Abstract
A child presenting with Mainzer-Saldino syndrome (MZSDS), characterized by renal, retinal and skeletal involvements, was also diagnosed with lung infections and airway ciliary dyskinesia. ...These manifestations suggested dysfunction of both primary and motile cilia, respectively. Targeted exome sequencing identified biallelic mutations in WDR19, encoding an IFT-A subunit previously associated with MZSDS-related chondrodysplasia, Jeune asphyxiating thoracic dysplasia and cranioectodermal dysplasia, linked to primary cilia dysfunction, and in TEKT1 which encodes tektin-1 an uncharacterized member of the tektin family, mutations of which may cause ciliary dyskinesia. Tektin-1 localizes at the centrosome in cycling cells, at basal bodies of both primary and motile cilia and to the axoneme of motile cilia in airway cells. The identified mutations impaired these localizations. In addition, airway cells from the affected individual showed severe motility defects without major ultrastructural changes. Knockdown of tekt1 in zebrafish resulted in phenotypes consistent with a function for tektin-1 in ciliary motility, which was confirmed by live imaging. Finally, experiments in the zebrafish also revealed a synergistic effect of tekt1 and wdr19. Altogether, our data show genetic interactions between WDR19 and TEKT1 likely contributing to the overall clinical phenotype observed in the affected individual and provide strong evidence for TEKT1 as a new candidate gene for primary ciliary dyskinesia.
Ciliopathies are a group of genetic multi-systemic disorders related to dysfunction of the primary cilium, a sensory organelle present at the cell surface that regulates key signaling pathways during ...development and tissue homeostasis. In order to identify novel genes whose mutations would cause severe developmental ciliopathies, >500 patients/fetuses were analyzed by a targeted high throughput sequencing approach allowing exome sequencing of >1200 ciliary genes. NEK8/NPHP9 mutations were identified in five cases with severe overlapping phenotypes including renal cystic dysplasia/hypodysplasia, situs inversus, cardiopathy with hypertrophic septum and bile duct paucity. These cases highlight a genotype-phenotype correlation, with missense and nonsense mutations associated with hypodysplasia and enlarged cystic organs, respectively. Functional analyses of NEK8 mutations in patient fibroblasts and mIMCD3 cells showed that these mutations differentially affect ciliogenesis, proliferation/apoptosis/DNA damage response, as well as epithelial morphogenesis. Notably, missense mutations exacerbated some of the defects due to NEK8 loss of function, highlighting their likely gain-of-function effect. We also showed that NEK8 missense and loss-of-function mutations differentially affect the regulation of the main Hippo signaling effector, YAP, as well as the expression of its target genes in patient fibroblasts and renal cells. YAP imbalance was also observed in enlarged spheroids of Nek8-invalidated renal epithelial cells grown in 3D culture, as well as in cystic kidneys of Jck mice. Moreover, co-injection of nek8 MO with WT or mutated NEK8-GFP RNA in zebrafish embryos led to shortened dorsally curved body axis, similar to embryos injected with human YAP RNA. Finally, treatment with Verteporfin, an inhibitor of YAP transcriptional activity, partially rescued the 3D spheroid defects of Nek8-invalidated cells and the abnormalities of NEK8-overexpressing zebrafish embryos. Altogether, our study demonstrates that NEK8 human mutations cause major organ developmental defects due to altered ciliogenesis and cell differentiation/proliferation through deregulation of the Hippo pathway.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Biallelic variants in RNU4ATAC, a non-coding gene transcribed into the minor spliceosome component U4atac snRNA, are responsible for three rare recessive developmental diseases, namely ...Taybi-Linder/MOPD1, Roifman and Lowry-Wood syndromes. Next-generation sequencing of clinically heterogeneous cohorts (children with either a suspected genetic disorder or a congenital microcephaly) recently identified mutations in this gene, illustrating how profoundly these technologies are modifying genetic testing and assessment. As RNU4ATAC has a single non-coding exon, the bioinformatic prediction algorithms assessing the effect of sequence variants on splicing or protein function are irrelevant, which makes variant interpretation challenging to molecular diagnostic laboratories. In order to facilitate and improve clinical diagnostic assessment and genetic counseling, we present i) an update of the previously reported RNU4ATAC mutations and an analysis of the genetic variations affecting this gene using the Genome Aggregation Database (gnomAD) resource; ii) the pathogenicity prediction performances of scores computed based on an RNA structure prediction tool and of those produced by the Combined Annotation Dependent Depletion tool for the 285 RNU4ATAC variants identified in patients or in large-scale sequencing projects; iii) a method, based on a cellular assay, that allows to measure the effect of RNU4ATAC variants on splicing efficiency of a minor (U12-type) reporter intron. Lastly, the concordance of bioinformatic predictions and cellular assay results was investigated.