Gene Interaction in Development and Disease

University Of Michigan At Ann Arbor, Ann Arbor MI

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Abstract

ABSTRACT Neurodegeneration and peripheral neuropathy are the major clinical consequence of human mutations affecting biosynthesis of the low abundance signaling lipid PI(3,5)P2. We have identified human and mouse mutations in the 3 major proteins of the PI(3,5)P2 biosynthetic complex: the phosphatase FIG4, the phosphokinase FAB1 (gene symbol PIKFYVE), and the scaffold protein VAC14. Patients with Yunis-Varón Syndrome, a multi-system disorder that can affect cardiac function, are null for FIG4. We will examine cardiac function in Fig4 mutant mice using physiological and metabolic phenotyping. Missense mutations of FIG4 result in peripheral neuropathy and epilepsy with polymicrogyria. We will evaluate the functional effects of missense variants of FIG4, VAC14 and PIKFYVE that have been identified in exome sequences of affected individuals, using novel assays in HAP1 human cells with targeted mutations of FIG4 and VAC14. FACS sorting will be used to identify cells containing enlarged acidic endolysosomes. The proportion of cells with enlarged vesicles can be reproducibly quantitated to assess the effectiveness of genetic or pharmacological intervention. We will use CRISPR/Cas9 in combination with the GecKO sgRNA library to individually inactivate human genes and test their effect on lysosome morphology. Similarly, we will use the Synergistic Activation Mediator (SAM) library to individually overexpress human genes and test their ability to rescue the enlarged lysosomes in FIG4 null cells. These experiments will identify novel genes that cause or suppress cellular vacuolization, both of which are candidates for therapeutic targeting and pathogenic characterization. Genes identified in both screens will be strong candidates for functional characterization in mutant mice. We will use proximity-dependent biotinylation by BirA to identify proteins that interact directly with FIG4 and VAC14. These proteins will be evaluated as candidate genes for inherited disorders. The proposed experiments will integrate genetic analysis of pathogenic human mutations with functional dissection of the PI(3,5)P2 signaling pathway to advance our understanding of known and newly identified human disorders.