Wednesday, February 5, 2025
2/5/2025
PROJECT SUMMARY/ABSTRACT A single mutation in a gene can lead to a disease. Many mutations, however, are benign. How do we determine which mutations are pathogenic and which are benign? There are hundreds of millions of single nucleotide variants in the human genome that have been identified by sequencing, with 5 million of these in protein-coding DNA. Despite this wealth of data, only ~2% of these variants have clinical annotations, half of which are “variants of uncertain significance”. High throughput functional screening of variants is thus needed for the millions of variants that have not been annotated. One approach for studying such large variant libraries is to use a method called deep mutational scanning, in which a library containing millions of gene variants is screened in parallel. This approach has proven useful in classifying variants of BRCA1, PTEN, and others by carrying out screens in cell culture. While this approach has the benefit of scale, it does not enable mutations to be studied in a developmental or tissue-specific context. However, human genetic diseases, including cancer, are tissue- specific. It is therefore desirable to study mutations in the context of whole organisms. We have recently shown in our lab that we can generate many Drosophila melanogaster with genomically integrated gene variants on the order of 1,000 or more variants at a cost of ~$1/variant for DNA delivery. Using this new ability to generate large numbers of transgenic D. melanogaster, we propose use a D. melanogaster seizure disease model to phenotype over 1,000 gene variants of para, a closely related ortholog to the human gene SCN1A, the most commonly implicated gene in epilepsy. Aim 1a: Generate over 1,000 D. melanogaster harboring different para variants. Aim 1b: Phenotype the variant flies in parallel using a seizure assay. Pool flies by their seizure severity, sequence them, and report findings to ClinVar to improve clinical variant annotation. Aim 2a: Increase the scale of variant libraries that can be made in D. melanogaster by characterizing more highly efficiency DNA recombinases for genomic integration of DNA into the genome. Aim 2b: Use a gene gun to increase the rate at which DNA is delivered to embryos, up to a thousand or more embryos per second. In this work, we will use our recently developed approach for generating large numbers of transgenic D. melanogaster to perform mutational scanning of over 1,000 variants of para. These results will provide supporting evidence for variant classification of the analogous variants of the human gene SCN1A, which is very closely related to para. In this work we will also increase the scale of DNA delivery and efficiency of integration into the fly genome so that larger numbers of gene variants can be screened in the future. We believe our approaches will prove very useful for assigning clinical annotations for the vast majority of variants of disease-related genes that show tissue-specific effects. We also expect the technology to be widely adopted by the developmental biology community, as it will enable rapid phenotyping of thousands of variants of developmentally important genes in a whole organism context.
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