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Functionalization of ASD Variants of PTEN in Yeast and Fly
Objectives: To address this problem, a two-stage strategy was used to capitalize on high-throughput biological systems to screen for gene mutations most likely to provide strong phenotypes that are then forwarded to secondary, slower throughput but higher resolution assays. The first gene chosen to be studied in this paradigm was PTEN (phosphatase and tensin homolog) which is a crucial negative regulator of the PI3K/mTOR pathway.
Methods: Wildtype, human PTEN and its multiple variants, selected based on bioinformatics assessment of association with ASD or expression in the normal population, were expressed in high-throughput assays in yeast and Drosophila. In yeast, Synthetic Genetic Array (SGA) technology was used to screen ~5,000 yeast strains to identify deletion mutants that were sensitive to overexpression of wild type human PTEN. Strains identified through the yeast SGA with wildtype PTEN were then used to assay the activity of over one hundred PTEN variants. Using GAL4-mediated human gene/variant expression in Drosophila, we identify gain of function phenotypes and variants of altered function. Combined with assays for protein stability in both yeast and Drosophila, these experiments gave quantitative information on variants that likely result in loss of PTEN function in vivo.
Results: From the original SGA screen, a small subset of yeast deletion strains were identified as having genetic interactions with wildtype PTEN. Through the creation of a 'mini array' containing these deletion strains, it was possible to determine an interaction profile for each of the over one hundred PTEN variants selected for study. The results from this screen were then compared to the stability data to identify PTEN variants with varying stability and interaction states as compared to wildtype.
Conclusions: Through these assays we were able to classify PTEN variants based on their protein stability, genetic interactions interactions, and effects on nervous system function in an in vivo model. ASD-associated PTEN variants fell into distinct classes based on these measures. Results guided selection of variants for high-resolution assays.