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.