Advancing our understanding of the ciliopathy, Bardet-Biedl Syndrome: an omics approach

Bardet-Biedl Syndrome (BBS) is a rare pleiotropic disorder, characterised by loss of vision, obesity, renal dysfunction, learning difficulties, and hypogonadism. This multisystem phenotype is caused by defects in genes that localize to the basal body of the primary cilia, where over 20 genes have now been attributed to cause BBS. However, ~32% of patients are affected by a recurrent missense variant, namely BBS1 p.M390R, which contributes greatly to the overall burden of BBS. Despite recent advances in understanding the syndrome’s genetic aetiology, much of the pathobiology of BBS1 p.M390R remains elusive. This thesis aimed to use an innovative multi-omic strategy, implementing genomic, transcriptomic, and proteomic technologies, to uncover the molecular pathology of a cohort of 15 BBS patients, each carrying the common BBS1 p.M390R variant. Phenotypic variability is a hallmark feature of BBS, where clinical heterogeneity exists within the BBS1 genotype, even if patients have the same underlying pathogenic mutation. It has been suggested that differences in disease expressivity are linked to secondary mutations that modify the manifestation of the primary locus. Here, the objective was to identify putative genetic modifying alleles from whole genome sequencing data generated from BBS1 p.M390R patients expressing discordant disease presentation. A novel 4-tier variant categorisation system was developed where 37 candidate modifiers were detected in 13 patients. This included a known modifying variant previously shown to have a high penetrance with ciliopathies, TTC21B p.L1002V found in 2 patients. Furthermore, it was investigated whether the presence of these modifying variants contributed to the overall mutational burden of BBS. Mutational burden analysis determined that there was no significant enrichment of variants in primary cilia genes in BBS patients compared to control individuals. There is a definitive lack of molecular biomarkers for rare diseases, such as BBS. Untargeted and targeted proteomic profiling assays were applied to plasma and urine, which aimed to uncover novel biomarkers specific to BBS. 8 significantly differentially expressed proteins were identified from urine, including PEDF, a secreted factor that is linked to fatty acid metabolism and insulin resistance in obesity (Log2FC: 2.56, p = 0.015). Similarly, plasma proteomic analysis identified putative biomarkers that are linked to secondary metabolic features of BBS, such as LEP and ApoM. Markers, such as these, will become subsequent targets for further validation in larger cohorts. BBS patient-derived fibroblasts were profiled by transcriptomic and proteomic technologies, which aimed to identify dysregulated pathways at a cellular level compared to control cultures. Pathway analysis uncovered discordant expression of centrosomal genes between BBS and control cells, as well as a significant enrichment of genes associated with adipogenesis, which may provide insight into obesity manifested by BBS patients. Analysis of protein profiling data revealed dysregulation of processes not detected by RNA-seq, including actin cytoskeleton remodelling and hedgehog signalling. Finally, pathways were integrated to increase the power of analysis, identifying 17 pathways that were found to be impaired at both transcript and protein levels. The phenotype of retinal dystrophy is one of the most detrimental effects on patient welfare, and affects over 90% of BBS patients. As retinal degeneration of BBS patients cannot be investigated in vivo, this project utilised BBS patient-derived induced pluripotent stem cells with the aim of developing a model for the study of retinal degeneration in vitro. For the first time, it was shown that BBS patient cells can differentiate into three-dimensional optic cups, which recapitulated the temporal expression of key retinal markers of in vivo mammalian eye development. Furthermore, immunohistochemistry and electron microscopy assays determined that BBS-derived optic cups could successfully undergo ciliogenesis, which was demonstrated by the formation of nascent photoreceptor outer segments

De-Suppression of Mesenchymal Cell Identities and Variable Phenotypic Outcomes Associated with Knockout of Bbs1

