DNA Methylation Based Biomarkers and Epigenetic Regulation in IBS

 描述(申请人提供)：肠易激综合征(IBS)是一种慢性胃肠道疾病，在美国，以女性为主，影响高达15%的成年人。症状包括慢性或复发性腹痛与腹泻、便秘或两者兼而有之。它被认为是一种与大脑-肠道相互作用改变有关的应激敏感障碍。目前缺乏能够可靠诊断IBS或监测治疗反应的生物标记物。目前的建议是基于我们的初步数据，即表观遗传标记，即DNA甲基化，可以区分IBS患者和健康个体。DNA甲基化是控制脊椎动物基因功能的关键表观遗传机制。它已经成为将基因-环境相互作用与长期行为发展联系起来的主要机制，特别是在IBS等复杂疾病中。我们对有限数量的受试者(IBS：n=12，健康对照组：n=12)外周血单个核细胞(PBMC)全基因组DNA甲基化的初步数据发现，一组表观遗传标记位于SSPO、RNF39、GSTM1、GSTM5、TPPP和SNCAIP等基因上，可能会将IBS患者与健康对照组区分开来。基因本体论分析表明，参与神经肽途径的基因丰富。我们使用亚硫酸氢盐测序验证了这些基因中CpG位点的差异甲基化。然而，这些发现需要在更大的队列中重复和验证。因此，在这里提出的研究旨在确定强大的表观遗传生物标志物，以准确诊断IBS，并获得对这种疾病的发病机制的关键见解。我们的目标是：1)利用全基因组DNA甲基化图谱在IBS患者的外周血单个核细胞(PBMC)中识别基于DNA甲基化的生物标志物。2)研究IBS患者和HCS患者结肠组织全基因组甲基化及表达差异。在目标1中，我们将在更大的独立队列中对IBS患者(N=108)和对照组(N=36)进行全基因组DNA甲基化分析，并使用一种可用于IBS常规诊断的负担得起的技术(甲基光聚合酶链式反应)在样本子集中复制选定标记的差异甲基化。在目标2中，我们对收集自IBS患者(N=108)和对照组(N=36)的大肠粘膜活检组织进行全基因组DNA甲基化和基因表达谱分析，以研究表观基因失控及其相关途径，这将为IBS的病理生理学研究提供重要的见解。

 DESCRIPTION (provided by applicant): Irritable bowel syndrome (IBS) is a chronic gastrointestinal disorder that affects up to 15% of adults, predominantly women, in the United States. Symptoms include chronic or recurrent abdominal pain associated with diarrhea, constipation or both. It is considered a stress-sensitive disorder that is associated with altered brain-gut interactions. Biomarkers that can reliably diagnose IBS or monitor treatment response are currently lacking. The current proposal is based on our preliminary data that epigenetic markers, namely DNA methylation, can distinguish IBS patients from healthy individuals. DNA methylation is a key epigenetic mechanism that governs vertebrate gene function. It has emerged as a leading mechanism linking gene- environment interactions to long-term behavioral development, particularly in complex disorders such as IBS. Our preliminary data on genome-wide DNA methylation for peripheral blood mononuclear cells (PBMCs) in a limited number of subjects (IBS: N=12, healthy controls: N=12) identified a set of epigenetic markers located on genes including SSPO, RNF39, GSTM1, GSTM5, TPPP and SNCAIP that can potentially distinguish IBS patients from healthy controls. Gene ontology analysis showed an enrichment of genes involved in neuropeptide pathways. We validated the differential methylation of CpG sites in these genes using bisulphite sequencing. However, these findings need to be replicated and validated in a larger cohort. Therefore, the studies proposed here are intended to identify robust epigenetic biomarkers for an accurate diagnosis of IBS and to gain critical insights into the pathogenesis of this disorder. Our aims are to: 1) Identify DNA methylation based biomarkers using genome-wide DNA methylation profiling in peripheral blood mononuclear cells (PBMCs) in IBS. 2) Study genome-wide methylation and expression differences in colon tissue of IBS patients and HCs. In Aim 1, we will conduct a genome-wide DNA methylation profiling in a larger, independent cohort of IBS patients (N=108) and controls (N=36) replicate the differential methylation of selected markers in a subset of samples using an affordable technique (MethyLight PCR) that can be employed for the routine diagnosis of IBS. In Aim 2, we propose genome-wide DNA methylation and gene expression profiling of banked colonic mucosal biopsies collected from IBS patients (N=108) and controls (N=36) to investigate epigenetically deregulated genes and associated pathways, which can give important insights on pathophysiology of IBS.