Abstract
The multiple genetic approaches available for molecular diagnosis of human diseases have made possible to identify an increasing number of pathogenic genetic changes, particularly with the advent of next generation sequencing (NGS) technologies. However, the main challenge lies in the interpretation of their functional impact, which has resulted in the widespread use of animal models. We describe here the functional modelling of seven BBS loci variants, most of them novel, in zebrafish embryos to validate their in silico prediction of pathogenicity. We show that target knockdown (KD) of known BBS (BBS1, BB5 or BBS6) loci leads to developmental defects commonly associated with ciliopathies, as previously described. These KD pleiotropic phenotypes were rescued by co-injecting human wild type (WT) loci sequence but not with the equivalent mutated mRNAs, providing evidence of the pathogenic effect of these BBS changes. Furthermore, direct assessment of cilia located in Kupffer’s vesicle (KV) showed a reduction of ciliary length associated with all the studied variants, thus confirming a deleterious effect. Taken together, our results seem to prove the pathogenicity of the already classified and unclassified new BBS variants, as well as highlight the usefulness of zebrafish as an animal model for in vivo assays in human ciliopathies.
Highlights
The multiple genetic approaches available for molecular diagnosis of human diseases have made possible to identify an increasing number of pathogenic genetic changes, with the advent of generation sequencing (NGS) technologies
Bardet-Biedl syndrome (BBS) proteins are necessary to maintain primary cilia structure and function[12], some of them through multiprotein complexes. Such is the case of the BBSome, composed by eight BBS proteins (BBS1, BBS2, BBS4, BBS5, BBS7-BBS9 and BBIP10/BBS18), which participates in the vesicle trafficking of membrane proteins to and inside the primary cilium to promote
The combination of several genetic approaches allowed us to confirm the molecular diagnosis of five BBS patients whereby seven predicted pathogenic variants, some of them novel, were found in three BBS genes: BBS1, BBS5 and BBS6
Summary
Bbs-MOs and WT human BBS capped mRNA co-injected embryos showed KV cilia length similar to controls (4.97 + 0.31 μm, 4.96 + 0.20 μm and 4.83 + 0.13 μm in bbs[1], bbs[5] and bbs[6] genes; p > 0.05; Figs 5B, 6B and 7B), confirming the rescue of the morphant phenotype in all cases. We compared bss morphants to controls and found that all injected capped mRNA mutant variants resulted in shorter KV cilia (values shown in Figs 5G, 6G and 7E), with the exception of BBS5:p.(Phe180Phefs*6), which in contrast fully rescued the morphant phenotype, being not statistically different from controls (Fig. 6G). No significant differences in cilia length of BBS5:p.(Phe180Phefs*6) rescued morphants were found, KV cilia appear to be dispersed, covering a slightly broader area compared to controls (Fig. 6E), as well as a significant reduced number of KV cilia (Supplementary Fig. S1). BBS6:p.(Gly411Ala) non-rescued morphant fish showed a portion of cilia with a curvy shape, not as frequently seen in other morphants (Fig. 7D), accompanied with a significant lower number of cilia (Supplementary Fig. S1)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
