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“Our findings improve the interpretability and rigor of epigenetic analyses using pediatric saliva.” – Meingold Chan, PhD
Understanding Best Practices to Address Saliva Cell Heterogeneity in Pediatric Epigenetic Research
Not all saliva samples are equal: The role of cellular heterogeneity in DNA methylation and epigenetic age analyses with biological and psychosocial factors
Meingold H. Chan, Mandy Meijer, Sarah M. Merrill, Maggie P.Y. Fu, David Lin, Julia L. MacIsaac, Jenna L. Riis, Douglas A. Granger, Elizabeth A. Thomas, Michael S. Kobor. (2025) Psychoneuroendocrinology
Research Highlights:
Saliva is widely used in biomedical population research, including epigenetic analyses to investigate gene-environment interplay and identify biomarkers. Its minimally invasive collection procedure makes it ideal for studies in pediatric populations. However, not all saliva samples are equal—specifically some may contain higher proportion of immune cells, while others may contain higher proportion of epithelial cells. This could make interpretation and replicability of saliva epigenetic findings challenging since DNA methylation, a commonly studied epigenetic marks in human, is highly cell type specific. Cell type composition can drive salivary DNA methylation associations with environments or health as well as epigenetic age acceleration, which is the discrepancy between chronological and biological age derived from DNA methylation. Therefore, whether DNA methylation and epigenetic age associations in saliva primarily originate from immune cells or epithelial cells, or if they persist across saliva samples despite varying cell type proportions still requires more investigations.
The current study used salivary DNA methylation samples obtained from 529 children (mean age=7.26 years, SD=0.26 years) in a community-based cohort, the Family Life Project. Upon stratifying the salivary DNA methylation, samples into three subsamples—primarily epithelial cells, primarily immune cells, and an approximately equal mix of both, we found significantly different EAAs across stratified samples when cell type proportions were not accounted for. In both the contexts of DNA methylation and epigenetic age associations, we detected stronger effects of cotinine concentrations, a tobacco smoke-exposure biomarker, in the subsample with primarily immune cells. We discussed the implications of our findings for the interpretation and replication of epigenetic research involving pediatric saliva samples.
Keywords: Saliva, pediatric epigenetics, DNA methylation, cell heterogeneity
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