Wednesday, May 8, 2024
5/8/2024
PROJECT SUMMARY/ABSTRACT Among individuals with autism spectrum disorder (ASD), some of the worst prognoses come from comorbidity with accelerated brain growth, known as disproportionate megalencephaly (DM). ASD-DM is associated with regressive autism, slower gains in IQ, greater difficulties with expressive language, and more severe cognitive defects. Recent genome sequencing studies of probands with ASD have identified an excess of rare de novo heterozygous mutations of genes expressed in early fetal development that impact cell cycle and proliferation. Although recurrent variants have been identified in a handful of well-known ASD-DM genes, including CHD8 and PTEN, many genes impacted by de novo variants in patients with ASD-DM have never before been reported, thus requiring sifting through hundreds to thousands of candidate genes with unknown significance. To ultimately confirm disease genes, experimental validation is necessary. The proposed study hypothesizes that knockout of ASD-DM candidate gene orthologs will result in alterations in the abundance of specific cell types in the developing zebrafish brain, reminiscent of those observed in human patients as well as mouse and cerebral organoid models. Due to their small size, robust reproduction, embryonic transparency, and rapid development, zebrafish are well suited for functional studies of developmental genes. Although knockouts of single genes in zebrafish have successfully pinpointed defects, no systematic study characterizing multiple genes in parallel has been performed for ASD. One limitation is the lack of higher-throughput quantitative assays to characterize neurodevelopment. Further, very few studies have assessed disease-causing missense substitutions using fish. The primary goal of the proposed project is to functionally characterize ASD-DM candidate genes and develop an in vivo strategy to rapidly assay identified patient mutations to measure their impact on neurodevelopment. To achieve this goal, the project will focus on the following aims: (1) functionally assay patient loss-of-function and missense variants of unknown significance in the conserved human/fish ortholog of a single ASD-DM gene, CHD8; and (2) target multiple ASD-DM candidate genes identified from disease sequencing studies using a higher-throughput gene editing method to characterize their impacts on brain development in zebrafish. As mutants are identified, future work includes developing small-molecule screens to rescue quantitative phenotypes of zebrafish carrying mutations of candidate genes generated from our study. These avenues of research differentiate our use of zebrafish from ongoing mouse studies. If successful, the developed approaches will significantly improve our ability to pinpoint disease genes critical in improving diagnostic measures facilitating earlier interventions and treatments as well as contributing to a better understanding of the etiology underlying megalencephaly in ASD.
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