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Mutations in genes encoding components of the intraflagellar transport (IFT) complexes have previously been associated with a spectrum of diseases collectively termed ciliopathies. Ciliopathies ...relate to defects in the formation or function of the cilium, a sensory or motile organelle present on the surface of most cell types. IFT52 is a key component of the IFT-B complex and ensures the interaction of the two subcomplexes, IFT-B1 and IFT-B2. Here, we report novel IFT52 biallelic mutations in cases with a short-rib thoracic dysplasia (SRTD) or a congenital anomaly of kidney and urinary tract (CAKUT). Combining in vitro and in vivo studies in zebrafish, we showed that SRTD-associated missense mutation impairs IFT-B complex assembly and IFT-B2 ciliary localization, resulting in decreased cilia length. In comparison, CAKUT-associated missense mutation has a mild pathogenicity, thus explaining the lack of skeletal defects in CAKUT case. In parallel, we demonstrated that the previously reported homozygous nonsense IFT52 mutation associated with Sensenbrenner syndrome Girisha et al. (2016) A homozygous nonsense variant in IFT52 is associated with a human skeletal ciliopathy. Clin. Genet., 90, 536-539 leads to exon skipping and results in a partially functional protein. Finally, our work uncovered a novel role for IFT52 in microtubule network regulation. We showed that IFT52 interacts and partially co-localized with centrin at the distal end of centrioles where it is involved in its recruitment and/or maintenance. Alteration of this function likely contributes to centriole splitting observed in Ift52-/- cells. Altogether, our findings allow a better comprehensive genotype-phenotype correlation among IFT52-related cases and revealed a novel, extra-ciliary role for IFT52, i.e. disruption may contribute to pathophysiological mechanisms.
The pancreaticobiliary ductal system connects the liver and pancreas to the intestine. It is composed of the hepatopancreatic ductal (HPD) system as well as the intrahepatic biliary ducts and the ...intrapancreatic ducts. Despite its physiological importance, the development of the pancreaticobiliary ductal system remains poorly understood. The SRY-related transcription factor SOX9 is expressed in the mammalian pancreaticobiliary ductal system, but the perinatal lethality of Sox9 heterozygous mice makes loss-of-function analyses challenging. We turned to the zebrafish to assess the role of SOX9 in pancreaticobiliary ductal system development. We first show that zebrafish sox9b recapitulates the expression pattern of mouse Sox9 in the pancreaticobiliary ductal system and use a nonsense allele of sox9b, sox9b(fh313), to dissect its function in the morphogenesis of this structure. Strikingly, sox9b(fh313) homozygous mutants survive to adulthood and exhibit cholestasis associated with hepatic and pancreatic duct proliferation, cyst formation, and fibrosis. Analysis of sox9b(fh313) mutant embryos and larvae reveals that the HPD cells appear to mis-differentiate towards hepatic and/or pancreatic fates, resulting in a dysmorphic structure. The intrahepatic biliary cells are specified but fail to assemble into a functional network. Similarly, intrapancreatic duct formation is severely impaired in sox9b(fh313) mutants, while the embryonic endocrine and acinar compartments appear unaffected. The defects in the intrahepatic and intrapancreatic ducts of sox9b(fh313) mutants worsen during larval and juvenile stages, prompting the adult phenotype. We further show that Sox9b interacts with Notch signaling to regulate intrahepatic biliary network formation: sox9b expression is positively regulated by Notch signaling, while Sox9b function is required to maintain Notch signaling in the intrahepatic biliary cells. Together, these data reveal key roles for SOX9 in the morphogenesis of the pancreaticobiliary ductal system, and they cast human Sox9 as a candidate gene for pancreaticobiliary duct malformation-related pathologies.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Renal epithelial cells are subjected to fluid shear stress of urine flow. Several cellular structures act as mechanosensors–the primary cilium, microvilli and cell adhesion complexes–that directly ...relay signals to the cytoskeleton to regulate various processes including cell differentiation and renal cell functions. Nephronophthisis (NPH) is an autosomal recessive tubulointerstitial nephropathy leading to end-stage kidney failure before adulthood.
