Mise en évidence et caractérisation de nouveaux gènes impliqués dans les ciliopathies rénales

The primary cilium is a sensory antenna present on the surface of most of the cells. It controls key signaling pathways during development and tissue homeostasis. Defects in cilia growth or activity are responsible for complex genetic diseases called ciliopathies. Nephronophthisis (NPH) is a ciliopathy characterized by chronic tubulointerstitial nephritis which usually progresses to end-stage renal disease (ESRD) before adulthood. NPH may be isolated or associated with extra-renal defects such as retinitis pigmentosa and skeleton involvement. The combination of these symptoms defines syndromes such as Saldino-Mainzer (MZSDS). NPH is an autosomal recessive disorder highly genetically heterogeneous and almost all of proteins encoded by the identified genes have been involved in ciliary function. The exome sequencing in patients, targeting up to 1300 ciliary genes (ciliome), highlighted new mutations in 2 NPH candidate genes: CEP83 and TEKT1. My work was to characterize the effects of the mutations and validate their involvement in patient phenotypes. CEP83 was found mutated in several unrelated patients with early-onset of NPH (IRT<5 years). CEP83 is a component of distal appendages on the mother centriole which play a crucial role in the early steps of cilia formation. I have shown that the identified mutations perturbed the distal appendages formation which might explain the defects in ciliogenesis observed in fibroblasts and kidney biopsies from patients. These results have demonstrated the involvement of a new centriolar protein in the pathophysiology of NPH severe forms. TEKT1 presents compound heterozygous mutations in a patient with a complex phenotype combining a MZSDS and primary ciliary dyskinesia (PCD) due to defects in motile cilia. The genetic analysis showed mutations in a second gene, WDR19, already characterized in NPH associated with bone defects. TEKT1 encodes the Tektin-1 protein, an uncharacterized member of the tektin family involved in motile cilia. The nasal multiciliated cells analysis showed that Tektin-1 was localized along the axoneme of control motile cilia and absent from the cilia in patient cells, which also had severe beating impairment. In parallel, defects in ciliogenesis, typical of WDR19 mutations, were observed in the fibroblasts from the patient. These results suggest that this dual ciliary phenotype is rather due to the additional effect of mutations in both TEKT1 and WDR19, responsible for the defects in motile and primary cilia, respectively.Le cil primaire est une antenne sensorielle présente à la surface de la plupart des cellules qui contrôle des voies de signalisation clés au cours du développement et de l’homéostasie tissulaire. Des défauts de formation ou de fonctionnement des cils sont responsables de maladies génétiques complexes appelées ciliopathies. La néphronophtise (NPH) est une ciliopathie caractérisée par une néphropathie tubulo-interstitielle chronique évoluant généralement vers l’insuffisance rénale terminale (IRT) avant l’âge adulte. La NPH peut être isolée ou associée à des signes extra-rénaux tels que la rétinite pigmentaire et des défauts du squelette permettant de définir des syndromes comme celui de Saldino-Mainzer (MZSDS). La NPH est une maladie à transmission autosomique récessive très hétérogène sur le plan génétique et les protéines codées par les gènes identifiés ont quasiment toutes été impliquées dans des fonctions ciliaires. Le séquençage d’exome de patients, ciblant plus de 1300 gènes ciliaires (ciliome), a permis de mettre en évidence des mutations dans deux nouveaux gènes candidats pour la NPH : CEP83 et TEKT1. Mon travail de thèse a consisté à caractériser l’effet des mutations et à valider leur implication dans les phénotypes des patients. CEP83 a été retrouvé muté chez plusieurs patients non-apparentés présentant une NPH avec IRT précoce (< 5 ans). CEP83 est un composant des appendices distaux du centriole père qui joue un rôle clé dans les étapes précoces de la formation du cil. J’ai montré que les mutations identifiées entraînaient une désorganisation des appendices distaux qui pourrait expliquer les défauts de ciliogénèse observés dans les fibroblastes et les biopsies rénales de patients. Ces résultats ont permis de démontrer l’implication d’une nouvelle protéine centriolaire dans la physiopathologie des formes sévères de NPH. TEKT1 présente des mutations hétérozygotes composites chez un patient ayant un tableau clinique complexe associant un MZSDS et une dyskinésie ciliaire primitive (PCD) due à des défauts de cils motiles. Une analyse génétique détaillée a mis en évidence des mutations sévères dans un second gène, WDR19, déjà caractérisé dans les formes de NPH associées à des défauts osseux. TEKT1 code la protéine Tektine-1, un membre encore non caractérisé de la famille des tektines impliquées dans les cils motiles. L’analyse de cellules nasales multiciliées a montré que Tektine-1 était localisée le long de l’axoneme des cils motiles contrôles et absent des cils des cellules du patient qui présentaient aussi des anomalies sévères de battement. En parallèle, des défauts de ciliogénèse, typiques de mutations de WDR19, ont été observés dans les fibroblastes du patient. Ces résultats suggèrent que ce phénotype complexe est dû aux effets complémentaires des mutations des deux gènes TEKT1 et WDR19, responsables des défauts dans les cils motiles et primaires, respectivement

Whole-genome screen identifies diverse pathways that negatively regulate ciliogenesis.

