Integrin Alpha 8 Recessive Mutations Are Responsible for Bilateral Renal Agenesis in Humans

Renal hypodysplasia (RHD) is a heterogeneous condition encompassing a spectrum of kidney development defects including renal agenesis, hypoplasia, and (cystic) dysplasia. Heterozygous mutations of several genes have been identified as genetic causes of RHD with various severity. However, these genes and mutations are not associated with bilateral renal agenesis, except for RET mutations, which could be involved in a few cases. The pathophysiological mechanisms leading to total absence of kidney development thus remain largely elusive. By using a whole-exome sequencing approach in families with several fetuses with bilateral renal agenesis, we identified recessive mutations in the integrin α8-encoding gene ITGA8 in two families. Itga8 homozygous knockout in mice is known to result in absence of kidney development. We provide evidence of a damaging effect of the human ITGA8 mutations. These results demonstrate that mutations of ITGA8 are a genetic cause of bilateral renal agenesis and that, at least in some cases, bilateral renal agenesis is an autosomal-recessive disease

Characterization of the NPHP1 Locus: Mutational Mechanism Involved in Deletions in Familial Juvenile Nephronophthisis

Familial juvenile nephronophthisis is an autosomal recessive, genetically heterogeneous kidney disorder representing the most frequent inherited cause of chronic renal failure in children. A gene, NPHP1, responsible for ∼85% of the purely renal form of nephronophthisis, has been mapped to 2q13 and characterized. The major NPHP1 gene defect is a large homozygous deletion found in ∼80% of the patients. In this study, by large-scale genomic sequencing and pulsed-field gel electrophoresis analysis, we characterized the complex organization of the NPHP1 locus and determined the mutational mechanism that results in the large deletion observed in most patients. We showed that the deletion is 290 kb in size and that NPHP1 is flanked by two large inverted repeats of ∼330 kb. In addition, a second sequence of 45 kb located adjacent to the proximal 330-kb repeat was shown to be directly repeated 250 kb away within the distal 330-kb repeat deleting the sequence tag site (STS) 804H10R present in the proximal copy. The patients' deletion breakpoints appear to be located within the 45-kb repeat, suggesting an unequal recombination between the two homologous copies of this smaller repeat. Moreover, we demonstrated a nonpathologic rearrangement involving the two 330-kb inverted repeats found in 11 patients and, in the homozygous state, in 2 (1.3%) control individuals. This could be explained by interchromosomal mispairing of the 330-kb inverted repeat, followed by double recombination or by a prior intrachromosomal mispairing of these repeats, leading to an inversion of the NPHP1 region, followed by an interchromosomal unequal crossover event. This complex rearrangement, as well as the common deletion found in most patients, illustrates the high level of rearrangements occurring in the centromeric region of chromosome 2

Dishevelled stablisation at the cilium by RPGRIP1L is essential for planar cell polarity

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Dishevelled stabilization by the ciliopathy protein Rpgrip1l is essential for planar cell polarity.

International audienceCilia are at the core of planar polarity cellular events in many systems. However, the molecular mechanisms by which they influence the polarization process are unclear. Here, we identify the function of the ciliopathy protein Rpgrip1l in planar polarity. In the mouse cochlea and in the zebrafish floor plate, Rpgrip1l was required for positioning the basal body along the planar polarity axis. Rpgrip1l was also essential for stabilizing dishevelled at the cilium base in the zebrafish floor plate and in mammalian renal cells. In rescue experiments, we showed that in the zebrafish floor plate the function of Rpgrip1l in planar polarity was mediated by dishevelled stabilization. In cultured cells, Rpgrip1l participated in a complex with inversin and nephrocystin-4, two ciliopathy proteins known to target dishevelled to the proteasome, and, in this complex, Rpgrip1l prevented dishevelled degradation. We thus uncover a ciliopathy protein complex that finely tunes dishevelled levels, thereby modulating planar cell polarity processes

Nephrocystins play a crucial role in renal epithelial morphogenesis via the regulation of Wnt/PCP components Dishevelled and Rho GTPases

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Alteration of nephrocystins and IFT-A proteins causes similar ciliary phenotypes leading to Nephronophthisis

International audienceNephronophtisis (NPH) is a kidney ciliopathy often associated with extra-renal defects and for which 12 genes (NPHP1-12) have been identified. NPHP1 and NPHP4 control the ciliary access at the transition zone and the velocity of some intraflagellar transport (IFT)/BBS proteins in C.elegans. Recently, in a collaborative effort, we have identified, in families with isolated NPH, mutations in TTC21B as well as in WDR19, which encode the retrograde IFT-A proteins IFT139 and IFT144, respectively. By ciliome sequencing of 1600 candidate genes from 14 NPH patients followed by Sanger sequencing of a cohort of 52 patients, we have found respectively 8 and 7 patients carrying pathogenic missense mutations in genes coding IFT-A proteins, including WDR35, TTC21B and IFT140, which could partially affect their function. Together, these results indicate that IFT-A are involved in nephronophtisis. Moreover, alteration of cilia length was observed in patient kidney, Nphp4-/- mice kidney tubules and NPHP1 or NPHP4 knockdown IMCD3 cell lines. In these cells, primary cilia present swellings at the distal region accompanied by an accumulation of IFT-B at the base and the tip, similar to what was observed in IFT-A mutants, suggesting a possible alteration of retrograde transport. Additionally, ARL13B, a small GTPase required for proper cilium shape and IFT stability, is absent along the axoneme of NPHP4-KD-IMCD cells. By controlling the entry of ciliary components at the transition zone, NPHP1 and NPHP4 may modulate IFT-A cargos thus participating in the same pathway (i.e. Wnt/PCP), alteration of which would lead to renal lesions observed in nephronophthisis

The ciliary pocket: an endocytic membrane domain at the base of primary and motile cilia

International audienceCilia and flagella are eukaryotic organelles involved in multiple cellular functions. The primary cilium is generally non motile and found in numerous vertebrate cell types where it controls key signalling pathways. Despite a common architecture, ultrastructural data suggest some differences in their organisation. Here, we report the first detailed characterisation of the ciliary pocket, a depression of the plasma membrane in which the primary cilium is rooted. This structure is found at low frequency in kidney epithelial cells (IMCD3) but is associated with virtually all primary cilia in retinal pigment epithelial cells (RPE1). Transmission and scanning electron microscopy, immunofluorescence analysis and videomicroscopy revealed that the ciliary pocket establishes closed links with the actin-based cytoskeleton and that it is enriched in active and dynamic clathrin-coated pits. The existence of the ciliary pocket was confirmed in mouse tissues bearing primary cilia (cumulus), as well as motile cilia and flagella (ependymal cells and spermatids). The ciliary pocket shares striking morphological and functional similarities with the flagellar pocket of Trypanosomatids, a trafficking-specialised membrane domain at the base of the flagellum. Our data therefore highlight the conserved role of membrane trafficking in the vicinity of cilia