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

Author summary DNA sequencing is becoming commonplace in the clinic, as physicians read your DNA to determine if you have variations in gene sequence that may help with diagnosis and therapy planning. The assumption is that we would know a damaging sequence variation if we saw it. However, this is in fact extremely difficult, and to this day most sequence variations observed in people have unknown disease implication. We must turn to experimental assessments of the variant's impact on gene function. This current report shows that an organism widely used to understand human disease mechanisms, Drosophila melanogaster, is a valid option for testing the function of hundreds of variants in a human gene of interest. In this case, we tested the function of a hundred variants in the human PTEN gene, found in patients with cancer or autism spectrum disorder, and we were able to pinpoint which ones are likely to contribute to disease, and which were not. Our work provides evidence that Drosophila offers a powerful experimental platform for establishing assay to easily test the function of high numbers of gene variations, that can be used to complement and extend other similar assays. 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.

Automated summary

DNA sequencing is widely used in clinical practice, and the fruit fly Drosophila melanogaster has been proven to be a valuable tool for testing the function of hundreds of human gene variants. Experimental assessments of variant function are becoming more common, and Drosophila offers a powerful platform for clinical variant interpretation.