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: 8816784
• 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/8816784
• 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; Genomics; Goals; 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; public health relevance; small hairpin RNA; therapeutic target; 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.

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.