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Impact of Autism Spectrum Disorder associated PTEN point mutations on protein function and stability Post, Kathryn L.


ASD is a prevalent neurodevelopmental disorder characterized by social impairments and repetitive behaviors. Repeated studies indicate a role of genetics in the etiology of the disorder. In the search for genetic causes, whole exome sequencing has identified de novo likely gene disrupting and missense mutations in multiple, unrelated probands in many genes. The research community is now faced with the task of determining how dysfunction in genes caused by point mutations contribute to the pathophysiology of the disorder. From the high-confidence genes associated with ASD, we chose phosphatase and tensin homolog, PTEN, as our gene of interest. We first utilized an unbiased approach to identify novel genetic interactions of wildtype PTEN using a synthetic dosage lethality (SDL) screen in the Saccharomyces cerevisiae model system. Our screen yielded eight strong genetic interactions, ‘sentinels’ specific for PTEN catalytic function. We then tested for disruptions to these genetic interactions by designing a novel mini-SDL to screen a total of 97 PTEN point mutations. Functional scores obtained from this screen indicated the level of dysfunction caused by each point mutation. Interestingly, we found the dysfunction of variants to lie on a continuum with many variants displaying a range of partial loss of function (LoF). By testing the stability of all PTEN variants in both yeast and HEK293 cells we determined that many variants were dysfunctional due to a partial or complete loss of protein stability. Further, we also tested the ability of PTEN point mutations to negatively regulate the AKT pathway similar to wildtype PTEN. From this assay, we identified variants that were fully functional, partial LoF, complete LoF, and dominant negative. Through the creation of a biallelic PTEN knockout cell line we were able to deduce that the main mechanism of dominant negativity in this assay was likely due to interference of mutant PTEN with endogenous PTEN. Combined, our data indicates that point mutations of PTEN disrupt protein function via several mechanisms. Understanding how each mutation alters function will hopefully advance our understanding of the disorder and lead to precision therapeutics in the future.

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