BBS is a genetically heterogeneous disorder. To date, mutations in 18 genes (BBS1-18) are known to cause the phenotype
[14, 16]. Bioinformatics analysis of these genes demonstrates that the majority of BBS genes are not genetic duplications, but are distinct genes encoding for proteins in a linked pathway
. In BBS, the most commonly mutated genes are BBS1 and BBS10, accounting for 23% and 21% of cases respectively
. BBS5 mutations are rare, with only 0.4 to 2% of cases linked to a mutation in this gene
The intracellular role of BBS proteins has only recently begun to be understood. A breakthrough in understanding the function of BBS proteins came with the proposal by Nachury et al. which detailed that BBS1,2,4,5,7-9 form a functional complex named the BBSome
. They observed that these proteins were found in stoichiometric amounts after purification and identification with mass spectrometry, and that these proteins co-fractionate together
. Interestingly, the BBSome proteins are highly conserved across ciliated organisms
. The BBSome associates with both the ciliary membrane and with Rab8, a GTPase which is known to promote vesicular transport to the cell membrane
. BBS3 (or ARL6), while not a BBSome protein, is also a GTPase and thought to localise to the ciliary membrane assembly point at the base of the cilium
, and has been shown to associate with the BBSome
. The BBSome localises with intraflagellar transport (IFT) particles as it travels the length of C. reinhardtii flagella
 but does not seem to be a core component of the IFT complex
. Instead, it is proposed that the BBSome acts as an adaptor for protein cargo that is transported up the cilia by IFT
. These studies come together to present the current hypothesis that the BBSome, together with Rab8 and BBS3, function as adapters between vesicular bound proteins and IFT proteins to promote ciliary membrane biogenesis.
For many years, analysis of families with BBS showed evidence of a fifth locus for the disorder at position 2q31
[1, 36] but it was not until a study by Li et al. in 2004 that the BBS5 gene was discovered using an elegant comparative genomics method
. They hypothesised that unknown genes related to ciliary function will not be present in an organism without cilia, Arabidopsis, but present in organisms with cilia, humans and Chlamydomonas. BBS5 is conserved and present in at least 23 orthologues including Toxoplasma gondii. The molecular genetic diagnosis of patients with BBS is challenging because of genetic heterogeneity of BBS. We and others have shown that homozygosity mapping is a robust approach that is highly suited for genetically heterogeneous autosomal recessive disorders in populations in which consanguinity is highly prevalent. This approach of using genechip platforms of genome wide SNPs helped identify a disease locus. In the family we report, homozygosity mapping directly targeted BBS5 as the likely cause. Phenotype-genotype correlation is typically very poor in BBS. Although we detected a mutation in BBS5, the phenotype we report resembles reported phenotypes from same population who had mutations in BBS1, BBS3, and BBS4.
In our family, the BBS5 c.966dupT (p.Ala323CysfsX57) novel mutation led to a predicted elongation of the BBS protein following a change in the reading frame. To date, there are 17 previously reported mutations in BBS5, which include missense
[17, 38–43], splice site mutations
[17, 43] and small deletions/insertions
[17, 43, 44]. However, to our knowledge a mutation that leads to a predicted elongation of the BBS5 protein has not previously been reported. Additional studies including RT-PCR studies and Western blotting of BBS5 protein from affected patients would be required to confirm the predicted effect of the mutation on mRNA and protein, respectively. Unfortunately this was not feasible.
In order to determine the pathogenicity of the BBS5 mutation, we used MO injection of zebrafish embryos. The zebrafish bbs5 shares 90.9% protein identity with human BBS5 which allows specific modelling of human mutations. Data on a limited number (40 to 50) of bbs5 knockout zebrafish embryos have been previously reported
. These embryos demonstrated abnormal Kupffer’s vesicles (KV), altered heart looping and abnormal melanosome transport. These findings show convincing evidence that BBS5 is a ciliopathy gene with roles in multiple developmental processes.
Using mutant BBS5 mRNA to ‘rescue’ the phenotype of bbs5 ATG MO, we saw an enhanced number of embryos with a disease phenotype. In addition, the eye and retinal phenotype remained severe. These data suggest that the c.966dupT mutation is indeed pathogenic, although the exact mechanism is not clear. A number of possible mechanisms can be speculated. As BBS5 is part of the BBSome, the mutation may affect other BBSome proteins and the localisation and function of the BBSome. The loss of the WT C-terminal amino acids may lead to the mislocalisation of BBS5 protein and a series of C-terminal-truncating mutations could be used to identify such a centrosomal localisation motif.
