Ciliopathies are a group of heterogeneous diseases associated with ciliary dysfunction. Diseases in this group display considerable phenotypic variation within individual diseases as well as overlapping phenotypes among clinically distinct diseases. In particular, mutations in CEP290 cause phenotypically diverse ciliopathies ranging from isolated retinal degeneration, nephronophthisis (NPHP), and Bardet-Biedl Syndrome (BBS) to the neonatal lethal Meckel-Gruber syndrome (MKS). However, the underlying mechanisms of the variable expressivity in ciliopathies are not well understood. This thesis focuses on evaluating the molecular and biological processes behind the retinal degeneration and obesity observed in cilia disorders with respect to CEP290 and other ciliopathy genes using the zebrafish and mouse model systems. CEP290 is the most frequently mutated gene underlying the non-syndromic blinding disorder, Leber's congenital amaurosis (LCA). We first aimed to characterize the function of various CEP290 domains and to characterize a zebrafish model aimed at progressing towards future therapy for patients with CEP290 LCA. To this end, an antisense oligonucleotide [Morpholino(MO)] was used for gene knockdown. We showed that cep290 MO-injected embryos have reduced Kupffer's vesicle size and delays in melanosome transport, two phenotypes that are observed upon knockdown of BBS genes in zebrafish. More importantly, the embryos had a statistically significant reduction in visual function, and this vision impairment caused by the disruption of cep290 can be rescued by expressing only the N-terminal region of the human CEP290 protein. These data indicate a specific region of the CEP290 protein, which is necessary for visual function. We examined the contribution of BBS genes to the clinical variability of CEP290-associated ciliopathies. We demonstrated that the BBSome binds to the N-terminal region of CEP290 and co-localizes with CEP290 to the centriolar satellite in ciliated cells and to the connecting cilium of photoreceptor cells. We further showed that the BBSome is required for proper localization of CEP290 in these structures. Genetic interactions were tested using Cep290rd16, a Cep290 hypomorphic allele with an in-frame deletion of 299 residues, and Bbs4 null mutant mouse lines. Additional loss of Bbs4 alleles in Cep290rd/rd mutants results in increased body weight and accelerated photoreceptor degeneration compared to mice without Bbs4 mutations. Furthermore, double heterozygous mice (Cep290+/rd16; Bbs4+/-) have increased body weight compared to single heterozygous animals. Our data indicated that genetic interactions between the BBSome components and CEP290 underlie the variable expression and overlapping phenotypes of ciliopathies caused by CEP290 mutations. Finally, this work was extended to other cilia disorders through the characterization of genetic interactions between CEP290 and other ciliopathy genes. We found that different NPHP and MKS proteins interact with CEP290 via its different regions, suggesting the central role of CEP290 in CEP290 biological/cellular functions. To characterize the functional interaction between these proteins, we used in vitro systems to double knockdown CEP290 with other NPHP and MKS genes and showed that depletion of a certain combination set of these proteins disrupted the localization of proteins into the cilia. The data indicated that the phenotypic variability of human ciliopathies is associated with different degree of compromise of cilia function.

Mutations in BBS6 cause two clinically distinct syndromes, Bardet-Biedl syndrome (BBS), a syndrome caused by defects in cilia transport and function, as well as McKusick-Kaufman syndrome, a genetic disorder characterized by congenital heart defects. Congenital heart defects are rare in BBS, and McKusick-Kaufman syndrome patients do not develop retinitis pigmentosa. Therefore, the McKusick-Kaufman syndrome allele may highlight cellular functions of BBS6 distinct from the presently understood functions in the cilia. In support, we find that the McKusick-Kaufman syndrome disease-associated allele, BBS6H84Y; A242S, maintains cilia function. We demonstrate that BBS6 is actively transported between the cytoplasm and nucleus, and that BBS6H84Y; A242S, is defective in this transport. We developed a transgenic zebrafish with inducible bbs6 to identify novel binding partners of BBS6, and we find interaction with the SWI/SNF chromatin remodeling protein Smarcc1a (SMARCC1 in humans). We demonstrate that through this interaction, BBS6 modulates the sub-cellular localization of SMARCC1 and find, by transcriptional profiling, similar transcriptional changes following smarcc1a and bbs6 manipulation. Our work identifies a new function for BBS6 in nuclear-cytoplasmic transport, and provides insight into the disease mechanism underlying the congenital heart defects in McKusick-Kaufman syndrome patients.

