Background: Adverse Childhood Experiences (ACEs), which include traumatic injury, are associated with poor health outcomes in later life, yet the biological mechanisms mediating this association are unknown. Neurocircuitry, immune system and hormone regulation differ from normal in adults reporting ACEs. These systems could be affected by epigenetic changes, including methylation of cytosine (5mC) in genomic DNA, activated by ACEs. Since 5mC levels influence gene expression and can be long-lasting, altered 5mC status at specific sites or throughout the genome is hypothesized to influence mental and physical outcomes after ACE(s). Human and animal studies support this, with animal models allowing experiments for attributing causality. Here we provide a lengthy introduction and background on 5mC and the impact of early life adversity. Objective: Next we address the issue of a mixture of cell types in saliva, the most accessible biospecimen for 5mC analysis. Typical human bio-specimens for 5mC analysis include saliva or buccal swabs, whole blood or types of blood cells, tumors and post-mortem brain. In children saliva is the most accessible biospecimen, but contains a mixture of keratinocytes and white blood cells, as do buccal swabs. Even in saliva from the same individual at different time points, cell composition may differ widely. Similar issues affect analysis in blood, where nucleated cells represent a wide array of white blood cell types. Unless variations in ratios of these cells between each sample are included in the analysis, results can be unreliable. Methods: Several different biochemical assays are available to test for site-specific methylation levels genome-wide, each producing different information, with high-density arrays being the easiest to use, and bisulfite whole genome sequencing the most comprehensive. We compare results from different assays and use high-throughput computational processing to deconvolve cell composition in saliva samples. Results: Here we present examples demonstrating the critical importance of determining the relative contribution of blood cells versus keratinocytes to the 5mC profile found in saliva. We further describe a strategy to perform a reference-based computational correction for cell composition, and therefore to identify differential methylation patterns due to experience, or for the diagnosis of phenotypes that correlate between traits, such as hormone levels, trauma status and various mental health outcomes. Conclusion: Specific sites that respond to adversity with altered methylation levels in either blood cells, keratinocytes or both can be identified by this rigorous approach, which will then be useful as diagnostic biomarkers and therapeutic targets.

中文翻译：

---

与早期生活经历相关的表观遗传变化：唾液，DNA 甲基化特征的生物样本


背景：不良童年经历（ACE），包括创伤性损伤，与以后生活中不良的健康结果相关，但介导这种关联的生物学机制尚不清楚。报告 ACE 的成人的神经回路、免疫系统和激素调节与正常情况不同。这些系统可能会受到表观遗传变化的影响，包括由 ACE 激活的基因组 DNA 中胞嘧啶 (5mC) 的甲基化。由于 5mC 水平影响基因表达并且可能是持久的，因此假设特定位点或整个基因组中 5mC 状态的改变会影响 ACE 后的精神和身体结果。人类和动物研究支持了这一点，动物模型允许进行归因因果关系的实验。在这里，我们提供了有关 5mC 以及早年逆境影响的详细介绍和背景。目标：接下来我们解决唾液中细胞类型混合物的问题，唾液是最容易进行 5mC 分析的生物样本。用于 5mC 分析的典型人类生物样本包括唾液或口腔拭子、全血或血细胞类型、肿瘤和死后大脑。对于儿童来说，唾液是最容易获得的生物样本，但与口腔拭子一样，含有角质形成细胞和白细胞的混合物。即使在同一个体不同时间点的唾液中，细胞组成也可能存在很大差异。类似的问题影响血液分析，其中有核细胞代表多种白细胞类型。除非分析中包含每个样品之​​间这些细胞比例的变化，否则结果可能不可靠。 方法：有几种不同的生化检测可用于测试全基因组范围内的位点特异性甲基化水平，每种检测都会产生不同的信息，其中高密度阵列最容易使用，亚硫酸氢盐全基因组测序最全面。我们比较不同测定的结果，并使用高通量计算处理来解卷积唾液样本中的细胞组成。结果：在这里，我们提供的示例证明了确定血细胞与角质形成细胞对唾液中 5mC 谱的相对贡献的至关重要性。我们进一步描述了一种对细胞组成进行基于参考的计算校正的策略，从而识别由于经验而导致的差异甲基化模式，或用于诊断与性状之间相关的表型，例如激素水平、创伤状态和各种心理健康状况结果。结论：通过这种严格的方法可以识别血细胞、角质形成细胞或两者中甲基化水平改变的对逆境做出反应的特定位点，然后将其用作诊断生物标志物和治疗靶点。