Multi-model functionalization of disease-associated PTEN missense mutations identifies multiple molecular mechanisms underlying protein dysfunction

Abstract

<jats:title>ABSTRACT</jats:title><jats:p>Functional variomics provides the foundation for personalized medicine by linking genetic variation to disease expression, outcome and treatment, yet its utility is dependent on appropriate assays to evaluate mutation impact on protein function. To fully assess the effects of 106 missense and nonsense variants of PTEN associated with autism spectrum disorder, somatic cancer and PHTS, we take a deep phenotypic profiling approach using 18 assays in 5 model systems spanning diverse cellular environments ranging from molecular function to neuronal morphogenesis and behavior. Variants inducing instability occurred across the protein, resulting in partial to complete loss of function (LoF), which was well correlated across models. However, assays were selectively sensitive to variants located in substrate binding and catalytic domains, which exhibited complete LoF or dominant negativity independent of effects on stability. Our results indicate that full characterization of variant impact requires assays sensitive to instability and a range of protein functions.</jats:p>

Cross-level analysis of molecular and neurobehavioral function in a prospective series of patients with germline heterozygous PTEN mutations with and without autism

“…If a dominant negative process is operating, it may suggest that early interventions that focus on degrading mutant PTEN could be a useful strategy in some cases. Recent work has demonstrated substantial variability in impact on PTEN protein stability and function across different missense mutations [48,49,51]. This suggests that the above-described pattern of modest reduction in PTEN levels may mask substantial variability, potentially explaining the observed heterogeneity of outcomes across PTEN mutation cases.…”

“…Samples sizes were also insufficient to evaluate the functional impact of missense mutations in PTEN patients [51]. Future work in larger samples, computing fitness [48] and abundance [49] scores and identifying candidate dominant negative mutations, will be crucial to understanding whether different mutations influence peripheral protein levels and/or neurobehavioral outcomes.…”

Multi-parametric analysis of 57 SYNGAP1 variants reveal impacts on GTPase signaling, localization, and protein stability

“…We next explored whether a measure of protein stability may explain some of our findings, since protein instability is a major mechanism of missense variant dysfunction for other proteins 56,57,72,73 .…”

“…While many mutations in SYNGAP1 are identified as pathogenic early termination/truncating variants, there is a growing number of missense variants of unknown significance (VUS) for which potential contribution to disease development is unclear. To strengthen clinical relevance of in vitro findings, and allow for structure-function prediction, functional variomics provides multi-dimensional, deep phenotypic characterization of diseaseassociated missense variants [56][57][58][59][60] . Here, we use high-throughput multiplex-phospho flow cytometry and high-content screening to measure the impact of 58 variants on protein localization, stability and function in multiple disease-associated signaling pathways, including pERK, .…”

Sentinel Interaction Mapping (SIM) – A generic approach for the functional analysis of human disease gene variants using yeast

“…To demonstrate the ability of these sentinels to report on PTEN activity, we sought to measure the strength of the genetic interaction of each deletion mutant with 100 PTEN variants, comprising a mixture of disease-associated variants and population controls. This was done as part of a larger study using multiple model organisms to functionalize PTEN variants found in autism spectrum disorder, cancer and PTEN hamartoma tumor syndrome [25]. Based on the results of the mini-array experiments described above, we selected the eight strains that demonstrated repeated sensitivity to functional PTEN activity (vac14, fig4, vac7, vam3, vam7, ypt7, vps30 and vps38).…”