Accelerating Gene Discovery by Phenotyping Whole-Genome Sequenced Multi-mutation Strains and Using the Sequence Kernel Association Test (SKAT)

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.

Whole Exome Sequencing of Patients from Multicase Families with Systemic Lupus Erythematosus Identifies Multiple Rare Variants

“…For this, we used a large and independent imputed genome-wide association scan of 4,254 SLE patients and 4,349 controls with European ancestry 25 (Table 2 ). Each gene was analyzed using two procedures: the sequence kernel association test (SKAT) 32 and an aggregated case-control enrichment test. It is important to note that we performed targeted gene-based tests, that is, we did not test for association of rare variants neither at a genome-wide level nor tested individual variants.…”

Genes and molecular pathways underpinning ciliopathies

“…A promising technique is proximity-dependent protein identification, in which a given protein is fused to an enzyme that can tag (for example, biotinylate) nearby interaction partners for subsequent identification by mass spectrometry 98 . Similarly, model organism genetic or genome-wide RNA interference (RNAi) screens can uncover, in an unbiased manner, new genes that are required for cilia function 140,175,176 .…”

“…The endocrine-cerebroosteodysplasia syndrome was shown to result from mutations in ICK 178 , which encodes a kinase that is involved in the control of IFT 180 . Walker–Warburg (WWS) syndrome was a suspected but unproven ciliopathy; the B3GNT1 (also known as B4GNT1) glycosyltransferase implicated in this disorder is now known to influence ciliated cell function in C. elegans 175 . The 241 candidates listed in Supplementary information S1 (table) may reveal additional connections to known or novel ciliary disorders.…”

Systematical analyses of variants in DNase I hypersensitive sites to identify hepatocellular carcinoma susceptibility loci in a Chinese population

“…13 Meanwhile, pathway-based approaches are ideally suited for gene-based tests to interpreting the findings from GWAS. 14,15 Therefore, in the present study, gene-based analysis was performed by using the sequence kernel association test, 16 and then the data were bootstrapped with 10 000 replications to control family wise error rate. Pathway analysis was performed with database from DAVID Bioinformatics Resources 6.8 (https:// david.ncifcrf.gov/summary.jsp).…”