Bardet–Biedl syndrome (BBS) is an archetypal ciliopathy caused by dysfunction of primary cilia. BBS affects multiple tissues, including the kidney, eye and hypothalamic satiety response. Understanding pan-tissue mechanisms of pathogenesis versus those which are tissue-specific, as well as gauging their associated inter-individual variation owing to genetic background and stochastic processes, is of paramount importance in syndromology. The BBSome is a membrane-trafficking and intraflagellar transport (IFT) adaptor protein complex formed by eight BBS proteins, including BBS1, which is the most commonly mutated gene in BBS. To investigate disease pathogenesis, we generated a series of clonal renal collecting duct IMCD3 cell lines carrying defined biallelic nonsense or frameshift mutations in Bbs1, as well as a panel of matching wild-type CRISPR control clones. Using a phenotypic screen and an unbiased multi-omics approach, we note significant clonal variability for all assays, emphasising the importance of analysing panels of genetically defined clones. Our results suggest that BBS1 is required for the suppression of mesenchymal cell identities as the IMCD3 cell passage number increases. This was associated with a failure to express epithelial cell markers and tight junction formation, which was variable amongst clones. Transcriptomic analysis of hypothalamic preparations from BBS mutant mice, as well as BBS patient fibroblasts, suggested that dysregulation of epithelial-to-mesenchymal transition (EMT) genes is a general predisposing feature of BBS across tissues. Collectively, this work suggests that the dynamic stability of the BBSome is essential for the suppression of mesenchymal cell identities as epithelial cells differentiate

Effects of control interventions on Clostridium difficile infection in England: an observational study

Background: The control of Clostridium difficile infections is an international clinical challenge. The incidence of C difficile in England declined by roughly 80% after 2006, following the implementation of national control policies; we tested two hypotheses to investigate their role in this decline. First, if C difficile infection declines in England were driven by reductions in use of particular antibiotics, then incidence of C difficile infections caused by resistant isolates should decline faster than that caused by susceptible isolates across multiple genotypes. Second, if C difficile infection declines were driven by improvements in hospital infection control, then transmitted (secondary) cases should decline regardless of susceptibility. Methods: Regional (Oxfordshire and Leeds, UK) and national data for the incidence of C difficile infections and antimicrobial prescribing data (1998–2014) were combined with whole genome sequences from 4045 national and international C difficile isolates. Genotype (multilocus sequence type) and fluoroquinolone susceptibility were determined from whole genome sequences. The incidence of C difficile infections caused by fluoroquinolone-resistant and fluoroquinolone-susceptible isolates was estimated with negative-binomial regression, overall and per genotype. Selection and transmission were investigated with phylogenetic analyses. Findings: National fluoroquinolone and cephalosporin prescribing correlated highly with incidence of C difficile infections (cross-correlations >0·88), by contrast with total antibiotic prescribing (cross-correlations 0·2). Interpretation: Restricting fluoroquinolone prescribing appears to explain the decline in incidence of C difficile infections, above other measures, in Oxfordshire and Leeds, England. Antimicrobial stewardship should be a central component of C difficile infection control programmes

Multiple novel prostate cancer susceptibility signals identified by fine-mapping of known risk loci among Europeans

Genome-wide association studies (GWAS) have identified numerous common prostate cancer (PrCa) susceptibility loci. We have fine-mapped 64 GWAS regions known at the conclusion of the iCOGS study using large-scale genotyping and imputation in 25 723 PrCa cases and 26 274 controls of European ancestry. We detected evidence for multiple independent signals at 16 regions, 12 of which contained additional newly identified significant associations. A single signal comprising a spectrum of correlated variation was observed at 39 regions; 35 of which are now described by a novel more significantly associated lead SNP, while the originally reported variant remained as the lead SNP only in 4 regions. We also confirmed two association signals in Europeans that had been previously reported only in East-Asian GWAS. Based on statistical evidence and linkage disequilibrium (LD) structure, we have curated and narrowed down the list of the most likely candidate causal variants for each region. Functional annotation using data from ENCODE filtered for PrCa cell lines and eQTL analysis demonstrated significant enrichment for overlap with bio-features within this set. By incorporating the novel risk variants identified here alongside the refined data for existing association signals, we estimate that these loci now explain ∼38.9% of the familial relative risk of PrCa, an 8.9% improvement over the previously reported GWAS tag SNPs. This suggests that a significant fraction of the heritability of PrCa may have been hidden during the discovery phase of GWAS, in particular due to the presence of multiple independent signals within the same regio