NPHP1
and
NPHP4
are the major genes which code for proteins that form a complex at the transition zone of the primary cilium, a crucial region required for the maintenance of the ciliary composition integrity. These two proteins also interact with signaling components and proteins associated with the actin cytoskeleton at cell junctions. Due to their specific subcellular localization, we wondered whether NPHP1 and NPHP4 could ensure mechanosensory functions. Using a microfluidic set up, we showed that murine inner medullary collecting ductal cells invalidated for
Nphp1
or
Nphp4
are more responsive to immediate shear exposure with a fast calcium influx, and upon a prolonged shear condition, an inability to properly regulate cilium length and actin cytoskeleton remodeling. Following a transcriptomic study highlighting shear stress-induced gene expression changes, we showed that prolonged shear triggers both cholesterol biosynthesis pathway and uptake, processes that do not seem to involve neither NPHP1 nor NPHP4. To conclude, our study allowed us to determine a moderate role of NPHP1 and NPHP4 in flow sensation, and to highlight a new signaling pathway induced by shear stress, the cholesterol biosynthesis and uptake pathways, which would allow cells to cope with mechanical stress by strengthening their plasma membrane through the supply of cholesterol.
Nephrocystin and nephrocystin-4 are newly identified proteins involved in familial juvenile nephronophthisis, an autosomal recessive nephropathy characterized by cyst formation and renal fibrosis. ...Nephrocystin is an adaptor protein that is able to associate with signaling molecules involved in cell adhesion and actin cytoskeleton organization, such as p130Cas, Pyk2, tensin and filamins. Nephrocystin was recently shown to interact and to co-localize with the microtubule component β-tubulin to the primary cilia in renal epithelial cells, an organelle known to play a key role in the pathogenesis of cystic kidney diseases. In this study, we demonstrated that nephrocystin-4 also localizes to the primary cilia in polarized epithelial tubular cells, particularly at the basal bodies, and associates with microtubule component α-tubulin, suggesting a common role for the nephrocystin proteins in ciliary function. However, the co-localization of nephrocystin-4 with the microtubules is not restricted to the primary cilia, as nephrocystin-4 was also detected at the centrosomes of dividing cells and close to the cortical actin cytoskeleton in polarized cells. We also detected p130Cas and Pyk2 in the nephrocystin-4-containing complex, confirming the role of the nephrocystin proteins in cell–cell and cell–matrix adhesion signaling events. Finally, we refined the structural and functional regions involved in the interaction between nephrocystin and nephrocystin-4. These data suggest that nephrocystin and nephrocystin-4 belong to a multifunctional complex localized in actin- and microtubule-based structures involved in cell–cell and cell–matrix adhesion signaling as well as in cell division.