A systematic approach to using zebrafish to evaluate human mutations in BBS genes has previously been reported
 where a significant number of BBS-associated mutations were suggested to have a dominant-negative mode of action. Evidence was provided in zebrafish embryos that certain mutant mRNAs produced phenotypes significantly worse than MO alone
. However, it must be remembered that when using over-expression systems, there is the possibility of inducing an over-expression defect that is not relevant to the human condition.
Using zebrafish as a model for ciliopathies is well established, with reported models of Joubert syndrome
[21, 46, 47], Meckel-Gruber syndrome,
[48, 49] Jeune syndrome
 and nephronophthisis
[51–53]. There have also been several other reported zebrafish models of BBS
[26, 29, 54, 55] again demonstrating retinal defects, defective melanosome transport, abnormal left-right determination with defective heart looping, abnormal KVs with defective cilia and kidney anomalies
[26, 29, 54, 55]. The establishment of left-right asymmetry in the zebrafish is secondary to an asymmetric fluid flow in KV, resulting in asymmetric gene expression across the whole of the developing embryo
. The abnormal cardiac looping secondary to bbs gene knockdown shown here and by others
 is likely to be secondary to a cilial defect within KV and part of a generalised laterality defect.
In bbs5 morphant zebrafish, we have demonstrated dilatation as well as pronephric and cloacal cysts. Furthermore, in the bbs5 morphants we were able to demonstrate a reduction in renal excretory function. In zebrafish, cilia in the pronephros are motile and are important for driving fluid flow within these organs. Disruption of cilia structure or motility in the pronephros leads to fluid accumulation and cystic dilatation
. In bbs5 morphants, the pronephic ducts, as well being dilated, had a reduced and irregular pattern of cilia.
Within the family we describe there were renal anomalies in two out of the three affected siblings. Renal anomalies have a reported prevalence in BBS patients of 24%, although only 52% of patients had undergone an investigative renal examination
. Renal anomalies may be structural changes such as renal parenchymal cysts, calyceal clubbing, foetal lobulation, dysplastic kidneys, unilateral agenesis, hydronephrosis and horseshoe kidney implicating BBS5 in normal renal development
. A progressive decline in renal function in BBS patients may also be seen and may be secondary to vesicouretic reflux and obstruction leading to scarring. Renal disease is a major cause of mortality in BBS patients. Reviewing 20 BBS patients, it was revealed that all patients had either structural or functional renal abnormalities and three had established renal failure
. The functional defects included inability to concentrate urine and renal tubular acidification defects, implicating a dysfunctional renal collecting duct in a similar manner to nephronophthisis
. Chronic kidney disease progressing to established renal failure is a significant cause of morbidity and mortality in patients with BBS
. The findings of pronephric dilatation, together with reduced excretory function in bbs5 morphants, implicate bbs5 in both renal development and functional maintenance of the pronephros. We speculate that these defects may be secondary to renal ciliary dysfunction where bbs proteins coordinate ciliary proteins and ciliary signalling. Remarkably, these zebrafish morphant studies of bbs genes demonstrate a high degree of concordance and fit well with phenotypes reported in humans. Taken together with our data, we confirm that zebrafish are a valid model for the study of BBS genes.
At a cellular level, genes that are mutated in ciliopathies can affect ciliary signalling in different ways. This may be through changes in cilia structure, in mistargeting of signalling molecules or effecting the sensory role of primary cilia
. It is known that BBS5 protein is localized to basal bodies just beneath the cilia
. We confirm a basal body localisation for GFP-tagged BBS5, which became mislocalised upon introduction of the mutation. The (p.Ala323CysfsX57) mutation affects the C-terminal amino acids of the BBS5 protein and does not disrupt the PH domains of BBS5
. Previously, it has been shown that following BBS5 knockdown, centrin and pericentrin were still targeted to centrosomes and centriolar satellite function remained intact, however ciliogenesis was disrupted
. In HEK293 cells transfected with mutant BBS5, we confirm a normal expression pattern of pericentrin with complete loss of colocalisation with BBS5. This data is comparable to the BBS4 mutant allele L327P, which failed to colocalise with γ-tubulin in post-mitotic cells