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

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-Organism Developmental Expression Profiling Identifies RAB-28 as a Novel Ciliary GTPase Associated with the BBSome and Intraflagellar Transport.

Primary cilia are specialised sensory and developmental signalling devices extending from the surface of most eukaryotic cells. Defects in these organelles cause inherited human disorders (ciliopathies) such as retinitis pigmentosa and Bardet-Biedl syndrome (BBS), frequently affecting many physiological and developmental processes across multiple organs. Cilium formation, maintenance and function depend on intracellular transport systems such as intraflagellar transport (IFT), which is driven by kinesin-2 and IFT-dynein motors and regulated by the Bardet-Biedl syndrome (BBS) cargo-adaptor protein complex, or BBSome. To identify new cilium-associated genes, we employed the nematode C. elegans, where ciliogenesis occurs within a short timespan during late embryogenesis when most sensory neurons differentiate. Using whole-organism RNA-Seq libraries, we discovered a signature expression profile highly enriched for transcripts of known ciliary proteins, including FAM-161 (FAM161A orthologue), CCDC-104 (CCDC104), and RPI-1 (RP1/RP1L1), which we confirm are cilium-localised in worms. From a list of 185 candidate ciliary genes, we uncover orthologues of human MAP9, YAP, CCDC149, and RAB28 as conserved cilium-associated components. Further analyses of C. elegans RAB-28, recently associated with autosomal-recessive cone-rod dystrophy, reveal that this small GTPase is exclusively expressed in ciliated neurons where it dynamically associates with IFT trains. Whereas inactive GDP-bound RAB-28 displays no IFT movement and diffuse localisation, GTP-bound (activated) RAB-28 concentrates at the periciliary membrane in a BBSome-dependent manner and undergoes bidirectional IFT. Functional analyses reveal that whilst cilium structure, sensory function and IFT are seemingly normal in a rab-28 null allele, overexpression of predicted GDP or GTP locked variants of RAB-28 perturbs cilium and sensory pore morphogenesis and function. Collectively, our findings present a new approach for identifying ciliary proteins, and unveil RAB28, a GTPase most closely related to the BBS protein RABL4/IFT27, as an IFT-associated cargo with BBSome-dependent cell autonomous and non-autonomous functions at the ciliary base.

The impact of disclosing emotions on ratings of interpersonal closeness, warmth, competence, and leadership ability.

Introduction: Increasingly, business leaders and other professionals are called upon to be vulnerable and authentic in the workplace, which often includes disclosing emotions to others. While sharing emotions is known to enhance closeness, several questions remain underexplored. Specifically, disclosing personal facts about oneself and disclosing emotions have often been studied together, making it difficult to determine the effects of disclosing emotions per se. Moreover, not enough is known about factors that may influence effects of disclosing emotions, including recipients' attitudes toward emotion-sharing, the sharer's gender, and whether one considers the disclosure to be similar to one's own experiences. We examined the impact of disclosing positive and negative emotion on ratings of closeness, warmth, competence, and leadership ability. Methods: 119 participants (95 female) in the United States were shown headshots of individuals who were introduced in the first person in written format. For half of the pictures, an autobiographical fact about the individual's past was disclosed. For the other half, an autobiographical fact and an associated emotion were disclosed. Results: We found that sharing both positive and negative emotions increased feelings of closeness above and beyond the effects of autobiographical sharing alone. Sharing positive emotions also increased ratings of warmth, competence, and leadership ability. Male and female sharers benefited equally from disclosing emotions and effects were largely robust to recipients' attitudes toward emotional expression. Having something in common with the disclosed fact or emotion further increased all ratings. Conclusion: These findings indicate that disclosing emotions may improve interpersonal interactions, with potential management applications in business.

Old lessons learned anew: family-based methods for detecting genes responsible for quantitative and qualitative traits in the Genetic Analysis Workshop 17 mini-exome sequence data.

Family-based study designs are again becoming popular as new next-generation sequencing technologies make whole-exome and whole-genome sequencing projects economically and temporally feasible. Here we evaluate the statistical properties of linkage analyses and family-based tests of association for the Genetic Analysis Workshop 17 mini-exome sequence data. Based on our results, the linkage methods using relative pairs or nuclear families had low power, with the best results coming from variance components linkage analysis in nuclear families and Elston-Stewart model-based linkage analysis in extended pedigrees. For family-based tests of association, both ASSOC and ROMP performed well for genes with large effects, but ROMP had the advantage of not requiring parental genotypes in the analysis. For the linkage analyses we conclude that genome-wide significance levels appear to control type I error well but that "suggestive" significance levels do not. Methods that make use of the extended pedigrees are well powered to detect major loci segregating in the families even when there is substantial genetic heterogeneity and the trait is mainly polygenic. However, large numbers of such pedigrees will be necessary to detect all major loci. The family-based tests of association found the same major loci as the linkage analyses and detected low-frequency loci with moderate effect sizes, but control of type I error was not as stringent.

SAGE analysis from 1 microg of total RNA.

Serial analysis of gene expression (SAGE) is a powerful transcription-profiling method that allows simultaneous expression analysis of thousands of transcripts, provides absolute digital readout of the expression level, and identifies new genes. A disadvantage of SAGE is the relatively high amount of input RNA required. Consequently, several techniques have been developed to overcome this limitation, so that SAGE can be applied to very limited amounts of starting material, e.g., small biological samples such as tissue biopsies or microdissected materials. Here we describe a modified version of the original microSAGE protocol, which requires only 1 to 2 microg total RNA. This method avoids PCR amplification of cDNA and reamplification of ditags, procedures that potentially compromise the quantitative nature of this technique.