Development of a multiplex methylation SNaPshot assay for identification of saliva and analysis of sex-specific DNA methylation differences.

Govender, P.*¹, Ghai, M.¹, Kader, F.¹, Okpeku, M.¹, Zhou, M.²

¹ Discipline of Genetics, School of Life Sciences, University of KwaZulu-Natal, Westville, South Africa
² South African Research Institute, Mount Edgecombe, Durban, South Africa

Saliva is one of the most commonly encountered body fluid hence, its accurate identification is imperative for forensic investigations. DNA methylation (DNAm) is emerging as a sensitive method for body fluid identification and methylation SNaPshot (mSNaPshot) is a robust technique which allows rapid quantification of site-specific DNAm levels and can multiplex several markers in a single reaction. DNAm is affected by sex and has been well researched in humans, but mostly from a health perspective. The present study aimed to multiplex three saliva-specific DNAm markers in a mSNaPshot assay and to analyse sex-specific differences in DNAm patterns in saliva of 100 healthy Sub-Saharan Africans. Three saliva-specific CpG sites located in genes, FAM43A,/i> (cg09652652-55d), BCAS4 (Chr20:4844305) and FNDC1 (cg09107912) were selected for the multiplex assay. These markers were referred to as SAL-1 (FAM43A), SAL-2 (BCAS4) and SAL-3 (FNDC1). SAL-1 and SAL-3 displayed hypermethylation and hypomethylation, respectively in saliva. However, SAL-2 showed an unmethylation signal. Males displayed higher average methylation (50.07 %) than females (42.39 %) for all the three markers. Significant sex-specific differences were observed for SAL-1 and SAL-3 (p < 0.0001). Age did not affect DNAm at the three target sites. Our results indicated that sex-specific DNAm differences exist in methylation markers, and SAL-1 and SAL-3 could assist in forensic sex identification of the identified saliva sample by the mSNaPshot assay. DNAm varies in different populations; hence, future studies should target diverse populations from different geographical locations to ascertain the specificity of the findings.

Keywords: DNA methylation, multiplex methylation SNaPshot assay, saliva identification, sex-specific methylation