Sophie Saunier et Corinne Antignac

Laboratory of Hereditary Kidney Diseases

Publish at 18.11.2019


Sophie Saunier


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Molecular bases of hereditary kidney diseases: nephronopthisis and hypodysplasia

Our research aims at unraveling the pathogenesis of nephronophthisis (NPH) and renal hypodysplasia (RHD), two major genetic causes of renal insufficiency in children, using high throughput sequencing approaches and functional studies.

Nephronophthisis (NPH) is an autosomal recessive nephropathy, characterized by interstitial fibrosis and formation of tubular cysts, which represents the most common genetic cause of end-stage renal disease in children. NPH can be isolated or associated with extra- renal anomalies including retinal dystrophy, cerebellar vermis hypoplasia, skeletal dysmorphisms and/or situs inversus. The specific association of these anomalies defines complex syndromes called "ciliopathies". Based on large patient cohorts (>1000 NPH families) collected through a multicentric clinical network and thanks to the development of innovative NGS-based approaches, our group identified 13 of the 20 NPH causative genes known to date (NPHP1-20) as underlying NPH and associated syndromes. Most of the NPHP proteins localize at the primary cilium, an organelle which controls key signalling pathways during development and tissue homeostasis. Notably, over the past two years, we identified three new NPHP genes including two genes encoding IFTB components (IFT172 (Hallbritter et al, Am. J. Hum genet, 2014); IFT54 (Bizet et al, Nat. Comm.)) and CEP83 (Failler et al, Am J Hum Genet, 2014) encoding a component of the centrosome required for ciliogenesis. Based on the use of in vitro kidney epithelial cell models, patient fibroblasts and in vivo models including mouse and zebrafish, we demonstrated that the NPHP and IFT proteins are indeed critical for ciliary function and also for cell polarity (NPHP1, NPHP4) and epithelial morphogenesis through extraciliary functions related to regulation of cytoskeleton dynamics (IFT54; Bizet A., Nat Comm., 2015). Through national and international collaborations with have been implicated in the identification of mutations in several other ciliopathies genes, DCDC2 in neonatal sclerosing cholangitis (Girard et al., Hum Mutation, 2016), KIAA0586 in Joubert syndrome (Albi et al., Am J. Hum. Genet, 2015), C2CD3 and TMEM107 involved in OFD syndromes (Lambacher et al., 2015). These approaches have improved the molecular diagnosis of renal ciliopathies and broaden the spectrum phenotype associated with mutations of ciliary genes.

Renal hypodysplasia (RHD) is a phenotypically heterogeneous disorder that encompasses a spectrum of kidney development defects including renal agenesis, hypoplasia and dysplasia with or without cysts and belongs to the spectrum of CAKUT (Congenital Anomalies of the Kidney and Urinary Tract). It is also one of the most frequent causes of end-stage renal disease in children and the most severe forms (bilateral renal agenesis and multicystic dysplasia) are diagnosed in utero and justify medical termination of pregnancy. Although most RHD cases are isolated forms, familial and syndromic cases also exist and cystic kidney dysplasia can be associated with ciliopathy-like anomalies (situs inversus, skeletal and retinal defects, liver defects). To date, mutations in ~40 genes that play a role during kidney development have been reported, including two causative genes for isolated bilateral renal agenesis identified by whole exome sequencing by our group: FGF20 encoding fibroblast growth factor 20 (Barak et al, Dev Cell, 2012) and ITGA8 encoding the integrin alpha8 chain (Humbert et al, Am J. Hum. Genet., 2014), an adhesion molecule that plays a key role during nephron development. We have also recently characterized mutations in NPHP9/NEK8, a gene previously associated with NPH, in fetuses presenting a syndromic ciliopathy-like form of RHD (Grampa et al, Plos Genet, 2016), and demonstrated that they are associated with impaired Hippo signaling pathway. Finally, we have implemented a targeted exome sequencing strategy (“Cakutome”, Agilent SureSelect technology) focused on 388 selected genes, including known CAKUT genes, genes leading to kidney defects when knocked-out in the mouse, genes with a role in cellular processes and pathways relevant for kidney development, potential targets of relevant transcription factors as well as several candidates previously identified by whole exome sequencing, in order to identify new RHD causative genes and to improve genetic diagnosis. This approach notably led to the identification of a new CAKUT gene. We also identified variants of interest in several candidates that are currently being studied.

Our main project is to pursue the identification of NPHP/ RHD genes. The use of complementary exome sequencing approaches, including targeted exome sequencing [“Ciliome” (1300 genes) for NPH patients and ciliopathy-like RHD cases, “Cakutome” (388 genes) for RHD patients], as well as whole exome or genome sequencing for patients with no mutation identified by targeted exome sequencing, is generating a large amount of candidate genes that need to be validated. For validation of the mutated genes, we use a panel of functional studies of processes relevant for NPH and RHD, including ciliary functions, cell adhesion/migration, cell polarity and epithelium morphogenesis (3D culture), using cellular and animal models (mouse and zebrafish). We are using the powerful CRISPR/Cas9 technologies to invalidate or introduce specific patient mutations in cells, and zebrafish or mouse. Morpholinos in organotypic cultures of metanephros are used to analyse the effect of RHD candidate knockdown on kidney development (analysis by whole-mount immunofluorescence and multiphoton microscopy). We also characterize the effect of mutations on formation of protein complexes and RNA regulation using proteomic analysis and RNAseq. These combined approaches will help us to characterize the molecular mechanisms involved in renal development defects, interstitial fibrosis, cyst formation and renal failure observed in patients. In parallel, in collaboration with Alexion R&D Pharma France, we have developed new therapeutic approaches for patients with NPH, the only available treatments being dialysis and renal transplantation. Based on a drug screening, we identified several molecules that rescue cellular defects linked to NPHP dysfunctions. Effective compounds will be tested in vivo in the generated zebrafish or mouse mutants.

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