Emilie Filhol

Ciliopathies with Skeletal Anomalies and Renal Insufficiency due to Mutations in the IFT-A Gene WDR19

A subset of ciliopathies, including Sensenbrenner, Jeune, and short-rib polydactyly syndromes are characterized by skeletal anomalies accompanied by multiorgan defects such as chronic renal failure and retinitis pigmentosa. Through exome sequencing we identified compound heterozygous mutations in WDR19 in a Norwegian family with Sensenbrenner syndrome. In a Dutch family with the clinically overlapping Jeune syndrome, a homozygous missense mutation in the same gene was found. Both families displayed a nephronophthisis-like nephropathy. Independently, we also identified compound heterozygous WDR19 mutations by exome sequencing in a Moroccan family with isolated nephronophthisis. WDR19 encodes IFT144, a member of the intraflagellar transport (IFT) complex A that drives retrograde ciliary transport. We show that IFT144 is absent from the cilia of fibroblasts from one of the Sensenbrenner patients and that ciliary abundance and morphology is perturbed, demonstrating the ciliary pathogenesis. Our results suggest that isolated nephronophthisis, Jeune, and Sensenbrenner syndromes are clinically overlapping disorders that can result from a similar molecular cause.

Defects in the IFT-B Component IFT172 Cause Jeune and Mainzer-Saldino Syndromes in Humans

Intraflagellar transport (IFT) depends on two evolutionarily conserved modules, subcomplexes A (IFT-A) and B (IFT-B), to drive ciliary assembly and maintenance. All six IFT-A components and their motor protein, DYNC2H1, have been linked to human skeletal ciliopathies, including asphyxiating thoracic dystrophy (ATD; also known as Jeune syndrome), Sensenbrenner syndrome, and Mainzer-Saldino syndrome (MZSDS). Conversely, the 14 subunits in the IFT-B module, with the exception of IFT80, have unknown roles in human disease. To identify additional IFT-B components defective in ciliopathies, we independently performed different mutation analyses: candidate-based sequencing of all IFT-B-encoding genes in 1,467 individuals with a nephronophthisis-related ciliopathy or whole-exome resequencing in 63 individuals with ATD. We thereby detected biallelic mutations in the IFT-B-encoding gene IFT172 in 12 families. All affected individuals displayed abnormalities of the thorax and/or long bones, as well as renal, hepatic, or retinal involvement, consistent with the diagnosis of ATD or MZSDS. Additionally, cerebellar aplasia or hypoplasia characteristic of Joubert syndrome was present in 2 out of 12 families. Fibroblasts from affected individuals showed disturbed ciliary composition, suggesting alteration of ciliary transport and signaling. Knockdown of ift172 in zebrafish recapitulated the human phenotype and demonstrated a genetic interaction between ift172 and ift80. In summary, we have identified defects in IFT172 as a cause of complex ATD and MZSDS. Our findings link the group of skeletal ciliopathies to an additional IFT-B component, IFT172, similar to what has been shown for IFT-A.

ANKS6 is a central component of a nephronophthisis module linking NEK8 to INVS and NPHP3

Nephronophthisis is an autosomal recessive cystic kidney disease that leads to renal failure in childhood or adolescence. Most NPHP gene products form molecular networks. Here we identify ANKS6 as a new NPHP family member that connects NEK8 (NPHP9) to INVS (NPHP2) and NPHP3. We show that ANKS6 localizes to the proximal cilium and confirm its role in renal development through knockdown experiments in zebrafish and Xenopus laevis. We also identify six families with ANKS6 mutations affected by nephronophthisis, including severe cardiovascular abnormalities, liver fibrosis and situs inversus. The oxygen sensor HIF1AN hydroxylates ANKS6 and INVS and alters the composition of the ANKS6-INVS-NPHP3 module. Knockdown of Hif1an in Xenopus results in a phenotype that resembles loss of other NPHP proteins. Network analyses uncovered additional putative NPHP proteins and placed ANKS6 at the center of this NPHP module, explaining the overlapping disease manifestation caused by mutation in ANKS6, NEK8, INVS or NPHP3.

