A scalable Drosophila assay for clinical interpretation of human PTEN variants in suppression of PI3K/AKT induced cellular proliferation

Abstract

Gene variant discovery is becoming routine, but it remains difficult to usefully interpret the functional consequence or disease relevance of most variants. To fill this interpretation gap, experimental assays of variant function are becoming common place. Yet, it remains challenging to make these assays reproducible, scalable to high numbers of variants, and capable of assessing defined gene-disease mechanism for clinical interpretation aligned to the ClinGen Sequence Variant Interpretation (SVI) Working Group guidelines for ‘well-established assays’. Drosophila melanogaster offers great potential as an assay platform, but was untested for high numbers of human variants adherent to these guidelines. Here, we wished to test the utility of Drosophila as a platform for scalable well-established assays. We took a genetic interaction approach to test the function of ~100 human PTEN variants in cancer-relevant suppression of PI3K/AKT signaling in cellular growth and proliferation. We validated the assay using biochemically characterized PTEN mutants as well as 23 total known pathogenic and benign PTEN variants, all of which the assay correctly assigned into predicted functional categories. Additionally, function calls for these variants correlated very well with our recent published data from a human cell line. Finally, using these pathogenic and benign variants to calibrate the assay, we could set readout thresholds for clinical interpretation of the pathogenicity of 70 other PTEN variants. Overall, we demonstrate that Drosophila offers a powerful assay platform for clinical variant interpretation, that can be used in conjunction with other well-established assays, to increase confidence in the accurate assessment of variant function and pathogenicity.

Structural and Dynamic Effects of PTEN C-Terminal Tail Phosphorylation

“…For the PTEN 4A system, we found coupling of the P loop to be enhanced with the residues in the AHS, IDR, and core of the C2 domain, whereas a dramatic increase in coupling of the CTT is seen throughout the entire protein structure (Figure S12C), consistent with the regions identified in PRS analyses (Figure 6E,F). Previous reports indicate that PTEN 4A overrides autoinhibitory activity, 17,77 which increases membrane localization 29,62,63 to produce a pronounced tumor suppressive effect due to its enhanced lipid phosphatase activity. 78 Importantly, in a recent study, PTEN 4A acts as a gain of function variant causing a substantial increase in the WT PTEN function.…”

Structural and Dynamic Effects of PTEN C-terminal Tail Phosphorylation

“…Previous reports indicate that PTEN 4A overrides autoinhibitory activity 21,94 , which increases membrane localization 33,81,82 to produce a pronounced tumor suppressive effect due to its enhanced lipid phosphatase activity 95 . Importantly, in a recent study PTEN 4A acts as a gain of function variant causing a substantial increase of WT PTEN function 94 . When comparing the DCI profiles of the catalytic P loop and CTT across each system, fluctuations in DCI imply that the long-distance communication pathways do not follow similar channels to the active site and may be the result of altered dynamics ( Figure S11D ).…”

“…Importantly, in a recent study PTEN 4A acts as a gain of function variant causing a substantial increase of WT PTEN function 94 . When comparing the DCI profiles of the catalytic P loop and CTT across each system, fluctuations in DCI imply that the long-distance communication pathways do not follow similar channels to the active site and may be the result of altered dynamics (Figure S11D).…”

“…For the PTEN 4A system we found coupling of the P loop to be enhanced with the residues in the AHS, IDR, and core of the C2 domain, whereas a dramatic increase in coupling of the CTT is seen throughout the entire protein structure (Figure S11C), consistent with the regions identified in PRS analyses (Figure 6E and 6F). Previous reports indicate that PTEN 4A overrides autoinhibitory activity 21,94 , which increases membrane localization 33,81,82 to produce a pronounced tumor suppressive effect due to its enhanced lipid phosphatase activity 95 .…”

Inter-Species Rescue of Mutant Phenotype—The Standard for Genetic Analysis of Human Genetic Disorders in Drosophila melanogaster Model

“…In D. melanogaster , Pten 100 / Pten 117 represents a strong hypomorphic heteroallelic combination that leads to increased larval growth and, consequently, increased pupal volume and adult weights. Expression of the PTEN WT allele in embryos and larvae successfully rescued the hypomorphic mutants [ 63 ]. Within the same study, the authors devised a scalable experimental platform to functionally test about 100 human PTEN alleles with potential clinical relevance.…”