A saturation mutagenesis approach to understanding PTEN lipid phosphatase activity and genotype-phenotypes relationships

Abstract

Phosphatase and tensin homolog (PTEN) is a tumor suppressor frequently mutated in diverse cancers. Germline PTEN mutations are also associated with a range of clinical outcomes, including PTEN hamartoma tumor syndrome (PHTS) and autism spectrum disorder (ASD). To empower new insights into PTEN function and clinically relevant genotype-phenotype relationships, we systematically evaluated the effect of PTEN mutations on lipid phosphatase activity in vivo. Using a massively parallel approach that leverages an artificial humanized yeast model, we derived high-confidence estimates of functional impact for 7,244 single amino acid PTEN variants (86% of possible). These data uncovered novel insights into PTEN protein structure, biochemistry, and mutation tolerance. Variant functional scores can reliably discriminate likely pathogenic from benign alleles. Further, 32% of ClinVar unclassified missense variants are phosphatase deficient in our assay, supporting their reclassification. ASD associated mutations generally had less severe fitness scores relative to PHTS associated mutations (p = 7.16x10 -5 ) and a higher fraction of hypomorphic mutations, arguing for continued genotype-phenotype studies in larger clinical datasets that can further leverage these rich functional data. MAIN TEXTRecent large-scale exome sequencing studies have highlighted the abundance of protein-coding variation in the human population 1 . It remains challenging to predict variant pathogenicity and clinical outcomes, especially for genes with pleiotropic effects. With most rare variants private to a single family or individual, using traditional approaches to establish pathogenicity such as variant segregation within a pedigree or identification in independent patients is infeasible. Even for well-studied genes, hundreds of variants are currently defined as variants of uncertain significance (VUS). Moreover, purely computational approaches still suffer from high false positive rates 2 and subjective interpretations that limit the clinical utility of these predictions. To address these challenges for genes of clinical importance, one proposed approach is to prospectively measure the functional effects of all possible mutations, allowing these empirical data to be integrated into the clinical assessment of novel rare variants 3,4 . Historically, these types of functional assays have been conducted in a serial nature, which limits scalability, and often only within a portion of the protein of interest. While there are some notable examples of whole-gene brute force saturation mutagenesis, e.g., TP535 , new more scalable experimental paradigms are being developed that allow the functional dissection of the effects of thousands of genetic mutations in parallel 6 . These approaches leverage recent advances in DNA synthesis and sequencing technologies and have proven particularly valuable in understanding the effects of mutations in cancer-associated genes 7,8 . With these issues in mind, we have developed a saturation mutagenesis approach to...

PTEN-opathies: from biological insights to evidence-based precision medicine

“…Finally, because it is technically challenging to functionally interrogate all germline and somatic PTEN mutations, research efforts have focused on devising high-throughput methods to evaluate pathogenicity. Surprisingly, several residues within the catalytic pocket are shown to be tolerant to mutations, with solvent exposure playing a critical role in dictating tolerance (80). Moreover, several uncharacterized PTEN variants result in decreased PTEN thermodynamic stability and abundance, thus expanding the list of potentially functional variants (81).…”

“…Such effects cannot be extrapolated from PTEN's secondary structure alone and indeed provide an important dimension to consider for assessing PTEN genotype-PHTS phenotype associations. Additionally, studies have shown that ASD-associated mutations tend to retain higher PTEN activity relative to non-ASD-associated mutations (80,89,90). Whether assayed in vivo in yeast or in vitro in mammalian stable or primary cell lines, partial hypomorphic PTEN lipid phosphatase activity is retained in individuals with ASD, versus total loss of PTEN lipid phosphatase activity in individuals with more severe PHTS-related phenotypes (80,(89)(90)(91).…”

“…Additionally, studies have shown that ASD-associated mutations tend to retain higher PTEN activity relative to non-ASD-associated mutations (80,89,90). Whether assayed in vivo in yeast or in vitro in mammalian stable or primary cell lines, partial hypomorphic PTEN lipid phosphatase activity is retained in individuals with ASD, versus total loss of PTEN lipid phosphatase activity in individuals with more severe PHTS-related phenotypes (80,(89)(90)(91). Relatedly, it is predicted that PTEN mutations that result in the accumulation of stable inactive PTEN protein would lead to more severe PHTS-related developmental phenotypes and malignancies (90).…”

CRISPR-Cas9 human gene replacement and phenomic characterization inCaenorhabditis elegansto understand the functional conservation of human genes and decipher variants of uncertain significance

“…Several such exhaustive sequence-function maps have been recently been generated in 448 yeast and human cell culture systems (Findlay et al, 2018(Findlay et al, , 2014Majithia et al, 2016;449 Matreyek et al, 2018;Mighell et al, 2018;Weile et al, 2017). These tools offer amazing 450 resources that serve as 'lookup tables' of functional missense variation in human genes, to 451 enable experimentally confirmed variant interpretation immediately upon first clinical 452 presentation (Starita et al, 2017;Weile et al, 2017).…”

“…130 phenotypes (collectively called PTEN harmatoma tumor syndrome), intellectual 139 disability, and Autism Spectrum Disorders (Hobert et al, 2014;Li et al, 1997;Danny 140 Liaw et al, 1997;McBride et al, 2010;O'Roak et al, 2012;Orrico et al, 2009;Sanders 141 et al, 2015;Varga et al, 2009). Despite extensive study, it is currently impossible to 142 predict the clinical outcome of a PTEN mutation carrier using sequence data alone 143 (Matreyek et al, 2018;Mighell et al, 2018). PTEN also has several technical advantages 144 that make it an ideal test case: 1) PTEN functions in the highly conserved insulin 145 signaling pathway that is well characterized in C. elegans (Ozes et al 2001;Ogg & 146 Ruvkun 1998;Mihaylova et al 1999, and Fig.…”