Transcriptional Profiling of Metabolic Pathways in Mitochondrial Disease

描述(由申请人提供)： 原发性线粒体呼吸链(MRC)疾病导致一系列广泛的多系统表现，其特征是能量代谢受损，一生中每5000人中就有1人受到影响。不幸的是，这类疾病的诊断因缺乏一个生物标记物而变得复杂，该生物标记物能够以足够的敏感性或特异性揭示所有病例。因此，尽管进行了艰苦的诊断努力，但对于临床怀疑线粒体疾病的患者，通常无法获得线粒体功能障碍的客观证据。重大的生物信息学进展使得考虑从新的系统生物学角度解决这一诊断挑战是可行的。提出的方法是基于这样的假设，即人类的MRC功能障碍伴随着在生化途径表达变化水平上可识别的细胞适应。这项建议的具体目的是确定在确诊的MRC疾病患者中是否发生跨越生化途径的转录变化。识别人类组织中生物相关途径之间基因表达模式的特定适应性变化将允许追求两个总体目标。首先，阐明原发MRC功能障碍导致临床疾病的生化机制。这将提供对基于基因的线粒体疾病的次生代谢后果的洞察，这可能是治疗干预的结果。第二，根据这些生化途径的表达变化，确定人类原发MRC功能障碍的“特征”。这将允许开发一种基于系统生物学的“生物标记物”，有可能指导人类MRC疾病的分子诊断。长期目标是开发一种微创筛查试验，用于评估可疑患者发生原发MRC疾病的可能性，而不考虑个别病因。这笔赠款建议将生化途径聚类分析应用于临床上有症状的MRC疾病患者的组织中的全球基因组转录图谱。全球基因组表达模式将通过Affymetrix微阵列分析，使用从骨骼肌组织(SubAim A)和从经活检证实的MRC功能障碍并在可能的情况下确认致病突变的患者的微创标本(培养的成纤维细胞系和血液淋巴母细胞系)(SubAim B)中分离的RNA来研究。将对给定的MRC病患者的多个组织进行研究，以努力确定已发现的表达变化是临床上受影响的组织所特有的，还是微创获得的、无症状的组织所共有的。对于每种组织类型，将仔细选择年龄、性别和种族匹配的对照标本。数据分析将主要利用从Silico数据库中精选的生化途径簇的基因集浓缩分析。

DESCRIPTION (provided by applicant): Primary mitochondrial respiratory chain (MRC) disease causes an extensive array of multi-system findings characterized by impaired energy metabolism that affects 1 in 5,000 individuals across a lifetime. Unfortunately, diagnosis of this class of disorders is complicated by the absence of a biomarker that divulges all cases with sufficient sensitivity or specificity. Thus, despite arduous diagnostic efforts, objective evidence of mitochondrial dysfunction is commonly not obtained for clinically suspected mitochondrial disease patients. Significant bioinformatic advances have made it feasible to consider addressing this diagnostic challenge from a novel, systems-biology perspective. The proposed approach is based on the hypothesis that MRC dysfunction in humans is accompanied by cellular adaptations identifiable at the level of biochemical pathway expression alterations. The specific aim of this proposal is to determine if transcriptional alterations across biochemical pathways occur in patients with confirmed MRC disease. Recognition of specific adaptive changes in gene expression patterns among biologically-relevant pathways in human tissues will permit the pursuit of two overall goals. First, is to elucidate biochemical mechanisms by which primary MRC dysfunction results in clinical disease. This will provide insight into secondary metabolic consequences of genetically-based mitochondrial disease which may be amenable to therapeutic intervention. Second, is to identify a "signature" of primary MRC dysfunction in humans based on these biochemical pathway expression alterations. This will permit the development of a systems biology-based "biomarker" with the potential to guide the molecular diagnosis of human MRC disease. The long-term objective is to develop a minimally-invasive screening assay used to estimate the likelihood of primary MRC disease in suspected patients regardless of individual pathogenic cause. This grant proposes to apply biochemical pathway cluster analysis to global genome transcriptional profiling in tissues from clinically symptomatic patients with confirmed MRC disease. Global genome expression patterns will be studied by Affymetrix microarray analysis using RNA isolated both from skeletal muscle tissue (subaim A) and from minimally-invasively obtained specimens (cultured fibroblast cell lines and blood lymphoblastoid cell lines) (subaim B) in patients with biopsy-proven MRC dysfunction and, when possible, confirmed pathogenic mutations. Multiple tissues will be studied from a given MRC disease patient in an effort to determine if identified expression alterations are unique to clinically affected tissue or common to minimally-invasively obtained, asymptomatic tissues as well. Age-, gender-, and race-matched control specimens will be carefully selected for each tissue type. Data analysis will primarily utilize gene set enrichment analysis of biochemical pathway clusters curated from in silico databases.