Personalized functional genomics for mitochondrial encephalopathy gene discovery

描述(申请人提供)：线粒体疾病是一种常见的遗传性疾病，发病率为1/5000，可影响每个器官系统，因此表现出广泛的临床表型。最常见的是神经和神经肌肉功能障碍，表现为神经变性、癫痫发作、共济失调、慢性进行性眼外肌瘫痪(CPEO)和低眼压。儿童期起病的线粒体疾病最常见的原因是核基因组中的隐性e突变；然而，绝大多数病例仍然没有得到分子诊断，也没有有效的治疗方法，因此强调了确定导致这些疾病的遗传异常的迫切需要。我们提出了一种个性化的功能基因组学方法，结合全基因组测序、患者细胞线粒体功能图谱和功能基因组学来识别有效的新线粒体疾病基因。我们将通过对200例患者的全部外显子进行测序，全面评估导致儿童期起病的线粒体脑病的基因变异谱。这些病例将从我们的800多个成纤维细胞系中挑选出来，这些成纤维细胞系来自已经进行了电子传输链活性(ETC)评估的患者，并且已经过预筛选，显示出已知的线粒体和核基因突变为阴性。序列数据将由我们定制的生物信息学管道AthenaVar进行分析，该管道为功能研究注释和区分变体的优先顺序。基因因果关系将通过RNAi敲除、cDNA互补研究和患者和被拯救细胞的线粒体功能图谱来确定。此外，我们还创新了一种首创的慢病毒载体，它可以传递shRNA和cDNA，我们将使用它来同时击倒目标基因的内源性“健康”拷贝，并将同一基因的突变拷贝传递到健康细胞中。我们将利用这项技术来测试在我们的测序工作中发现的不确定意义的变体以及通过合作者、BCM诊断实验室和公共领域获得的那些变体的功能。我们将患者外显子组测序与线粒体功能图谱和功能基因组研究的创新结合在一起，将推动这项工作超越大多数疾病基因发现研究所经历的生物信息学权宜之计。这项工作将产生一个史无前例的原始线粒体疾病患者资源，具有完整的外显子组序列数据，系统地描述细胞线粒体功能，并在功能上确认致病分子缺陷。这些致病基因的阐明将立即提高对疑似线粒体疾病儿童的分子诊断潜力。此外，通过识别原发线粒体脑病的致病基因，我们将通过提供基因和途径，进一步研究这些全球健康问题的致病机制，从而增强专注于神经和神经退行性疾病的科学界的力量，这些疾病具有更复杂的病因。

DESCRIPTION (provided by applicant): Mitochondrial disease is a commonly occurring inherited condition, incidence 1/5000, which can affect every organ system and thus exhibits a broad range of clinical phenotypes. The most common are neurological and neuromuscular dysfunction that manifest as neurodegeneration, seizures, ataxia, chronic progressive external opthalmoplegia (CPEO), and hypotonia. Childhood-onset mitochondrial disease most often results from recessive e mutations in the nuclear genome; however, the vast majority of cases remain without a molecular diagnosis and no effective treatments thus underscoring the critical need to identify the genetic aberrations driving these disorders. We propose a personalized functional genomics approach combining genome-wide sequencing, mitochondrial functional profiling in patient cells, and functional genomics to identify validated novel mitochondrial disease genes. We will comprehensively assess the spectrum of genetic variation contributing to childhood-onset mitochondrial encephalopathy through sequencing whole exomes in 200 cases. These cases will be selected from our cohort of over 800 fibroblast cell lines from patients that have been assessed for electron transport chain activity (ETC) and have been pre-screened and shown to be negative for known mitochondrial and nuclear gene mutations. Sequence data will be analyzed by our custom bioinformatics pipeline, AthenaVar, that annotates and prioritizes variants for functional studies. Gene causality will be determined through RNAi knock down, cDNA complementation studies and mitochondrial functional profiling in patient and rescued cells. Additionally, we have innovated a first-in-kind lentiviral vector that delivers a shRNA and cDNA which we will use to simultaneously knock down the endogenous 'healthy' copy of a gene of interest and deliver a mutant copy of the same gene into healthy cells. We will utilize this technology to test the functionality of variants of uncertain significance identified in our sequencing efforts as well as those obtained through collaborators, the BCM diagnostic laboratory, and the public domain. The power of our innovative combination of patient exome sequencing with mitochondrial functional profiling and functional genomics studies will propel this work beyond the bioinformatics stop gap that most disease gene discovery studies experience. This work will generate an unprecedented resource of primary mitochondrial disease patients with complete exome sequence data, systematic profiling of cellular mitochondrial function, and functionally-confirmed pathogenic molecular defects. The elucidation of these pathogenic genes will immediately improve the molecular diagnostic potential for children with suspected mitochondrial disease. Moreover, by identifying the pathogenic genes for primary mitochondrial encephalopathy we will empower the scientific community focused on neurological and neurodegenerative disorders, which have a more complex etiology, by delivering genes and pathways for further study of the pathogenetic mechanisms of these global health problems.