Document Type


Date of Degree

Summer 2013

Degree Name

PhD (Doctor of Philosophy)

Degree In


First Advisor

Sheffield, Val C

First Committee Member

Smith, Richard

Second Committee Member

Mullins, Robert

Third Committee Member

Kwitek, Anne E

Fourth Committee Member

Rahmouni, Kamal


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.


Bardet-Biedl Syndrome, CEP290, cilia


xiii, 138 pages


Includes bibliographical references (pages 126-138).


Copyright 2013 Yan Zhang

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Genetics Commons