Date of Degree
PhD (Doctor of Philosophy)
Val C. Sheffield
Diane C. Slusarski
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.
Copyright 2010 Pamela Reed Pretorius
Pretorius, Pamela Reed. "Elucidating a role for BBS3 in syndromic and non-syndromic retinal disease." dissertation, University of Iowa, 2010.