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/肌肉痛脑脊髓炎(CFS/ME)的治疗完全依赖症状管理来提高生活质量，但没有解决疾病发生和发展的潜在机制。为了深入了解CFS/ME涉及的关键生物学靶点，本研究提案的主要目的是确定CFS/ME的新生物标记物和治疗靶点，并为疾病的发生和发展提供洞察力。这项研究提案旨在使用我们最近由NIH资助的研究招募的患者的外周血单个核细胞(PBMC)。从CFS/ME患者和健康对照组的三个时间点--运动前、最大摄氧量(VO2 Max)和运动后4小时分离出这些PBMC。这项建议的具体目标包括：具体目标1.确定CFS/ME受影响的代谢途径和可能的调控RNA。根据我们以前的发现，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。这导致了CFS/ME疾病活动的变化。我们的目标是扩大我们的研究努力，通过使用RNA-SEQ方法来确定转录本的丰度，发现新的剪接事件，以及新转录本的存在。这将允许识别生物标记物作为差异表达转录本和选择性剪接和非编码RNA的结果，以建立一组CFS/ME特定的候选转录本。然后，我们将使用NanoString Technologies的nCounter系统验证它们，并创建一个可能用于CFS/ME诊断和管理的转录本小组。具体目的2.探讨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特异的候选CNV。我们将使用Illumina Infinium人类甲基450珠芯片微阵列以及基于实时探针的聚合酶链式反应来评估CFS/ME患者和健康对照组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研究相关性来确定CFS/ME可能的转录调控机制，这将导致更好的靶向治疗干预。

 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.