Sex-specific DNA methylation in saliva from the multi-ethnic Future of Families and Child Wellbeing Study.

The prevalence and severity of many diseases differs by sex, potentially due to sex-specific patterns in DNA methylation. Autosomal sex-specific differences in DNA methylation have been observed in cord blood and placental tissue but are not well studied in saliva or in diverse populations. We sought to characterize sex-specific DNA methylation on autosomal chromosomes in saliva samples from children in the Future of Families and Child Wellbeing Study, a multi-ethnic prospective birth cohort containing an oversampling of Black, Hispanic and low-income families. DNA methylation from saliva samples was analysed on 796 children (50.6% male) at both ages 9 and 15 with DNA methylation measured using the Illumina HumanMethylation 450k array. An epigenome-wide association analysis of the age 9 samples identified 8,430 sex-differentiated autosomal DNA methylation sites (P < 2.4 × 10-7), of which 76.2% had higher DNA methylation in female children. The strongest sex-difference was in the cg26921482 probe, in the AMDHD2 gene, with 30.6% higher DNA methylation in female compared to male children (P < 1 × 10-300). Treating the age 15 samples as an internal replication set, we observed highly consistent results between the ages 9 and 15 measurements, indicating stable and replicable sex-differentiation. Further, we directly compared our results to previously published DNA methylation sex differences in both cord blood and saliva and again found strong consistency. Our findings support widespread and robust sex-differential DNA methylation across age, human tissues, and populations. These findings help inform our understanding of potential biological processes contributing to sex differences in human physiology and disease.

Polymethylation scores for prenatal maternal smoke exposure persist until age 15 and are detected in saliva in the Fragile Families and Child Wellbeing cohort.

Prenatal maternal smoking is associated with low birthweight, neurological disorders, and asthma in exposed children. DNA methylation signatures can function as biomarkers of prenatal smoke exposure. However, the robustness of DNA methylation signatures across child ages, genetic ancestry groups, or tissues is not clear. Using coefficients from a meta-analysis of prenatal smoke exposure and DNA methylation in newborn cord blood, we created polymethylation scores of saliva DNA methylation from children at ages 9 and 15 in the Fragile Families and Child Wellbeing study. In the full sample at age 9 (n = 753), prenatal smoke exposure was associated with a 0.51 (95%CI: 0.35, 0.66) standard deviation higher polymethylation score. The direction and magnitude of the association was consistent in European and African genetic ancestry samples. In the full sample at age 15 (n = 747), prenatal smoke exposure was associated with a 0.48 (95%CI: 0.32, 0.63) standard deviation higher polymethylation score, and the association was attenuated among the European and Admixed-Latin genetic ancestry samples. The polymethylation score classified prenatal smoke exposure accurately (AUC age 9 = 0.77, age 15 = 0.76). Including the polymethylation score increased the AUC of base model covariates by 5 (95% CI: (2.1, 7.2)) percentage points, while including a single candidate site in the AHRR gene did not (P-value = 0.19). Polymethylation scores for prenatal smoking were portable across genetic ancestries and more accurate than an individual DNA methylation site. Polymethylation scores from saliva samples could serve as robust and practical biomarkers of prenatal smoke exposure.

Saliva cell type DNA methylation reference panel for epidemiological studies in children.

Saliva is a widely used biological sample, especially in pediatric research, containing a heterogenous mixture of immune and epithelial cells. Associations of exposure or disease with saliva DNA methylation can be influenced by cell-type proportions. Here, we developed a saliva cell-type DNA methylation reference panel to estimate interindividual cell-type heterogeneity in whole saliva studies. Saliva was collected from 22 children (7-16 years) and sorted into immune and epithelial cells, using size exclusion filtration and magnetic bead sorting. DNA methylation was measured using the Illumina MethylationEPIC BeadChip. We assessed cell-type differences in DNA methylation profiles and tested for enriched biological pathways. Immune and epithelial cells differed at 181,577 (22.8%) DNA methylation sites (t-test p < 6.28 × 10-8). Immune cell hypomethylated sites are mapped to genes enriched for immune pathways (p < 3.2 × 10-5). Epithelial cell hypomethylated sites were enriched for cornification (p = 5.2 × 10-4), a key process for hard palette formation. Saliva immune and epithelial cells have distinct DNA methylation profiles which can drive whole-saliva DNA methylation measures. A primary saliva DNA methylation reference panel, easily implemented with an R package, will allow estimates of cell proportions from whole saliva samples and improve epigenetic epidemiology studies by accounting for measurement heterogeneity by cell-type proportions.