Department of Cellular and Physiological Sciences, Life Sciences Institute, Djavad Mowafaghian Centre for Brain Health, University of British Columbia, Vancouver, British Columbia, Canada.
Department of Molecular Biology and Biochemistry, Centre for Cell Biology, Development and Disease, Simon Fraser University, Burnaby, British Columbia, Canada.
PLoS Genet. 2021 Sep 7;17(9):e1009774. doi: 10.1371/journal.pgen.1009774. eCollection 2021 Sep.
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.
基因变异的发现已变得常规化，但要有效地解释大多数变异的功能后果或与疾病的关联仍然具有挑战性。为了填补这一解释空白，对变异功能的实验检测变得越来越普遍。然而，要使这些检测具有可重复性、能够大规模应用于大量变体，并且能够评估与 ClinGen 序列变异解释 (SVI) 工作组针对“成熟检测”的指南相一致的特定基因疾病机制，仍然具有挑战性。黑腹果蝇作为一种检测平台具有很大的潜力，但尚未经过严格测试，以适应大量符合这些指南的人类变体。在这里，我们希望测试黑腹果蝇作为可扩展的成熟检测平台的实用性。我们采用遗传相互作用的方法来测试 100 多种人类 PTEN 变体在癌症相关的 PI3K/AKT 信号抑制中的功能，这些变体与细胞生长和增殖有关。我们使用生化特征明确的 PTEN 突变体以及 23 种已知的致病性和良性 PTEN 变体来验证该检测，该检测正确地将所有变体分配到预测的功能类别中。此外，这些变体的功能调用与我们最近发表的人类细胞系数据非常吻合。最后，我们使用这些致病性和良性变体来校准检测，为 70 种其他 PTEN 变体的致病性临床解释设定了读出阈值。总的来说，我们证明了黑腹果蝇为临床变异解释提供了一种强大的检测平台，可与其他成熟的检测方法结合使用，以提高对变异功能和致病性进行准确评估的信心。
