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

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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).