Analysis of sex-specific DNA methylation differences in saliva by multiplex SNaPshot assay

DNA methylation (DNAm) analysis is emerging as a sensitive method for studying sex-specific differences in humans. 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. Sex-specific DNAm differences have been observed in blood, placental and brain tissue, but it is not well studied in saliva and African populations. Therefore, this study was undertaken to multiplex three saliva-specific CpG sites in a mSNaPshot assay to analyze sex-specific differences in DNAm patterns of 100 healthy Sub-Saharan Africans. Three saliva-specific CpG sites located in genes, FAM43A (cg09652652-55d), BCA S 4 (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. Overall, males displayed higher average methylation (50.07 %) than females (42.39 %) for all three markers. Significant sex-specific DNAm 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 body fluids, and SAL-1 and SAL-3 could assist in sex identification of a saliva sample by 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. We also highlight that sex-specific differential methylation holds great potential for identifying sex-specific biomarkers, improving diagnostics, early disease detection and monitoring, and development of target therapeutics. • Three CpG sites were multiplexed in a mSNaPshot assay for analysis of sex-specific methylation. • Cg09652652-55d ( FAM43A ) displayed hypermethylation in saliva. • Cg09107912 ( FNDC1 ) displayed hypomethylation in saliva. • Chr20:4844305 ( BCAS4 ) showed an unmethylation signal. • FAM43A and FNDC1 exhibited significant sex-specific DNA methylation differences.