Functional analysis of new human Bardet-Biedl syndrome loci specific variants in the zebrafish model.

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.

[Patho-physiology of renal dysfunction in Bardet-Biedl Syndrome]. / DIl coinvolgimento renale nella sindrome di Bardet-Biedl.

Bardet-Biedl Syndrome (BBS) is a rare autosomal recessive disorder with renal and extra-renal involvement. The wide spectrum of clinical manifestations is associated to the high genetic heterogeneity. To date 21 genes have been identified in humans and the majority of them encode proteins located on the basal body of the primary cilium. For this reason the disease is has been included among the 'ciliopathies'. The renal involvement is extremely heterogeneous in BBS and is considered the main cause of morbidity and mortality. Recent evidences have suggested that mutations in BBS6, 10 and 12 are associated with a more severe renal dysfunction. The most common renal dysfunction is the urine concentrating defect, even though the underlying mechanism is not completely known. Recently we have demonstrated that hyposthenuria in BBS patients has a renal origin, and depends on desmopessin resistance. The majority of hyposthenuric BBS patients have a combined defect to both concentrate and dilute the urine. The combined defect is associated with a blunted increased urine Aquaproine-2 (u-AQP2) excretion in antidiuresis. A ccordingly, in vitro BBS10 silencing prevented AQP2 trafficking to the apical plasma membrane. However, after long term water restriction hyposthenuric BBS patients showed the same u-AQP2 excretion compared with controls, suggesting that other mechanisms are implicated into the pathogenesis of hyposhtenuria. The complete molecular mechanism underlying hyposhtenuria remains largely unknown in BBS. Whether this defect may represent a predictor factor for poor renal outcome remains to be elucidated.