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Abstract

Next-generation sequencing has made genetic variant discovery routine in clinical practice. Assessing the pathogenicity of newly identified variants traditionally relies on classical clinical genetics approaches, such as co-segregation analysis or case–control studies. However, most variants detected in patients are rare, often de novo, and present in very few individuals, making these classical approaches infeasible. Although computational tools can predict the potential impact of thousands of variants, their reliability remains limited. Thus, experimental functional assays are essential for accurately interpreting variant effects. Drosophila melanogaster provides a fast, inexpensive, and robust platform for variant functional testing. In this thesis, I developed a Drosophila-based assay to evaluate six STXBP1 variants identified in individuals with autism spectrum disorder (ASD). Because complete loss of the fly STXBP1 ortholog, Rop, causes embryonic lethality, I established a calibrated lethality-rescue assay using Rop variants that mimic human STXBP1 mutations. In this system, I found that clinically validated pathogenic variants failed to rescue lethality (complete loss-of-function) or rescue only partially (partial loss-of-function), whereas benign variants fully rescued the phenotype. Using this calibrated assay, I tested the six STXBP1 variants found in ASD patients. Of the six variants two were complete loss-of-function which we interpreted as likely pathogenic, and three functioned normally which we interpreted as benign. One variant exhibited partial loss of function phenotype similar to a known pathogenic variant. The functional impact of this variant was further assessed using a climbing assay, where its pathogenicity became evident, supporting its classification as likely pathogenic.