Using cell and fly models to understand gene function in undiagnosed diseases

使用细胞和果蝇模型了解未确诊疾病的基因功能

• 批准号: 8679838
• 负责人: Brett Harrison Graham
• 金额: $ 23.48万
• 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: 2014-09-16 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8679838
• 关键词: Alleles; Biological; Biological Models; Biological Process; Candidate Disease Gene; Cell Line; Cell model; Cells; Characteristics; Clinical; Complex; DNA Sequence; Development; Diagnostic; Diagnostic tests; Disease; Disease model; Drosophila genus; Drosophila melanogaster; Ectopic Expression; Engineering; Fibroblasts; Future; Gene Expression Profile; Gene Targeting; Generations; Genes; Genetic; Genetic Engineering; Genetic Models; Genomics; Goals; Hela Cells; Human; Metabolic; Mitochondria; Modeling; Mutation; Nervous system structure; Neurologic; Pathway interactions; Patients; Phenotype; Population; Proteins; Proteomics; Published Comment; RNA Interference; Reagent; Research; Technology; Time; United States National Institutes of Health; Validation; Variant; Writing; abstracting; base; clinically significant; cohort; exome sequencing; fly; gain of function; gene function; genetic manipulation; genetic variant; improved; innovation; insight; interest; meetings; member; metabolomics; mutant; next generation sequencing; novel; pleiotropism; programs; public health relevance; research study; tool; transcriptomics

DESCRIPTION (provided by applicant): Abstract With the rapid development and widespread availability of next generation sequencing over the past decade, whole exome sequencing (WES) has become a popular and quite effective tool for identifying novel disease genes. The more recent emergence of WES as a clinical diagnostic tool over the past couple of years is now broadening the appreciation for wide phenotypic spectrums and pleiotropy for disease genes through the identification of known or strongly-suspected pathogenic alleles in patients with atypical or novel phenotypic presentations. However, a significant challenge for WES in either the research or clinical arena is the interpretation and validation of novel variants of uncertain clinical significance (VUS) in known or candidate disease genes. Study of gene function in patient cells and model systems are important components of the validation of disease genes, but the tools for generation of allele-specific mutants in multicellular model systems has historically been cumbersome and time consuming. Recent advancements in Drosophila melanogaster genomic tools pioneered by members of the applicant team now make it feasible to engineer specific mutations in almost any locus of interest in a high throughput fashion. This proposal is based on the hypothesis that combining and integrating phenotypic profiles of both human fibroblasts from patients with undiagnosed disorders manifesting neurological and/or metabolic phenotypes, and engineered candidate disease allele-specific Drosophila mutants will provide novel insights into gene function(s) and illuminate disease mechanisms. Genes with candidate disease alleles in patients manifesting neurological and/or metabolic phenotypes identified by WES through the NIH Undiagnosed Diseases Program and for which primary fibroblast cell lines are available will be prioritized for study. Specific Aim #: Characterize the transcriptomic, metabolomic, and mitochondrial energetic profiles of patient primary fibroblasts compared to a cohort of normal primary fibroblast cell lines. Specific Aim #2: Using state of the art technologies available for Drosophila, generate candidate allele-specific fl mutants using gene targeting or genomic engineering, and characterize cell biological, neurological, mitochondrial energetic and electrophysiological phenotypes in comparison to isogenic wild type and deficiency null mutants. Specific Aim #3: Integrate the phenotypic characterizations of patient cell lines and the orthologous allele-specific Drosophila mutants to identify conserved gene functions and elucidate disease mechanisms. The ultimate goal is to develop and optimize a pipeline that can be used in future projects as part of the Undiagnosed Diseases Gene Network to obtain large sets of patient-specific cell lines together with orthologous fly disease models that can be analyzed in depth to elucidate gene function(s).

描述（由申请人提供）：摘要随着在过去十年中下一代测序的快速发展和广泛可用性，全外显子组测序（WES）已经成为用于鉴定新疾病基因的流行且相当有效的工具。在过去的几年中，WES作为一种临床诊断工具的最近出现，现在通过在具有非典型或新表型表现的患者中鉴定已知或强烈怀疑的致病等位基因，扩大了对疾病基因的广泛表型谱和多效性的认识。然而，WES在研究或临床竞技场中的一个重大挑战是对已知或候选疾病基因中具有不确定临床意义（VUS）的新变体的解释和验证。患者细胞和模型系统中基因功能的研究是疾病基因验证的重要组成部分，但在多细胞模型系统中产生等位基因特异性突变体的工具历来是繁琐和耗时的。由申请人团队的成员开创的黑腹果蝇基因组工具的最新进展现在使得以高通量方式在几乎任何感兴趣的基因座中工程化特定突变成为可能。该提议基于以下假设：组合和整合来自患有表现出神经和/或代谢表型的未诊断疾病的患者的人成纤维细胞的表型谱，以及工程化的候选疾病等位基因特异性果蝇突变体，将提供对基因功能的新见解并阐明疾病机制。WES通过NIH未诊断疾病项目鉴定出的表现出神经和/或代谢表型的患者中具有候选疾病等位基因的基因，以及原代成纤维细胞系可用的基因将优先用于研究。具体目标编号：与一组正常原代成纤维细胞系相比，表征患者原代成纤维细胞的转录组学、代谢组学和线粒体能量谱。具体目标#2：使用可用于果蝇的现有技术，使用基因靶向或基因组工程产生候选等位基因特异性fl突变体，并与同基因野生型和缺陷无效突变体相比表征细胞生物学、神经学、线粒体能量和电生理学表型。具体目标#3：整合患者细胞系的表型特征和正交等位基因特异性果蝇突变体，以确定保守的基因功能和阐明疾病机制。最终目标是开发和优化可用于未来项目的管道，作为未诊断疾病基因网络的一部分，以获得大量患者特异性细胞系以及可以深入分析以阐明基因功能的直链果蝇疾病模型。