A homozygous PDE6D mutation in Joubert syndrome impairs targeting of farnesylated INPP5E protein to the primary cilium

Joubert syndrome (JS) is characterized by a distinctive cerebellar structural defect, namely the « molar tooth sign ». JS is genetically heterogeneous, involving 20 genes identified to date, which are all required for cilia biogenesis and/or function. In a consanguineous family with JS associated with optic nerve coloboma, kidney hypoplasia, and polydactyly, combined exome sequencing and mapping identified a homozygous splice‐site mutation in PDE6D, encoding a prenyl‐binding protein. We found that pde6d depletion in zebrafish leads to renal and retinal developmental anomalies and wild‐type but not mutant PDE6D is able to rescue this phenotype. Proteomic analysis identified INPP5E, whose mutations also lead to JS or mental retardation, obesity, congenital retinal dystrophy, and micropenis syndromes, as novel prenyl‐dependent cargo of PDE6D. Mutant PDE6D shows reduced binding to INPP5E, which fails to localize to primary cilia in patient fibroblasts and tissues. Furthermore, mutant PDE6D is unable to bind to GTP‐bound ARL3, which acts as a cargo‐release factor for PDE6D‐bound INPP5E. Altogether, these results indicate that PDE6D is required for INPP5E ciliary targeting and suggest a broader role for PDE6D in targeting other prenylated proteins to the cilia. This study identifies PDE6D as a novel JS disease gene and provides the first evidence of prenyl‐binding‐dependent trafficking in ciliopathies.

A Homozygous Missense Mutation in the Ciliary Gene TTC21B Causes Familial FSGS

Several genes, mainly involved in podocyte cytoskeleton regulation, have been implicated in familial forms of primary FSGS. We identified a homozygous missense mutation (p.P209L) in the TTC21B gene in seven families with FSGS. Mutations in this ciliary gene were previously reported to cause nephronophthisis, a chronic tubulointerstitial nephropathy. Notably, tubular basement membrane thickening reminiscent of that observed in nephronophthisis was present in patients with FSGS and the p.P209L mutation. We demonstrated that the TTC21B gene product IFT139, an intraflagellar transport-A component, mainly localizes at the base of the primary cilium in developing podocytes from human fetal tissue and in undifferentiated cultured podocytes. In contrast, in nonciliated adult podocytes and differentiated cultured cells, IFT139 relocalized along the extended microtubule network. We further showed that knockdown of IFT139 in podocytes leads to primary cilia defects, abnormal cell migration, and cytoskeleton alterations, which can be partially rescued by p.P209L overexpression, indicating its hypomorphic effect. Our results demonstrate the involvement of a ciliary gene in a glomerular disorder and point to a critical function of IFT139 in podocytes. Altogether, these data suggest that this homozygous TTC21B p.P209L mutation leads to a novel hereditary kidney disorder with both glomerular and tubulointerstitial damages.

Mutations in TRAF3IP1/IFT54 reveal a new role for IFT proteins in microtubule stabilization

Ciliopathies are a large group of clinically and genetically heterogeneous disorders caused by defects in primary cilia. Here we identified mutations in TRAF3IP1 (TNF Receptor-Associated Factor Interacting Protein 1) in eight patients from five families with nephronophthisis (NPH) and retinal degeneration, two of the most common manifestations of ciliopathies. TRAF3IP1 encodes IFT54, a subunit of the IFT-B complex required for ciliogenesis. The identified mutations result in mild ciliary defects in patients but also reveal an unexpected role of IFT54 as a negative regulator of microtubule stability via MAP4 (microtubule-associated protein 4). Microtubule defects are associated with altered epithelialization/polarity in renal cells and with pronephric cysts and microphthalmia in zebrafish embryos. Our findings highlight the regulation of cytoplasmic microtubule dynamics as a role of the IFT54 protein beyond the cilium, contributing to the development of NPH-related ciliopathies.