Retrospective evaluation of whole exome and genome mutation calls in 746 cancer samples

Funder: NCI U24CA211006Abstract: The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) curated consensus somatic mutation calls using whole exome sequencing (WES) and whole genome sequencing (WGS), respectively. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2,658 cancers across 38 tumour types, we compare WES and WGS side-by-side from 746 TCGA samples, finding that ~80% of mutations overlap in covered exonic regions. We estimate that low variant allele fraction (VAF < 15%) and clonal heterogeneity contribute up to 68% of private WGS mutations and 71% of private WES mutations. We observe that ~30% of private WGS mutations trace to mutations identified by a single variant caller in WES consensus efforts. WGS captures both ~50% more variation in exonic regions and un-observed mutations in loci with variable GC-content. Together, our analysis highlights technological divergences between two reproducible somatic variant detection efforts

De-Suppression of Mesenchymal Cell Identities and Variable Phenotypic Outcomes Associated with Knockout of <i>Bbs1</i>

Bardet–Biedl syndrome (BBS) is an archetypal ciliopathy caused by dysfunction of primary cilia. BBS affects multiple tissues, including the kidney, eye and hypothalamic satiety response. Understanding pan-tissue mechanisms of pathogenesis versus those which are tissue-specific, as well as gauging their associated inter-individual variation owing to genetic background and stochastic processes, is of paramount importance in syndromology. The BBSome is a membrane-trafficking and intraflagellar transport (IFT) adaptor protein complex formed by eight BBS proteins, including BBS1, which is the most commonly mutated gene in BBS. To investigate disease pathogenesis, we generated a series of clonal renal collecting duct IMCD3 cell lines carrying defined biallelic nonsense or frameshift mutations in Bbs1, as well as a panel of matching wild-type CRISPR control clones. Using a phenotypic screen and an unbiased multi-omics approach, we note significant clonal variability for all assays, emphasising the importance of analysing panels of genetically defined clones. Our results suggest that BBS1 is required for the suppression of mesenchymal cell identities as the IMCD3 cell passage number increases. This was associated with a failure to express epithelial cell markers and tight junction formation, which was variable amongst clones. Transcriptomic analysis of hypothalamic preparations from BBS mutant mice, as well as BBS patient fibroblasts, suggested that dysregulation of epithelial-to-mesenchymal transition (EMT) genes is a general predisposing feature of BBS across tissues. Collectively, this work suggests that the dynamic stability of the BBSome is essential for the suppression of mesenchymal cell identities as epithelial cells differentiate

Wench Tactics? Openings in Conditions of Closure

Picking up the question of what FLaK might be, this editorial considers the relationship between openness and closure in feminist legal studies. How do we draw on feminist struggles for openness in common resources, from security to knowledge, as we inhabit a compromised space in commercial publishing? We think about this first in relation to the content of this issue: on image-based abuse continuums, asylum struggles, trials of protestors, customary justice, and not-so-timely reparations. Our thoughts take us through the different ways that openness and closure work in struggles against violence, cruel welcomes, and re-arrangements of code and custom. Secondly, we share some reflections on methodological openness and closure as the roundtable conversation on asylum, and the interview with Riles, remind us of #FLaK2016 and its method of scattering sources as we think about how best to mix knowledges. Thirdly, prompted by the FLaK kitchen table conversations on openness, publishing and ‘getting the word out’, we respond to Kember’s call to ‘open up open access’. We explain the different current arrangements for opening up FLS content and how green open access, the sharedit initiative, author request and publisher discretion present alternatives to gold open access. Finally drawing on Franklin and Spade, we show how there are a range of ‘wench tactics’—adapting gifts, stalling and resting—which we deploy as academic editors who are trying to have an impact on the access, use and circulation of our journal, even though we do not own the journal we edit. These wench tactics are alternatives to the more obvious or reported tactic of resignation, or withdrawing academic labour from editing and reviewing altogether. They help us think about brewing editorial time, what ambivalence over our 25th birthday might mean, and how to inhabit painful places. In this, we respond in our own impure, compromised way to da Silva’s call not to forget the native and slave as we do FLaK, and repurpose shrapnel, in our common commitments