In a forward genetic screen for regulators of pancreas development in zebrafish, we identified donut(s908) , a mutant which exhibits failed outgrowth of the exocrine pancreas. The s908 mutation leads ...to a leucine to arginine substitution in the ectodomain of the hepatocyte growth factor (HGF) tyrosine kinase receptor, Met. This missense mutation impedes the proteolytic maturation of the receptor, its trafficking to the plasma membrane, and diminishes the phospho-activation of its kinase domain. Interestingly, during pancreatogenesis, met and its hgf ligands are expressed in pancreatic epithelia and mesenchyme, respectively. Although Met signaling elicits mitogenic and migratory responses in varied contexts, normal proliferation rates in donut mutant pancreata together with dysmorphic, mislocalized ductal cells suggest that met primarily functions motogenically in pancreatic tail formation. Treatment with PI3K and STAT3 inhibitors, but not with MAPK inhibitors, phenocopies the donut pancreatic defect, further indicating that Met signals through migratory pathways during pancreas development. Chimera analyses showed that Met-deficient cells were excluded from the duct, but not acinar, compartment in the pancreatic tail. Conversely, wild-type intrapancreatic duct and "tip cells" at the leading edge of the growing pancreas rescued the donut phenotype. Altogether, these results reveal a novel and essential role for HGF signaling in the intrapancreatic ducts during exocrine morphogenesis.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Hepatic cysts are fluid‐filled lesions in the liver that are estimated to occur in 5% of the population. They may cause hepatomegaly and abdominal pain. Progression to secondary fibrosis, cirrhosis, ...or cholangiocarcinoma can lead to morbidity and mortality. Previous studies of patients and rodent models have associated hepatic cyst formation with increased proliferation and fluid secretion in cholangiocytes, which are partially due to impaired primary cilia. Congenital hepatic cysts are thought to originate from faulty bile duct development, but the underlying mechanisms are not fully understood. In a forward genetic screen, we identified a zebrafish mutant that developed hepatic cysts during larval stages. The cyst formation was not due to changes in biliary cell proliferation, bile secretion, or impairment of primary cilia. Instead, time‐lapse live imaging data showed that the mutant biliary cells failed to form interconnecting bile ducts because of defects in motility and protrusive activity. Accordingly, immunostaining revealed a disorganized actin and microtubule cytoskeleton in the mutant biliary cells. By whole‐genome sequencing, we determined that the cystic phenotype in the mutant was caused by a missense mutation in the furinb gene, which encodes a proprotein convertase. The mutation altered Furinb localization and caused endoplasmic reticulum (ER) stress. The cystic phenotype could be suppressed by treatment with the ER stress inhibitor 4‐phenylbutyric acid and exacerbated by treatment with the ER stress inducer tunicamycin. The mutant liver also exhibited increased mammalian target of rapamycin (mTOR) signaling. Treatment with mTOR inhibitors halted cyst formation at least partially through reducing ER stress. Conclusion: Our study has established a vertebrate model for studying hepatic cystogenesis and illustrated the contribution of ER stress in the disease pathogenesis.
In a forward genetic screen, we identified the first zebrafish genetic mutant that develops hepatic cysts during liver development. Cystogenesis in these animals is due to a missense mutation in the furinb gene. The mutation alters Furinb protein expression that subsequently induces endoplasmic reticulum stress to impair biliary cell behaviors.
Various genetic diseases associated with microcephaly and developmental defects are due to pathogenic variants in the U4atac small nuclear RNA (snRNA), a component of the minor spliceosome essential ...for the removal of U12-type introns from eukaryotic mRNAs. While it has been shown that a few RNU4ATAC mutations result in impaired binding of essential protein components, the molecular defects of the vast majority of variants are still unknown. Here, we used lymphoblastoid cells derived from RNU4ATAC compound heterozygous (g.108_126del;g.111G>A) twin patients with MOPD1 phenotypes to analyze the molecular consequences of the mutations on small nuclear ribonucleoproteins (snRNPs) formation and on splicing. We found that the U4atac108_126del mutant is unstable and that the U4atac111G>A mutant as well as the minor di- and tri-snRNPs are present at reduced levels. Our results also reveal the existence of 3'-extended snRNA transcripts in patients' cells. Moreover, we show that the mutant cells have alterations in splicing of INTS7 and INTS10 minor introns, contain lower levels of the INTS7 and INTS10 proteins and display changes in the assembly of Integrator subunits. Altogether, our results show that compound heterozygous g.108_126del;g.111G>A mutations induce splicing defects and affect the homeostasis and function of the Integrator complex.
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