We performed a high-throughput whole-genome RNAi screen to identify novel inhibitors of ciliogenesis in normal and basal breast cancer cells. Our screen uncovered a previously undisclosed, extensive network of genes linking integrin signaling and cellular adhesion to the extracellular matrix (ECM) with inhibition of ciliation in both normal and cancer cells. Surprisingly, a cohort of genes encoding ECM proteins was also identified. We characterized several ciliation inhibitory genes and showed that their silencing was accompanied by altered cytoskeletal organization and induction of ciliation, which restricts cell growth and migration in normal and breast cancer cells. Conversely, supplying an integrin ligand, vitronectin, to the ECM rescued the enhanced ciliation observed on silencing this gene. Aberrant ciliation could also be suppressed through hyperactivation of the YAP/TAZ pathway, indicating a potential mechanistic basis for our findings. Our findings suggest an unanticipated reciprocal relationship between ciliation and cellular adhesion to the ECM and provide a resource that could vastly expand our understanding of controls involving outside-in and inside-out signaling that restrain cilium assembly

Characterization of new genes involved in renal ciliopathies

Le cil primaire est une antenne sensorielle présente à la surface de la plupart des cellules qui contrôle des voies de signalisation clés au cours du développement et de l’homéostasie tissulaire. Des défauts de formation ou de fonctionnement des cils sont responsables de maladies génétiques complexes appelées ciliopathies. La néphronophtise (NPH) est une ciliopathie caractérisée par une néphropathie tubulo-interstitielle chronique évoluant généralement vers l’insuffisance rénale terminale (IRT) avant l’âge adulte. La NPH peut être isolée ou associée à des signes extra-rénaux tels que la rétinite pigmentaire et des défauts du squelette permettant de définir des syndromes comme celui de Saldino-Mainzer (MZSDS). La NPH est une maladie à transmission autosomique récessive très hétérogène sur le plan génétique et les protéines codées par les gènes identifiés ont quasiment toutes été impliquées dans des fonctions ciliaires. Le séquençage d’exome de patients, ciblant plus de 1300 gènes ciliaires (ciliome), a permis de mettre en évidence des mutations dans deux nouveaux gènes candidats pour la NPH : CEP83 et TEKT1. Mon travail de thèse a consisté à caractériser l’effet des mutations et à valider leur implication dans les phénotypes des patients. CEP83 a été retrouvé muté chez plusieurs patients non-apparentés présentant une NPH avec IRT précoce (< 5 ans). CEP83 est un composant des appendices distaux du centriole père qui joue un rôle clé dans les étapes précoces de la formation du cil. J’ai montré que les mutations identifiées entraînaient une désorganisation des appendices distaux qui pourrait expliquer les défauts de ciliogénèse observés dans les fibroblastes et les biopsies rénales de patients. Ces résultats ont permis de démontrer l’implication d’une nouvelle protéine centriolaire dans la physiopathologie des formes sévères de NPH. TEKT1 présente des mutations hétérozygotes composites chez un patient ayant un tableau clinique complexe associant un MZSDS et une dyskinésie ciliaire primitive (PCD) due à des défauts de cils motiles. Une analyse génétique détaillée a mis en évidence des mutations sévères dans un second gène, WDR19, déjà caractérisé dans les formes de NPH associées à des défauts osseux. TEKT1 code la protéine Tektine-1, un membre encore non caractérisé de la famille des tektines impliquées dans les cils motiles. L’analyse de cellules nasales multiciliées a montré que Tektine-1 était localisée le long de l’axoneme des cils motiles contrôles et absent des cils des cellules du patient qui présentaient aussi des anomalies sévères de battement. En parallèle, des défauts de ciliogénèse, typiques de mutations de WDR19, ont été observés dans les fibroblastes du patient. Ces résultats suggèrent que ce phénotype complexe est dû aux effets complémentaires des mutations des deux gènes TEKT1 et WDR19, responsables des défauts dans les cils motiles et primaires, respectivement.The primary cilium is a sensory antenna present on the surface of most of the cells. It controls key signaling pathways during development and tissue homeostasis. Defects in cilia growth or activity are responsible for complex genetic diseases called ciliopathies. Nephronophthisis (NPH) is a ciliopathy characterized by chronic tubulointerstitial nephritis which usually progresses to end-stage renal disease (ESRD) before adulthood. NPH may be isolated or associated with extra-renal defects such as retinitis pigmentosa and skeleton involvement. The combination of these symptoms defines syndromes such as Saldino-Mainzer (MZSDS). NPH is an autosomal recessive disorder highly genetically heterogeneous and almost all of proteins encoded by the identified genes have been involved in ciliary function. The exome sequencing in patients, targeting up to 1300 ciliary genes (ciliome), highlighted new mutations in 2 NPH candidate genes: CEP83 and TEKT1. My work was to characterize the effects of the mutations and validate their involvement in patient phenotypes. CEP83 was found mutated in several unrelated patients with early-onset of NPH (IRT<5 years). CEP83 is a component of distal appendages on the mother centriole which play a crucial role in the early steps of cilia formation. I have shown that the identified mutations perturbed the distal appendages formation which might explain the defects in ciliogenesis observed in fibroblasts and kidney biopsies from patients. These results have demonstrated the involvement of a new centriolar protein in the pathophysiology of NPH severe forms. TEKT1 presents compound heterozygous mutations in a patient with a complex phenotype combining a MZSDS and primary ciliary dyskinesia (PCD) due to defects in motile cilia. The genetic analysis showed mutations in a second gene, WDR19, already characterized in NPH associated with bone defects. TEKT1 encodes the Tektin-1 protein, an uncharacterized member of the tektin family involved in motile cilia. The nasal multiciliated cells analysis showed that Tektin-1 was localized along the axoneme of control motile cilia and absent from the cilia in patient cells, which also had severe beating impairment. In parallel, defects in ciliogenesis, typical of WDR19 mutations, were observed in the fibroblasts from the patient. These results suggest that this dual ciliary phenotype is rather due to the additional effect of mutations in both TEKT1 and WDR19, responsible for the defects in motile and primary cilia, respectively

