Characterization of Whole Genome DNA Methylation Profile Associated with Post-Traumatic Stress Disorder in OIF/OEF Veterans

Emerging knowledge suggests that post-traumatic stress disorder (PTSD) is causally associated with epigenetic changes although its molecular underpinnings are still largely elusive. We postulate that differentially methylated probes mined from peripheral whole blood could be candidates for potential PTSD diagnostic signatures. Working within the Systems Biology PTSD Biomarker Consortium (SBPBC), we investigated a training set comprising of 48 PTSD male veterans (CAPS > 40) and 51 age/ethnicity matched controls (CAPS < 20). Agilent whole genome array detected ~5,600 differentially methylated CpG islands (CpGI) annotated to ~2,800 differentially methylated genes (DMG). The majority (84.5%) of these DMGs was hypermethylated in PTSD veterans. Thereof ~30% promoter-bound DMGs were used for functional analysis. Taking cues from the clinical information, the curated networks were enlisted into four major clusters, namely PTSD-associated complications, PTSD-relevant endocrine signaling, nervous system development and nervous system functions. Enduring impacts of PTSD was manifested by differentially methylated genes enriching networks associated with LTP, fear memory architecture and complications linked to insulin resistance and innate immunity. These networks were further validated by an independent test set comprising of 31/29 PTSD+/- veteran selected using aforementioned screening protocol. Two independent assay platforms presented technical validations. Probing the combined 83/83 PTSD+/- cohort, whole genome array from Illumina, Inc. validated most of the networks of interest. Methylation statuses of eight DMGs relevant to PTSD and comorbidities were confirmed by targeted bisulfite sequencing. This list presents a potential set of PTSD biomarkers of translational potential. Working within the Systems Biology PTSD Biomarker Consortium (SBPBC), we investigated a training set comprising of 48 PTSD male veterans (CAPS > 40) and 51 age/ethnicity matched controls (CAPS < 20). Agilent whole genome array detected ~5,600 differentially methylated CpG islands (CpGI) annotated to ~2,800 differentially methylated genes (DMG). In this study, further validation was conducted by an independent test set comprising of 31/29 PTSD+/- veteran. Clinician-Administered PTSD Scale (CAPS); The IP (sample of interest) samples labeled with Cy5 and the WCE (whole cell extract; reference) labeled with Cy3.

新兴研究证据表明，创伤后应激障碍（post-traumatic stress disorder, PTSD）与表观遗传改变（epigenetic changes）存在因果关联，但其分子基础在很大程度上仍未明晰。我们推测，从外周全血（peripheral whole blood）中挖掘的差异甲基化探针（differentially methylated probes），可作为潜在PTSD诊断特征的候选标志物。本研究依托系统生物学PTSD生物标志物联盟（Systems Biology PTSD Biomarker Consortium, SBPBC）展开工作，对训练集进行分析：该训练集包含48名符合PTSD诊断标准（临床医师PTSD量表（Clinician-Administered PTSD Scale, CAPS）评分>40）的男性退伍军人，以及51名年龄、种族匹配的健康对照（CAPS评分<20）。通过安捷伦（Agilent）全基因组芯片（whole genome array）检测，共检出约5600个差异甲基化CpG岛（differentially methylated CpG islands, CpGI），这些CpG岛对应约2800个差异甲基化基因（differentially methylated genes, DMG）。其中84.5%的差异甲基化基因在PTSD退伍军人样本中呈高甲基化（hypermethylated）状态。我们选取其中约30%的启动子结合型差异甲基化基因（promoter-bound DMGs）开展功能分析。结合临床信息，我们将整理得到的调控网络（curated networks）划分为四大主要聚类（four major clusters）：PTSD相关并发症、PTSD相关内分泌信号通路、神经系统发育及神经系统功能。差异甲基化基因富集的网络涉及长时程增强（long-term potentiation, LTP）、恐惧记忆结构（fear memory architecture）以及与胰岛素抵抗（insulin resistance）和固有免疫（innate immunity）相关的并发症，这体现了PTSD的持久影响。上述调控网络通过独立测试集（independent test set）得到进一步验证：该测试集包含31名PTSD阳性、29名PTSD阴性的退伍军人，均采用前述筛选流程招募。两个独立的检测平台完成了技术验证。针对联合队列（共83名PTSD阳性、83名PTSD阴性受试者）的分析显示，来自Illumina公司（Illumina, Inc.）的全基因组芯片验证了大部分目标调控网络。通过靶向亚硫酸氢盐测序（targeted bisulfite sequencing），我们确认了8个与PTSD及其共病症相关的差异甲基化基因的甲基化状态。本研究提出了一组具备转化应用潜力（translational potential）的PTSD生物标志物候选物。
本研究再次依托系统生物学PTSD生物标志物联盟（SBPBC）开展分析，对包含48名PTSD男性退伍军人（CAPS评分>40）及51名年龄、种族匹配对照（CAPS评分<20）的训练集进行研究。通过安捷伦全基因组芯片检测，共检出约5600个差异甲基化CpG岛（CpGI），对应约2800个差异甲基化基因（DMG）。本研究中，进一步验证工作由独立测试集完成，该测试集包含31名PTSD阳性、29名PTSD阴性的退伍军人。
临床医师PTSD量表（CAPS）；将目标样本（IP，感兴趣的样本，sample of interest, IP）用Cy5标记，全细胞提取物（WCE，参照样本，whole cell extract, WCE）用Cy3标记。