficient and reliable technique to identify biological fluids and tissues and to differentiate between human populations. Future prospects involve validation of new tDMRs based markers on a wider population size and to determine methylation differences in other forensically relevant body fluids among ethnic groups of South Africa.