Using Drosophila Models to Investigate Human Disease Variants Implicated in Neurodevelopmental Disorders

Abstract

As exome and genome sequencing is becoming increasingly routine in clinical practice, the identification of rare variants is also increasing. Individuals with unique disease phenotypes who undergo whole-genome or -exome sequencing are often found to have a number of candidate disease-causing variants. However, the ability to make confident predictions on the pathogenicity of rare variants remains a challenge. In modern medicine, a molecular diagnosis holds paramount importance for effective disease treatment and management for patients and their families. To address the discrepancy between the number of identified variants and their classifications of pathogenicity, functional assays in model organisms are being utilized. In this thesis, I describe how I utilized Drosophila humanized model systems to effectively study the functional effect of patient-identified missense variants in the context of Neurodevelopmental Disorders (NDDs). Chromatin regulation proves to be an important mechanism in the etiology of NDDs and chromatin regulator genes carry a high mutational burden in NDDs. I developed a ‘humanized’ fly model where human SMARCD1 rescues lethality caused by null Bap60 mutations and demonstrate the clinical utility of this assay for future SMARCD1 variant interpretation studies. Next, using an innovative dual gain-of-function assay, the functional screening of KDM6B missense variants contributed to an accurate redefinition of the KDM6B-related NDD. Overexpression of WDR5 reference and variant proteins in neurons of flies reveal differential effects on behaviours including increased locomotion, seizure susceptibility, and social behaviours providing functional evidence of WDR5 variants in disease-relevant contexts. As an alternative approach to expressing human genes in flies, I describe a CRISPR Escape approach that can be applied to study conserved human disease variants in the context of the fly protein. Each fly model has its own set of advantages and limitations. Considerations, such as the primary research goal, the nature of the allele, and the availability of resources should be taken into account when choosing the optimal fly model for human disease modeling. Fly models have proven to be effective in addressing the impact of patient-identified variants on chromatin regulators involved in NDDs and have contributed to the characterization of novel NDDs.