ABSTRACT

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.

摘要

唾液是一种广泛使用的生物样本，尤其是在儿科研究中，它含有免疫细胞和上皮细胞的异质混合物。暴露或疾病与唾液 DNA 甲基化的关联可能受细胞类型比例的影响。在这里，我们开发了一个唾液细胞类型 DNA 甲基化参考面板来估计整个唾液研究中的个体间细胞类型异质性。从 22 名儿童（7-16 岁）中收集唾液，并使用尺寸排阻过滤和磁珠分选将其分选为免疫细胞和上皮细胞。使用 Illumina MethylationEPIC BeadChip 测量 DNA 甲基化。我们评估了 DNA 甲基化谱中的细胞类型差异，并测试了丰富的生物途径。免疫细胞和上皮细胞在 181,577 (22.8%) 个 DNA 甲基化位点处存在差异（t 检验 p < 6.28 × 10^-8 )。免疫细胞低甲基化位点被映射到富含免疫途径的基因（p < 3.2 × 10 ^-5）。上皮细胞低甲基化位点富集角化（p = 5.2 × 10 ^-4），这是硬调色板形成的关键过程。唾液免疫细胞和上皮细胞具有不同的 DNA 甲基化谱，可以驱动全唾液 DNA 甲基化测量。使用 R 软件包轻松实施的主要唾液 DNA 甲基化参考面板将允许估计整个唾液样本中的细胞比例，并通过考虑细胞类型比例的测量异质性来改进表观遗传流行病学研究。