Considerations for Cell Type Heterogeneity in Pediatric Salivary DNA Methylation Analyses: Comparison of Reference Panels & Stratification by Estimated Cell Type Proportion.

Saliva is a widely used sample in epigenetic research with children due to its non-invasive nature. Since DNA methylation (DNAm) profile is cell type (CT) specific, salivary DNAm associations with exposures may be influenced by CT compositions, which is highly variable in saliva as it contains immune and buccal epithelial cells (BEC). Reference-based CT deconvolution and statistically adjusting estimated CT in DNAm analyses have become an increasingly common practice. However, careful examinations of how different reference panels may affect DNAm results and alternative approaches (e.g., stratification) are lacking. To scrutinize the best analytical strategies on pediatric salivary DNAm, the current study used 529 salivary DNAm samples obtained from children (mean age = 7.26 years, SD = 0.26 years) in the Family Life Project. Our results showed higher estimated CT variability with child than adult reference panels and highlighted the impact of these estimated CT discrepancies on DNAm associations with social variables (socioeconomic status). Stratifying salivary DNAm samples by BEC proportions detected a larger number of significant associations with biological variables (sex) and tissue-specific effect with cotinine level, a tobacco smoke-exposure biomarker. We provide analytical recommendations for future epigenetic research involving pediatric saliva samples.