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Jan 30, 2011

Functional analysis of BBS3 A89V that results in non-syndromic retinal degeneration

Pamela R Pretorius, Mohammed A Aldahmesh, Fowzan S Alkuraya, Val C Sheffield, Diane C Slusarski | Human Molecular Genetics | 2011 Jan 31 | 20(8) | 1625–1632 | doi:10.1093/hmg/ddr039


Abstract

Bardet–Biedl syndrome (BBS) is a syndromic form of retinal degeneration. Recently, homozygosity mapping with a consanguineous family with isolated retinitis pigmentosa identified a missense mutation in BBS3, a known BBS gene. The mutation in BBS3 encodes a single amino acid change at position 89 from alanine to valine. Since this amino acid is conserved in a wide range of vertebrates, we utilized the zebrafish model system to functionally characterize the BBS3 A89V mutation. Knockdown of bbs3 in zebrafish alters intracellular transport, a phenotype observed with knockdown of all BBS genes in the zebrafish, as well as visual impairment. Here, we find that BBS3 A89V is sufficient to rescue the transport delays induced by the loss of bbs3, indicating that this mutation does not affect the function of BBS3 as it relates to syndromic disease. BBS3L A89V, however, was unable to rescue vision impairment, highlighting a role for a specific amino acid within BBS3 that is necessary for visual function, but dispensable in other cell types. These data aid in our understanding of why patients with the BBS3 A89V missense mutation only present with isolated retinitis pigmentosa.


Introduction

Bardet–Biedl syndrome (BBS, OMIM 209900) is a genetically heterogeneous autosomal recessive disorder characterized by retinitis pigmentosa, obesity, polydactyly, renal abnormalities, hypogenitalism and cognitive impairment (1–4). Moreover, BBS is associated with an increased risk for hypertension, diabetes and heart defects (1,2,5). BBS patients present with early and progressive photoreceptor degeneration and are blind by the third decade of life (2,6–13). To date, 12 BBS (BBS1–12) genes are reported to individually cause BBS (14–27). Additionally, hypomorphic mutations in MKS1 and CEP290 have been associated with BBS, representing BBS13 and BBS14, respectively (28). The BBS genes belong to multiple protein families and function cannot be defined based on homology; however, recent advances in molecular pathophysiology and animal models have helped to elucidate why 14 different genes can lead to the same phenotype. Work in mouse, zebrafish, Caenorhabditis elegans and Chlamydomonas has provided multiple lines of evidence supporting a role for BBS proteins in cilia function and intraflagellar and/or intracellular transport (19,22,23,26,29–36). Although progress has been made in understanding the pathophysiology of BBS, there are major gaps in our understanding of the precise cellular function of the BBS proteins.


BBS3 (ARL6, ADP-ribosylation factor-like), a member of the Ras family of small GTP-binding proteins, was initially identified as a BBS gene through computational genomics and high-density single nucleotide polymorphism (SNP) genotyping (21,22). Several mutations (G2X, T31M, T31R, P108L, R122X, G169A and L170W) leading to BBS have been reported throughout BBS3 (21,22,37). Knockdown of bbs3 using an antisense oligonucleotide [Morpholino (MO)] results in two cardinal features of BBS in the zebrafish: reduced size of the ciliated Kupffer's Vesicle and delays in intracellular melanosome transport (35,38). These prototypical phenotypes are preset with knockdown of all BBS genes in the zebrafish (26,34,35,38). Recently, we identified a second longer eye-specific transcript of BBS3, BBS3L, which is required for retinal organization and function in both the mouse and zebrafish (38). Knockdown of either both bbs3 transcripts or bbs3L alone leads to vision impairment in zebrafish. To determine the functional requirement of each transcript, RNA encoding either human BBS3 or BBS3L was co-injected with the bbs3 aug MO, which targets both transcripts. We determined that human BBS3 RNA is sufficient to suppress the melanosome transport delays, but not the vision defect. In contrast, BBS3L RNA was sufficient to rescue the vision defect; however, it was unable to suppress the cardinal phenotypes of BBS seen in the zebrafish, supporting a retina specific role for BBS3L (38).


BBS is rare in the general population; however, the study of this disease can offer insight into normal retinal development as well as provide an understanding of the pathophysiology involved in non-syndromic forms of BBS. Homozygosity mapping of a consanguineous Saudi Arabian family has identified a missense mutation (A89V) in BBS3 that leads to non-syndromic retinitis pigmentosa (39,40). The identification of specific mutations in the same gene that results in either syndromic or non-syndromic retinitis pigmentosa will provide insight into tissue-specific functional regions of BBS3 in the retina. Moreover, understanding the functional domains of proteins involved in vision aids in our understanding of not only the disease state, but also normal vision development.


Here we report the functional characterization of the BBS3 missense mutation (A89V), which occurs in a highly conserved region of BBS3. The function of the BBS3 A89V mutation was evaluated by utilizing gene knockdown of bbs3 coupled with RNA rescue in the zebrafish. We examined the intracellular transport of melanosomes, a cardinal feature of BBS gene knockdown in the zebrafish, and visual function using a vision startle assay. The A89V mutation can suppress the melanosome transport defects, but not the vision impairment observed with the loss of bbs3. Thus, the missense mutation identified in patients with non-syndromic retinal degeneration has uncovered an amino acid in BBS3 that is necessary for vision. The A89V mutation is able to function in melanosome transport, demonstrating that the mutant form of the protein retains the ability to function in tissues typically affected by BBS.




 

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