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.