Functionalization of human gene variants In Drosophila

Next-generation sequencing has made genetic variant discovery routine clinical practice. To assess a variant’s pathogenicity classical clinical genetics methods, require familial transmission or the identification of the variant in multiple affected individuals. However, most identified variants are rare, often de novo, or present in few individuals. Therefore, classical genetics methods such as case-control or co-segregation studies cannot be applied. Computational methods can be used to predict the function of thousands of gene variants, but these methods are not reliable. Experimental assays to assess the relative function of variants can provide a path to interpretation of variant activity. Drosophila melanogaster offers a robust platform for variant functional testing at scale, but remains unvalidated for accurate assessment of the function of high numbers of variants in well-established assays. The first section of my thesis describes my role in a multi-model organism variant functionalization project characterizing 100 human PTEN variants. In flies, ubiquitous overexpression of PTEN-WT resulted in developmental delay. By comparing eclosion delay for overexpressed PTEN variants, we categorized variants as wildtype, gain of function or loss of function. This work demonstrates the utility of Drosophila as a powerful platform for high volume screening for the function of human gene variants from healthy and diseased individuals. However, clinically interpreting the functional consequence of the identified variation is challenging. Therefore, my third chapter aimed to establish a structured ‘well-established’ assay calibrated against known pathogenic and benign PTEN variants to convert variant function in an assay into a clinically-relevant interpretation in adherence to ClinGen Sequence Variant Interpretation (SVI) Working Group recommended guidelines. We screened ~100 human PTEN variants for suppression of PI3K/AKT signaling dependent cellular proliferation in Drosophila, a pathway that underlies the primary cause of PTEN’s contribution to cancer. The assay correctly assigned the function of known pathogenic and benign variants and exhibited a high correlation with human cell line functional data. We also showed that PTEN functionally replaces its Drosophila ortholog in developmental growth. Our work provides evidence that well- established assays, directly testing disease-relevant protein activity in Drosophila, can be used to generate reliable functional data appropriate for clinical variant interpretation.Medicine, Faculty ofGraduat