Analyse synaptoproteomique d'un modèle animal de dépression avec interaction gène-environnement

PARIS-BIUP (751062107) / SudocSudocFranceF

Functional characterization of tektin-1 in motile cilia and evidence for TEKT1 as a new candidate gene for motile ciliopathies

International audienceA 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

Septins 2, 7 and 9 and MAP4 colocalize along the axoneme in the primary cilium and control ciliary length

Septins are a large, evolutionarily conserved family of GTPases that form hetero-oligomers and interact with the actin-based cytoskeleton and microtubules. They are involved in scaffolding functions, and form diffusion barriers in budding yeast, the sperm flagellum and the base of primary cilia of kidney epithelial cells. We investigated the role of septins in the primary cilium of retinal pigmented epithelial (RPE) cells, and found that SEPT2 forms a 1:1:1 complex with SEPT7 and SEPT9 and that the three members of this complex colocalize along the length of the axoneme. Similar to observations in kidney epithelial cells, depletion of cilium-localized septins by siRNA-based approaches inhibited ciliogenesis. MAP4, which is a binding partner of SEPT2 and controls the accessibility of septins to microtubules, was also localized to the axoneme where it appeared to negatively regulate ciliary length. Taken together, our data provide new insights into the functions and regulation of septins and MAP4 in the organization of the primary cilium and microtubule-based activities in cells

Loss of function mutations in KIF14 cause severe microcephaly and kidney development defects in humans and zebrafish

International audienceMutations in KIF14 have previously been associated with either severe, isolated or syndromic microcephaly with renal hypodysplasia (RHD). Syndromic microcephaly-RHD was strongly reminiscent of clinical ciliopathies, relating to defects of the primary cilium, a signalling organelle present on the surface of many quiescent cells. KIF14 encodes a mitotic kinesin, which plays a key role at the midbody during cytokinesis and has not previously been shown to be involved in cilia-related functions. Here, we analysed four families with fetuses presenting with the syndromic form and harbouring biallelic variants in KIF14. Our functional analyses showed that the identified variants severely impact the activity of KIF14 and likely correspond to loss-of-function mutations. Analysis in human fetal tissues further revealed the accumulation of KIF14-positive midbody remnants in the lumen of ureteric bud tips indicating a shared function of KIF14 during brain and kidney development. Subsequently, analysis of a kif14 mutant zebrafish line showed a conserved role for this mitotic kinesin. Interestingly, ciliopathy-associated phenotypes were also present in mutant embryos, supporting a potential direct or indirect role for KIF14 at cilia. However, our in vitro and in vivo analyses did not provide evidence of a direct role for KIF14 in ciliogenesis and suggested that loss of kif14 causes ciliopathy-like phenotypes through an accumulation of mitotic cells in ciliated tissues. Altogether, our results demonstrate that KIF14 mutations result in a severe syndrome associating microcephaly and RHD through its conserved function in cytokinesis during kidney and brain development