Les maladies rétiniennes héréditaires représentent un groupe de pathologie hétérogène sur le plan phénotypique et génétique. Elles sont dues à une dysfonction ou une dégénérescence de la neurorétine ou de l’épithélium pigmentaire rétinien. Elles peuvent être présente de manière isolée ou être associée à des atteintes extraoculaires dans les formes syndromiques. Le travail de thèse porte sur l’identification moléculaire de gènes responsables de formes rares de pathologie rétinienne syndromique incluant les ciliopathies dont le syndrome de Bardet-Biedl, caractérisé notamment par une dégénérescence rétinienne constante, et les microcéphalies associées à une choriorétinopathie. Par une approche de séquençage exomique, nous avons pu identifier trois nouveaux gènes impliqués dans ces pathologies rétiniennes syndromiques et confirmer l’implication d’un gène dans le syndrome de Bardet-Biedl. L’analyse des phénotypes rétiniens d’une cohorte de patients présentant un syndrome de Bardet-Biedl a permis la description d’un phénotype atypique avec atteinte prédominante des cônes. / Inherited retinal diseases (IRDs) represent a clinically and genetically heterogeneous group of rare pathologies. These disorders result of a dysfunction or a degeneration of the photoreceptors or the retinal pigment epithelium. IRDs can be subdivided in isolated forms, and syndromic forms that involve non–ocular features. This work deals with the molecular identification of genes implicated in rare forms of syndromic retinal diseases, including the ciliopathies with the Bardet-Biedl syndrome (BBS), characterized by a constant retinal degeneration, and the microcephaly associated with chorioretinopathy.Using an exomic sequencing approach, we have identified three new genes involved in these rare syndromic retinal diseases and confirmed the implication of a gene in Bardet-Biedl syndrome. The analysis of the retinal phenotypes of a BBS patients’ cohort allowed the description of an atypical retinal phenotype with predominantly cone dysfunction.

Hundreds of individually rare, but collectively common Mendelian disorders result in visual impairment. One of these disorders is a heterogeneous syndromic form of retinal degeneration, Bardet-Biedl Syndrome (BBS). This disease is an autosomal recessive disorder characterized by retinal degeneration, obesity, learning disabilities, congenital anomalies, and an increased incidence of hypertension and diabetes. Typically, individuals with BBS experience vision loss during childhood leading to blindness by the third decade of life. At least fourteen genes (BBS1-BBS14) are reported to individual cause BBS. This thesis focuses on one of these genes, BBS3, with the overall goal of characterizig the function of BBS3 in terms of both syndromic and non-syndromic retinal degeneration using the zebrafish and mouse model systems. A member of the Ras family of small GTP-binding proteins, BBS3 is postulated to play a role in vesicular transport. A second highly conserved transcript of BBS3, BBS3L, has been identified and is expressed predominantly in the mouse and zebrafish eye. The eye-specific expression of BBS3L facilitates the dissection of BBS function in the retina independent of alterations to other tissues. To this end a Bbs3L knockout mouse was generated and histological analysis at 9 months reveals disorganization of the inner segments, indicative of retinal degeneration. To further evaluate the functional effects of BBS3 deficiency in the eye, an antisense oligonucleotide (Morpholino) approach was utilized to knockdown bbs3 gene expression in zebrafish. Consistent with an eye specific role, knockdown of bbs3L results in mislocalization of the photopigment green cone opsin and reduced visual function, but not abnormalities of the Kupffer's vesicle or delays in intracellular trafficking of melanosomes, both cardinal features of BBS in the zebrafish. To dissect the individual functions of BBS3 and BBS3L, in vitro transcribed wild-type human BBS3 or BBS3L RNA was co-injected with the bbs3 morpholinos. BBS3L RNA, but not BBS3 RNA, restores green opsin localization and vision. Moreover, only BBS3 RNA is sufficient to rescue melanosome transport, a cardinal feature of BBS in the zebrafish. Bbs3L knockout mice as well as a zebrafish bbs3 knockdown model demonstrate that BBS3L is both necessary and sufficient for retinal function and organization. This work was extended to humans by characterizing the A89V missense mutation in BBS3 that results in non-syndromic retinal degeneration. To evaluate the in vivo function of the A89V missense mutation in non-syndromic retinal degeneration and BBS, rescue experiments were performed in the zebrafish. Unlike wild-type BBS3L RNA, BBS3L A89V RNA does not rescue the vision defect seen with loss of bbs3 in zebrafish; however, BBS3 A89V RNA is able to suppress the cardinal zebrafish BBS phenotype of melanosome transport, similar to wild-type BBS3 RNA. These data demonstrate that the BBS3L A89V mutation identified in patients with non-syndromic retinal degeneration is critical and specific for the vision defect.

