Abstract
Forward genetic screens represent powerful, unbiased approaches to uncover novel components in any biological process. Such screens suffer from a major bottleneck, however, namely the cloning of corresponding genes causing the phenotypic variation. Reverse genetic screens have been employed as a way to circumvent this issue, but can often be limited in scope. Here we demonstrate an innovative approach to gene discovery. Using C. elegans as a model system, we used a whole-genome sequenced multi-mutation library, from the Million Mutation Project, together with the Sequence Kernel Association Test (SKAT), to rapidly screen for and identify genes associated with a phenotype of interest, namely defects in dye-filling of ciliated sensory neurons. Such anomalies in dye-filling are often associated with the disruption of cilia, organelles which in humans are implicated in sensory physiology (including vision, smell and hearing), development and disease. Beyond identifying several well characterised dye-filling genes, our approach uncovered three genes not previously linked to ciliated sensory neuron development or function. From these putative novel dye-filling genes, we confirmed the involvement of BGNT-1.1 in ciliated sensory neuron function and morphogenesis. BGNT-1.1 functions at the trans-Golgi network of sheath cells (glia) to influence dye-filling and cilium length, in a cell non-autonomous manner. Notably, BGNT-1.1 is the orthologue of human B3GNT1/B4GAT1, a glycosyltransferase associated with Walker-Warburg syndrome (WWS). WWS is a multigenic disorder characterised by muscular dystrophy as well as brain and eye anomalies. Together, our work unveils an effective and innovative approach to gene discovery, and provides the first evidence that B3GNT1-associated Walker-Warburg syndrome may be considered a ciliopathy.
Highlights
A powerful, tried and true approach to identify which genes function in a particular biological process is to create collections of organisms harbouring multiple mutations via random mutagenesis, followed by screening the mutant library for organisms that exhibit the desired altered phenotypes
When we applied Sequence Kernel Association Test (SKAT) to the phenotype data we collected from screening the Million Mutation Project (MMP) strains for dye-filling, we found that a previously uncharacterised C. elegans gene, bgnt-1.1/F01D4.9, plays an essential role in this process
We demonstrate that rare-variant association analysis (e.g., SKAT) is an efficient way to rapidly uncover novel genes for a phenotype of interest in whole-genome sequenced strains harbouring multiple mutations induced via random mutagenesis
Summary
A powerful, tried and true approach to identify which genes function in a particular biological process is to create collections of organisms harbouring multiple mutations via random mutagenesis, followed by screening the mutant library for organisms that exhibit the desired altered phenotypes. Such forward genetics strategies have produced numerous fundamental discoveries, a significant limitation of this approach in metazoans is the prolonged time required to identify the causative mutations. Various strategies for disrupting a collection of known genes (e.g., RNAi, homologous recombination, transposon mutagenesis, etc.) are combined with phenotypic screening to identify candidates. Reverse genetics approaches have drawbacks, including the need to handle and process tens of thousands of strains to assay the entire genome, off-target effects in the case of RNAi, and omission of essential genes
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