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.
Highlights
Discovery of disease-causing genetic variants has long served as a powerful tool in understanding and tackling disease
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
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
Summary
Discovery of disease-causing genetic variants has long served as a powerful tool in understanding and tackling disease. In spite of the many computational tools for variant effect prediction [6,7,8,9,10,11], making confident predictions of pathogenicity remains a challenge for many variants, and most remain ‘variants of uncertain significance’ (VUS) [12, 13]. To fill this interpretation gap, a wide variety of functional assays are being developed [14, 15]. Key criteria include: (i) Assessing variant function in an assay modeling a defined gene-disease mechanism. (ii) Calibrating the assay with a minimum of 11 known pathogenic and benign variants, preferably established on clinical or approved grounds. (iii) Assays must be optimized for rigor and reproducibility
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