Personalized functional genomics for mitochondrial encephalopathy gene discovery

线粒体脑病基因发现的个性化功能基因组学

• 批准号: 8912553
• 负责人: Penelope E Bonnen
• 金额: $ 56.86万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-15 至 2019-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8912553
• 关键词: Affect; Algorithms; Alleles; Ataxia; Automobile Driving; Bioinformatics; Biological Assay; Candidate Disease Gene; Cell Line; Cells; Child; Childhood; Chromosome Mapping; Chronic; Collaborations; Collection; Communities; Complementary DNA; Complex; Custom; Data; Databases; Defect; Diagnosis; Diagnostic; Diagnostics Research; Disease; Electron Transport; Encephalopathies; Etiology; Evaluation; Exhibits; Fibroblasts; Flow Cytometry; Functional disorder; Gene Mutation; Genes; Genetic; Genetic Variation; Genome; Genomic approach; Goals; Health; Human; Incidence; Individual; Inherited; Laboratories; Lentivirus Vector; Medicine; Membrane Potentials; Mitochondria; Mitochondrial DNA; Mitochondrial Diseases; Molecular; Molecular Diagnosis; Morphology; Muscle hypotonia; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Neurologic; Nuclear; Pathogenicity; Pathology; Pathway interactions; Patients; Penetrance; Physicians; Public Domains; RNA Interference; Reactive Oxygen Species; Relative (related person); Resources; Respiratory Chain; Scientist; Seizures; Technology; Testing; Validation; Variant; Work; base; biobank; body system; clinical phenotype; cohort; college; effective therapy; empowered; exome; exome sequencing; experience; functional genomics; gene discovery; genetic variant; genome-wide; global health; improved; innovation; insight; interest; knock-down; mitochondrial dysfunction; mitochondrial membrane; mutant; nervous system disorder; neuromuscular; novel; pediatric patients; personalized medicine; small hairpin RNA; therapeutic target; variant of unknown significance; vector

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.

描述（由申请人提供）：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，表现为神经退行性变、癫痫发作、共济失调、慢性进行性眼外麻痹（CPEO）和张力低下。儿童期线粒体疾病最常由核基因组的隐性突变引起；然而，绝大多数病例仍然没有分子诊断，也没有有效的治疗方法，因此强调了识别导致这些疾病的遗传畸变的迫切需要。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分析和功能基因组学来鉴定经过验证的新型线粒体疾病基因。我们将通过对200例病例的全外显子组测序，全面评估导致儿童期线粒体脑病的遗传变异谱。这些病例将从800多个患者的成纤维细胞系中选择，这些患者已进行了电子传递链活性（ETC）评估，并已进行了预筛选，并显示已知线粒体和核基因突变呈阴性。序列数据将通过我们定制的生物信息学管道AthenaVar进行分析，该管道注释并优先考虑用于功能研究的变体。基因因果关系将通过RNAi敲除、cDNA互补研究和线粒体功能分析来确定。此外，我们还创新了一种首款慢病毒载体，可传递shRNA和cDNA，我们将使用它同时敲除感兴趣基因的内源性“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异的功能，以及通过合作者、BCM诊断实验室和公共领域获得的变异。我们将患者外显子组测序与线粒体功能分析和功能基因组学研究的创新结合，将推动这项工作超越大多数疾病基因发现研究经历的生物信息学空白。这项工作将产生前所未有的原始线粒体疾病患者资源，包括完整的外显子组序列数据，细胞线粒体功能的系统分析，以及功能确认的致病分子缺陷。这些致病基因的阐明将立即提高疑似线粒体疾病儿童的分子诊断潜力。此外，通过鉴定原发性线粒体脑病的致病基因，我们将为进一步研究这些全球健康问题的发病机制提供基因和途径，从而使科学界能够专注于具有更复杂病因的神经和神经退行性疾病。