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-Varn 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.
We demonstrated previously that mutations of the FIG4 gene are responsibile for three human disorders: Charcot-Marie-Tooth Disease, Yunis-Varon syndrome, and Polymicrogyria with epilepsy. We developed mouse models of these disorders in order to investigate pathogenic mechanisms in the brain and heart. We will identify additional genes in this pathway by a genetic approach, using a genome-wide suppressor screen in cultured cells. We will also use a biochemical approach to identify proteins that interact directly with FIG4 and its scaffold protein VAC14. We will develop functional assays for VAC14 and PIKFYVE similar to the one in use for FIG4, and use these assays to evaluate patient mutations of unknown significance that have been identified in large-scale sequencing projects. This work extends from basic investigation of the PI(3,5)P2 regulatory pathway to evaluation of human variants that contribute to the burden of genetic disease.
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