Mutations of CEP83 Cause Infantile Nephronophthisis and Intellectual Disability

Ciliopathies are a group of hereditary disorders associated with defects in cilia structure and function. The distal appendages (DAPs) of centrioles are involved in the docking and anchoring of the mother centriole to the cellular membrane during ciliogenesis. The molecular composition of DAPs was recently elucidated and mutations in two genes encoding DAPs components (CEP164/NPHP15, SCLT1) have been associated with human ciliopathies, namely nephronophthisis and orofaciodigital syndrome. To identify additional DAP components defective in ciliopathies, we independently performed targeted exon sequencing of 1,221 genes associated with cilia and 5 known DAP protein-encoding genes in 1,255 individuals with a nephronophthisis-related ciliopathy. We thereby detected biallelic mutations in a key component of DAP-encoding gene, CEP83, in seven families. All affected individuals had early-onset nephronophthisis and four out of eight displayed learning disability and/or hydrocephalus. Fibroblasts and tubular renal cells from affected individuals showed an altered DAP composition and ciliary defects. In summary, we have identified mutations in CEP83, another DAP-component-encoding gene, as a cause of infantile nephronophthisis associated with central nervous system abnormalities in half of the individuals.

Novel NEK8 Mutations Cause Severe Syndromic Renal Cystic Dysplasia through YAP Dysregulation

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.

DCDC2 Mutations Cause Neonatal Sclerosing Cholangitis.

Neonatal sclerosing cholangitis (NSC) is a rare biliary disease leading to liver transplantation in childhood. Patients with NSC and ichtyosis have already been identified with a CLDN1 mutation, encoding a tight‐junction protein. However, for the majority of patients, the molecular basis of NSC remains unknown. We identified biallelic missense mutations or in‐frame deletion in DCDC2 in four affected children. Mutations involve highly conserved amino acids in the doublecortin domains of the protein. In cholangiocytes, DCDC2 protein is normally located in the cytoplasm and cilia, whereas in patients the mutated protein is accumulated in the cytoplasm, absent from cilia, and associated with ciliogenesis defect. This is the first report of DCDC2 mutations in NSC. This data expands the molecular spectrum of NSC, that can be considered as a ciliopathy and also expands the clinical spectrum of the DCDC2 mutations, previously reported in dyslexia, deafness, and nephronophtisis.

Mutations in MAPKBP1 Cause Juvenile or Late-Onset Cilia-Independent Nephronophthisis

Nephronophthisis (NPH), an autosomal-recessive tubulointerstitial nephritis, is the most common cause of hereditary end-stage renal disease in the first three decades of life. Since most NPH gene products (NPHP) function at the primary cilium, NPH is classified as a ciliopathy. We identified mutations in a candidate gene in eight individuals from five families presenting late-onset NPH with massive renal fibrosis. This gene encodes MAPKBP1, a poorly characterized scaffolding protein for JNK signaling. Immunofluorescence analyses showed that MAPKBP1 is not present at the primary cilium and that fibroblasts from affected individuals did not display ciliogenesis defects, indicating that MAPKBP1 may represent a new family of NPHP not involved in cilia-associated functions. Instead, MAPKBP1 is recruited to mitotic spindle poles (MSPs) during the early phases of mitosis where it colocalizes with its paralog WDR62, which plays a key role at MSP. Detected mutations compromise recruitment of MAPKBP1 to the MSP and/or its interaction with JNK2 or WDR62. Additionally, we show increased DNA damage response signaling in fibroblasts from affected individuals and upon knockdown of Mapkbp1 in murine cell lines, a phenotype previously associated with NPH. In conclusion, we identified mutations in MAPKBP1 as a genetic cause of juvenile or late-onset and cilia-independent NPH.