The Washington University (WU) Model Organism Screening Center (wuMOSC) will utilize four model organisms, C. elegans, Drosophila, zebrafish and hPSC, for functional analysis of gene-variants nominated by Undiagnosed Disease Network (UDN). C. elegans is a major model organism for studies of animal cell and developmental biology, excelling at gene function discovery and elaboration of pathways in which gene products act. A number of features make C. elegans appropriate for being the first line organism for experimental assessment of UDN variant on gene product function. These include short generation time (4 days), small size, inexpensive maintenance, self-fertile hermaphroditism, and optical transparency that permits detailed phenotypic analysis at all stages of the life cycle. About 60% of human genes have an ortholog in the C. elegans genome, indicating that a substantial portion of potential disease genes can be experimentally examined in the worm. C. elegans research has advanced our understanding of disease. For example, published work has demonstrated that variant knock-in can recapitulate the effect on gene product function for known diseases, provide support for phenotypic expansion and provide support for molecular diagnosis of a UDP gene. In a project paralleling the activities of Phase I UDN MOSC, we have been collaborating with the WU Pediatric Genetics Clinic to examine candidate gene-variants of uncertain significance found in patients that did not receive a molecular diagnosis, by CRISPR/Cas9 variant knock-in into the C. elegans ortholog and assessing if there is a change in function. This work indicated that ~1/3 of patient variants can be assessed by knock-in with the worm, given conservation of both the gene and variant residues. Importantly, CRISPR/Cas9 mediated homology directed knock-in of variants occurs at very high frequency, allowing us to obtain variant knock-in strains, out-crossed twice, in ~3 weeks. The wuMOSC Leadership Project, working in conjunction with the C. elegans Resource Core, will assign ~60 UDN gene-variants for function analysis in the worm. The variants will be knocked into the orthologous worm gene. To assess function, a prioritized phenotyping pipeline will be employed that includes 7 different phenotyping platforms, from simple morphological analysis, to high-throughput assays for viability, growth, reproduction, to assessment of movement/behavior and custom assays that are derived from published phenotypes for the gene of interest. The pipeline allows focusing on known phenotypes or broad searches for unknown phenotypes, increasing the likelihood of detecting a phenotype. High-throughput phenotypic assays are important because of the large number of genes and genotypes that will be analyzed. Results from phenotypic analysis will inform on the functional effect of the variant, which will be communicated to the UDN, providing information that can contribute to a diagnosis.
