Identification of novel semen and saliva specific methylation markers and its potential application in forensic analysis.

Differential DNA methylation in human tissues has been widely used to develop markers for body fluid identification in forensics. In the present study, identification of potential tissue specific differentially methylated regions (tDMRs) was based on mining differentially expressed genes in surrogate tissues for blood, saliva, semen and vaginal fluid. Genes specifically over expressed in one of the surrogate tissues viz: blood, salivary glands, testis, prostrate, cervix, uterus and ovary were identified from genome wide expression datasets. We hypothesized that over expression in surrogate tissues for body fluids could be correlated with differential methylation. Methylation information from two methylation datasets, NGSmethDB and ENCODE were integrated and heavily methylated gene body CpG islands (CGI) representing the body fluids were extracted. From a total of 53 potential genes the present study reports, two genes, ZNF282 and HPCAL1 which were preferentially expressed in cervix with comparatively reduced expression in other surrogate tissues. Methylated CGIs were targeted to design primers for methylation specific PCR (MSP) and bisulphite sequencing (BS). The ZNF282 CpG sites displayed semen-specific hypomethylation while HPCAL1 CpGs showed saliva-specific hypomethylation. Clone-based bisulphite sequencing also revealed significant hypomethylation in the target body fluids. To evaluate the stability of methylation profiles, the ZNF282 tDMR was tested and each body fluid was subjected to five different forensic simulated conditions (dry at room temperature, wet in an exicator, outside on the ground, sprayed with alcohol and sprayed with bleach) for 50 days. Under the condition "outside on the ground", saliva showed a significant decrease in methylation level by bisulphite sequencing analysis over time. Complete methylation profiles were obtained only for vaginal fluid under all conditions and no differences in methylation levels were observed for this fluid after 50 days. Thus, ZNF282 and HPCAL1 tDMRs can be used as reliable semen and saliva identification markers respectively.