RESUMEN
Joubert syndrome (JBTS) is a genetically heterogeneous autosomal-recessive neurodevelopmental ciliopathy. We investigated further the underlying genetic etiology of Joubert syndrome by studying two unrelated families in whom JBTS was not associated with pathogenic variants in known JBTS-associated genes. Combined autozygosity mapping of both families highlighted a candidate locus on chromosome 10 (chr10: 101569997-109106128, UCSC Genome Browser hg 19), and exome sequencing revealed two missense variants in ARL3 within the candidate locus. The encoded protein, ADP ribosylation factor-like GTPase 3 (ARL3), is a small GTP-binding protein that is involved in directing lipid-modified proteins into the cilium in a GTP-dependent manner. Both missense variants replace the highly conserved Arg149 residue, which we show to be necessary for the interaction with its guanine nucleotide exchange factor ARL13B, such that the mutant protein is associated with reduced INPP5E and NPHP3 localization in cilia. We propose that ARL3 provides a potential hub in the network of proteins implicated in ciliopathies, whereby perturbation of ARL3 leads to the mislocalization of multiple ciliary proteins as a result of abnormal displacement of lipidated protein cargo.
Asunto(s)
Factores de Ribosilacion-ADP/genética , Anomalías Múltiples/genética , Cerebelo/anomalías , Cilios/genética , Anomalías del Ojo/genética , Enfermedades Renales Quísticas/genética , Mutación Missense/genética , Retina/anomalías , Adulto , Niño , Preescolar , Cromosomas Humanos Par 10/genética , Exoma/genética , Femenino , Proteínas de Unión al GTP/genética , Factores de Intercambio de Guanina Nucleótido/genética , Humanos , Masculino , Transporte de Proteínas/genética , Adulto JovenRESUMEN
Zebrafish are a valuable vertebrate model in which to study development and characterize genes involved in cystic kidney disease. Zebrafish embryos and larvae are transparent, allowing non-invasive imaging during their rapid development, which takes place over the first 72 hours post fertilisation. Gene-specific knockdown of nephronophthisis-associated genes leads to ciliary phenotypes which can be assessed in various developmental structures. Here we describe in detail the methods used for imaging cilia within Kupffer's vesicle to assess nephronophthisis and related ciliopathy phenotypes.