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

描述(申请人提供)：摘要在过去的十年中，随着下一代测序技术的迅速发展和广泛应用，全外显子组测序(WES)已经成为一种流行且相当有效的工具来识别新的疾病基因。在过去的几年里，WES作为一种临床诊断工具的出现，通过在具有非典型或新的表型表现的患者中识别已知或强烈怀疑的致病等位基因，扩大了对疾病基因的广表型谱和多效性的认识。然而，WES在研究或临床领域面临的一个重大挑战是对已知或候选疾病基因中不确定临床意义(VUS)的新变体的解释和验证。研究患者细胞和模型系统中的基因功能是疾病基因验证的重要组成部分，但在多细胞模型系统中生成等位基因特异性突变的工具历来都是繁琐和耗时的。申请团队成员开创的黑腹果蝇基因组工具的最新进展，现在使以高通量方式设计几乎任何感兴趣基因座的特定突变成为可能。这一建议基于这样一个假设，即结合和整合来自表现为神经和/或代谢表型的未诊断疾病患者的人类成纤维细胞的表型特征，以及设计的候选疾病等位基因特异性果蝇突变体，将为基因功能提供新的见解并阐明疾病机制(S)。WES通过NIH未诊断疾病计划确定的具有神经和/或代谢表型的患者中具有候选疾病等位基因的基因将被优先进行研究。具体目标#：与一组正常的原代成纤维细胞系相比，研究患者原代成纤维细胞的转录、代谢和线粒体能量谱特征。具体目标#2：利用可用于果蝇的最新技术，通过基因打靶或基因组工程产生候选的等位基因特异性fl突变体，并与同基因野生型和缺陷型突变体进行比较，鉴定细胞生物学、神经学、线粒体能量和电生理表型。具体目标#3：整合患者细胞系和同源等位基因特异的果蝇突变体的表型特征，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一条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，以获得大量针对患者的细胞系以及可以深入分析以阐明基因功能的同源苍蝇疾病模型(S)。

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).