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

 描述（由申请人提供）：目前对慢性疲劳综合症/肌痛性脑脊髓炎（CFS/ME）的治疗仅依赖于症状管理来改善生活质量，但没有解决疾病发作和进展的根本机制。为了深入了解 CFS/ME 表现的关键生物学靶点，本研究提案的主要目标是确定 CFS/ME 的新生物标志物和治疗靶点，并深入了解疾病的发病和进展。该研究计划旨在使用为我们最近 NIH 资助的研究招募的患者的外周血单核细胞 (PBMC)。这些 PBMC 是从 CFS/ME 患者中分离出来的，并在三个时间点与健康对照相匹配：运动挑战前、最大摄氧量 (VO2 max) 和峰值后四小时。该提案的具体目标包括： 具体目标 1. 确定受 CFS/ME 影响的代谢途径和可能的调节 RNA。基于我们之前的发现，我们的假设是未知的转录本和选择性剪接事件调节负责炎症反应、免疫系统过程、白细胞迁移和细胞发育调节的基因表达的变化。这导致 CFS/ME 疾病活动的变化。我们的目标是通过使用 RNA-seq 方法来识别转录本的丰度、发现新的剪接事件以及新转录本的存在，从而扩大我们的研究工作。这将允许识别由于差异表达转录本和选择性剪接和非编码RNA而产生的生物标志物，以建立一组CFS/ME特异性候选转录本。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，以可能用于 CFS/ME 的诊断和管理。具体目标 2. 研究 CFS/ME 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，我们的目标是确定拷贝数变异 (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，并确定一组 CFS/ME 特异性候选 CNV。我们将使用 Illumina Infinium Human Mmethylation450 BeadChip 微阵列以及基于实时探针的 PCR 来评估 CFS/ME 患者和健康对照之间甲基化模式的差异。我们将结合RNA-seq、CNV和甲基化检测的结果以及相关研究来确定CFS/ME中可能的转录调控机制，从而更好地进行针对性的治疗干预。

 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.