It is estimated that 4000 genetic diseases/syndromes affect humans with one third of these diseases involving the eye. Many eye disorders, such as age-related macular degeneration that affects an estimated 170 million elderly adults worldwide, are associated with genetic variants. Since the conception of the human genome project we have learned a great deal about the genetic make-up of the human race and have identified over ~20,000 genes. Over 270 of these genes have been implicated in retinal diseases alone with many more genes involved in other forms of ocular disease. Though we have made a great deal of progress in understanding the genetics of eye disease, there remain many eye diseases with significant evidence of genetic components for which a disease-causing gene has not been identified. In my thesis research, I utilized computational tools and strategies to analyze microarrays and whole-exome sequencing to investigate the genetic causes of three different eye diseases. First, I utilized a combination of familial analyses and whole-exome sequencing to study the genetic cause of Keratoconus, a progressive cornea abnormality that can lead to distorted vision and light sensitivity. Second, I analyzed three different cohorts of patients with Bardet-Biedl syndrome (BBS), a syndromic retinopathy leading to blindness, using whole-exome sequencing to identify both known and novel genetic causes of BBS. Finally, I performed the largest whole-exome sequencing study at the time for Pigment Dispersion Syndrome (PDS), a disorder associated with glaucoma, and identified variants within previously established candidate genes and a novel candidate gene that is now the subject of further scientific investigation. By using computational tools and strategies in tandem with high-quality bench research performed by fellow lab members, we have identified both candidate and known eye disease-causing genes/mutations and furthered the goal to cure blindness.

Thesis (Ph.D.)--Memorial University of Newfoundland, 2001. / Bibliography: leaves 197-218.

Bardet-Biedl syndrome (BBS) is a pleiotropic and genetically heterogeneous disorder, and a well-known ciliopathy. Nineteen different genes have been reported for BBS, mutations in which cause characteristic phenotypes including retinal degeneration, obesity, polydactyly, renal abnormalities, hypogenitalism and cognitive impairment. Protein products of eleven BBS genes are part of two major complexes: the BBSome complex and a CCT/CTRiC/BBS complex. The CCT/CTRiC/BBS complex assists in the formation of the BBSome complex, which in turn traffics numerous receptor proteins to the cilia. However, the precise mechanism by which BBSome ciliary trafficking activity is regulated is not fully understood. In fact, a complete picture of the cellular functions of BBS proteins is still missing, and gaps remain in our understanding of the pleiotropy and heterogeneity of the disease. With the aim of bridging those gaps, this thesis project was designed to identify tissue specific cargoes of the BBSome and to characterize their BBS-related functions. To this end, we generated a transgenic LAP-BBS4 mouse, which expresses the transgene in various tissues including brain, eye, testis, heart, kidney, and adipose tissue. We found that despite tissue specific variable expression, LAP-BBS4 was able to complement the deficiency of Bbs4 and rescue all the BBS phenotypes in the Bbs4 null mice. The finding provides an encouraging prospective for gene therapy for BBS related phenotypes and potentially for other ciliopathies. We also utilized the transgenic mice to search for tissue specific BBSome cargo proteins and identified CEP131 as a novel BBSome interacting protein. Using in vitro cell culture models we show that CEP131 interacts with the BBSome through BBS4. CEP131 is not involved in BBSome assembly, but accumulation of the BBSome in cilia is enhanced upon CEP131 depletion. Our in vitro data implicate CEP131 as a negative regulator of ciliary BBSome trafficking. Finally, we show that cep131 knockdown in zebrafish embryos results in typical BBS phenotypes including Kupffer's vesicle abnormalities and melanosome transport delay. This finding confirms the association of CEP131 with the BBS pathway. Overall, the work performed for this thesis provides further insight into the regulation of BBSome ciliary trafficking and suggests CEP131 as a BBS candidate gene.

