Genomic approach to find novel biomarkers and mechanisms of CFS/ME

 描述（由申请人提供）：目前慢性疲劳综合征/肌痛性脑脊髓炎（CFS/ME）的管理仅依赖于症状管理来改善生活质量，但未解决疾病发作和进展的潜在机制。为了深入了解CFS/ME表现所涉及的关键生物学靶点，本研究提案的主要目标是确定CFS/ME的新型生物标志物和治疗靶点，并深入了解疾病的发病和进展。这项研究计划的目的是使用外周血单核细胞（PBMC）从我们最近的NIH资助的研究招募的患者。这些PBMC已在三个时间点从CFS/ME患者和匹配的健康对照中分离出来-在运动激发前，在最大努力（VO 2 max）和最大努力后4小时。本提案的具体目标包括：确定CFS/ME受影响的代谢途径和可能的调控RNA。基于我们以前的研究结果，我们的假设是，未知的转录本和选择性剪接事件调节基因表达的变化，负责炎症反应，免疫系统过程，白细胞迁移和细胞发育的调节。这导致CFS/ME疾病活动的变化。我们的目标是扩大我们的研究工作，通过使用RNA-seq方法来识别转录本的丰度，发现新的剪接事件和新转录本的存在。这将允许鉴定作为差异表达的转录物和选择性剪接和非编码RNA的结果的生物标志物，以建立一组CFS/ME特异性候选转录物。然后，我们将使用NanoString Technologies的nCounter系统验证它们，并创建一组转录本，可能用于CFS/ME的诊断和管理。为了研究CFS/ME中转录调控的可能机制，为了更好地了解CFS/ME患者基因表达变化的原因，我们的目的是确定拷贝数变异（CNV）和基因组DNA甲基化的变化。我们将使用Agilent SurePrint G3人类基因组CGH+SNP微阵列来确定CFS/ME中的CNV，并鉴定一组CFS/ME特异性候选CNV。我们将使用Illumina Infinium Human Methylation 450 BeadChip微阵列以及基于探针的实时PCR来评估CFS/ME患者和健康对照之间甲基化模式的差异。我们将联合收割机结合RNA-seq、CNV和甲基化检测的结果及相关性研究，探讨CFS/ME可能的转录调控机制，为更好的靶向治疗提供依据。

 DESCRIPTION (provided by applicant): Current management of Chronic Fatigue Syndrome/Myalgic Encephalomyelitis (CFS/ME) relies solely on symptom management to improve quality of life but does not address the underlying mechanisms of disease onset and progression. In an effort to provide insight into the key biological targets involved CFS/ME presentation, the main objective of this research proposal is to identify novel biomarkers and therapeutic targets of CFS/ME and provide insight into disease onset and progression. This research proposal aims to use Peripheral Blood Mononuclear Cells (PBMC) from patients recruited for our recently NIH funded research. These PBMC have been isolated from CFS/ME patients and matched healthy controls at three time points - before exercise challenge, at the peak of effort (VO2 max) and four hours after the peak effort. Specific Aims of this proposal include: Specific Aim 1. Identify metabolic pathways affected at CFS/ME and possible regulatory RNAs. Based on our previous findings, our hypothesis is that unknown transcripts and alternative splicing events regulate changes in expression of genes responsible for inflammatory response, immune system processes, leukocyte migration, and regulation of cell development. This is leading to changes in CFS/ME disease activity. We aim to expand our research efforts, by using an RNA-seq approach to identify the abundance of transcripts, discover new splicing events, and presence of novel transcripts. This will allow identifying biomarkers as a result of differentially expressed transcripts and alternative splicing and non-coding RNAs to establish a set of CFS/ME specific candidate transcripts. We will then validate them using NanoString Technologies' nCounter system and create a panel of transcripts to potentially be used in the diagnosis and management of CFS/ME. Specific Aim 2. To investigate possible mechanisms of transcriptional regulation in CFS/ME. To better understand causes of changes in gene expression in patients with CFS/ME, we aim to identify changes in copy number variation (CNV) and genomic DNA methylation. We will use Agilent SurePrint G3 Human Genome CGH+SNP microarrays to determine CNV in CFS/ME and identify a set of CFS/ME-specific candidate CNVs. We will use Illumina Infinium Human Methylation450 BeadChip microarrays as well as real-time probe-based PCR to evaluate differences in the methylation patterns between CFS/ME patients and healthy controls. We will combine the results of RNA-seq, CNV and methylation assays and research correlation to determine possible mechanisms of transcriptional regulation involved in CFS/ME, which will lead to the better targeted therapeutic intervention.