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

基本信息

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
项目类别：
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

项目摘要

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状况，发病率为1/5000，可以影响每个器官系统，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，它们表现为神经变性，癫痫发作，共济失调，慢性进行性外乳突外乳突（CPEO）和降低。儿童期性线粒体疾病通常是由于核基因组中隐性E突变引起的。但是，绝大多数病例仍然没有分子诊断，也没有有效的治疗方法，因此突显了确定驱动这些疾病的遗传畸变的关键需求。我们提出了一种个性化的功能基因组学方法，该方法结合了全基因组测序，患者细胞中的线粒体功能分析以及功能基因组学，以鉴定经过验证的新型线粒体疾病基因。我们将通过在200例中测序整个异常，全面评估导致儿童发作的线粒体脑病的遗传变异谱。这些病例将从我们的同类中从已评估电子传输链活性（ETC）的患者的800多名成纤维细胞系中进行选择，并已预先筛选并显示为已知的线粒体和核基因突变为阴性。序列数据将通过我们的自定义生物信息学管道Athenavar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敲低，cDNA互补研究以及患者和救出细胞中的线粒体功能分析确定。此外，我们还创新了一种植物向齿病毒载体，该载体提供了shRNA和cDNA，我们将使用它来同时将感兴趣基因的内源性“健康”副本击倒，并将同一基因的突变副本传递到健康细胞中。我们将利用这项技术来测试我们测序工作中确定不确定意义的变体的功能，以及通过合作者，BCM诊断实验室和公共领域获得的变体的功能。我们的创新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组合的力量将推动这项工作超出生物信息学停止差距，这是大多数疾病基因发现研究经验的经验。这项工作将产生具有完整外显子序列数据的原发性线粒体疾病患者的前所未有的资源，细胞线粒体功能的系统分析以及功能确定的致病分子缺陷。这些致病基因的阐明将立即改善可疑线粒体疾病儿童的分子诊断潜力。此外，通过确定原发性线粒体脑病的致病基因，我们将通过提供基因和途径来进一步研究这些全球健康问题的病原机制，从而增强关注神经学和神经退行性疾病的科学界，具有更为复杂的病因。