Thesis (M.Sc.)--Memorial University of Newfoundland, 2004. / Restricted until May 2005. Bibliography: leaves 62-68.

Le syndrome de Bardet-Biedl (BBS) est une ciliopathie syndromique associant une rétinopathie pigmentaire, une polydactylie post-axiale, une obésité, un hypogonadisme, des anomalies rénales et des troubles des apprentissages. Le cil primaire est présent à la surface de la quasi totalité des cellules de l’organisme et joue un rôle d’antenne cellulaire captant les signaux extérieurs pour les transmettre à la cellule. A ce jour 21 gènes BBS ont été identifiés codant des protéines ayant une fonction ciliaire. Au cours de ce travail, nous avons identifié 3 nouveaux gènes BBS (SDCCAG8/BBS16, LZTFL1/BBS17, BBIP1/BBS18) et confirmé l’implication de IFT172/BBS20. Nous avons également établi des corrélations génotype-phénotype : absence de polydactylie et insuffisance rénale associées aux mutations dans BBS16 ; polydactylie mésoaxiale et atteinte rénale associées aux mutations dans BBS17 ; possible association d’une polydactylie préaxiale aux mutations dans BBS20. Enfin, nous décrivons sur le plan clinique et moléculaire la plus grande cohorte de patients BBS à partir d’une base de données clinico-biologique mise en place au cours de ce travail. / Bardet-Biedl syndrome (BBS) is a syndromic ciliopathy associating with retinitis pigmentosa, postaxial polydactyly, obesity, hypogonadism, renal anomalies and learning difficulties. The primary cilium is antenna-like structure at the surface of the cell. 21 BBS genes are identified and the corresponding proteins are related to primary cilium structure and function. In this study, we identified 3 new BBS genes (SDCCAG8/BBS16, LZTFL1/BBS17, BBIP1/BBS18) and we confirmed the implication of IFT172/BBS20 in this syndrome. We also established strong genotype-phenotype correlations: absence of polydactyly and early renal failure in SDCCAG8/BBS16 patients; mesoaxial polydactyly and early renal failure in LZTFL1/BBS17 patients; possible preaxial polydactyly in IFT172/BBS20 patients. Finally, we reported the molecular and clinical description of the largest BBS cohort thanks to the clinical and biological database created in the Laboratory.

Le syndrome de Bardet-Biedl (BBS) est considéré comme l’une des causes les plus fréquentes de rétinopathie pigmentaire dite syndromique. Il a été démontré une connexion entre les protéines BBS et les structures du cil primaire. Le cil primaire est un organelle formé par une fine évagination de la membrane plasmique soutenu par une ossature de microtubules. Dans la rétine, le photorécepteur (PR) est une cellule ciliaire composée d’un segment interne et d’un segment externe reliés par un cil primaire modifié. Au cours de ce travail, nous avons mis en évidence que le stress du réticulum endoplasmique est à l’origine du processus apoptotique car un défaut ciliaire dans le PR entraine l’accumulation de protéines dans le segment interne et déclenche une réponse au stress cellulaire appelé unfolded protein response. Nous avons développé un traitement pharmacologique modulant ce stress cellulaire afin de ralentir l’apoptose des PR dans un modèle murin BBS. Cette approche pharmacologique a montré son efficacité dans le maintien et la fonctionnalité des PR. Elle pourrait potentiellement être applicable à d’autres ciliopathies rétiniennes. / Bardet-Biedl syndrome (BBS) is one of the most frequent cause of syndromic retinitis pigmentosa. BBS proteins are related to primary cilium structure and function. The primary cilium is microtubule-based antenna-like structure at the surface of the cell. In the retina, the photoreceptor (PR) is a ciliated cell composed of an inner and an outer segment linked by a modified primary cilium. In this study, we demonstrated that endoplasmic reticulum stress induces unfolded protein response due to protein accumulation in the inner segment in case of ciliary defect in the PR leading to apoptosis. We designed a pharmacological treatment to alleviate PR apoptosis in a BBS mouse model. This pharmacological approach was efficient to protect PR from apoptosis and maintain their functionality. This treatment could be applicable to others retinal ciliopathies.