KEY POINTS:Exome sequencing solved 26% of nephronophthisis cases, identifying nephropathy and extrarenal disease genes beyond classic ciliopathy panels. Exome sequencing uncovered GN and tubular nephropathy genes misdiagnosed as ciliopathy-associated nephropathy, underscoring diagnostic overlap in kidney diseases. Patients with nonciliary genetic variants may present with ciliopathy-like extrarenal symptoms, showing phenocopies in kidney ciliopathy diagnostics. BACKGROUND:Nephronophthisis (NPH) is an autosomal recessive tubulointerstitial kidney disease and a leading genetic cause of chronic kidney failure in children and young adults. As a ciliopathy, NPH is caused by biallelic variants in genes encoding proteins involved in the structure and function of primary cilia. The broad clinical spectrum of NPH results in a clinically and genetically heterogeneous disease, posing diagnostic challenges and leaving approximately 30% of cases unresolved with current gene panels. METHODS:After targeted gene panel for ciliopathy-associated genes failed to identify diagnostic variants, exome sequencing (ES) was conducted on 42 unrelated index patients with a clinical diagnosis of NPH, defined as cystic nephropathy progressing to kidney failure within the first two decades of life, or by unspecific CKD accompanied by extrarenal features indicative of a ciliopathy. RESULTS:Pathogenic or likely pathogenic variants were identified in 11 of the 42 patients (26%). Variants were detected in known nephropathy genes ( LAMB2 , COQ8B , COL4A3 , MUC1 ) and a multisystem disease gene with secondary kidney involvement ( AGXT ). In addition, ES elucidated deleterious variants explaining extrarenal phenotypes without corresponding kidney disease in six patients ( APTX , TUBB3 , DHX38 , IQCE, CRX , RPGR ). Variants of unknown significance were identified in three patients, while heterozygous variants in genes associated with recessive disease were observed in three others. A potential candidate gene for syndromic tubulointerstitial nephropathy, SSBP1 , was also identified, suggesting a novel pathway involving mitochondrial dysfunction. CONCLUSIONS:ES enabled the identification of pathogenic variants in known genes associated with kidney diseases, nonkidney conditions, and multisystem disorders with secondary kidney involvement, thereby improving diagnosis accuracy, even in incomplete or atypical cases, and guiding specific diagnostic and therapeutic approaches. The identification of SSBP1 in association with tubulointerstitial nephropathy may provide new insights into the pathogenesis of ciliopathies.

Ciliopathies are a class of multi-systemic genetic diseases characterized by ciliary dysfunction. Here, we report a novel ANKS3 variant in patients with a renal ciliopathy known as nephronophthisis (NPH) associated with hepatic defects. ANKS3 is an ankyrin and sterile alpha motif domain-containing protein that interacts with many NPH proteins as well as with BICC1, an RNA-binding protein involved in renal cystic diseases. The pathogenic effect of the ANKS3 mutation was validated in the zebrafish mutant and knock-in rat model, the latter showing urine concentration defect and tubular dilatations similar to NPH patients. In addition, cilia morphology and function as well as epithelialization of kidney tubular cells was affected by loss or mutation of ANKS3 . Finally, our results evidenced that these classically renal ciliopathy-associated phenotypes were linked to the negative regulation of BICC1 by ANKS3 which binds to transcripts of the major NPH gene NPHP1 and mediates their decay through the AGO2-RISC complex and recruitment into P-bodies. Altogether, our findings suggest that the ANKS3/BICC1 complex is a key post-transcriptional regulator of NPHP1 transcript stability, providing another level of regulation of cilium biogenesis and kidney homeostasis, as well as an unusual mechanism leading to NPH-related ciliopathies. ### Competing Interest Statement The authors have declared no competing interest.

Congenital anomalies of the kidney and urinary tract (CAKUT) are the predominant cause for chronic kidney disease below age 30 years. Many monogenic forms have been discovered due to comprehensive genetic testing like exome sequencing. However, disease-causing variants in known disease-associated genes only explain a proportion of cases. Here, we aim to unravel underlying molecular mechanisms of syndromic CAKUT in three unrelated multiplex families with presumed autosomal recessive inheritance. Exome sequencing in the index individuals revealed three different rare homozygous variants in FOXD2, encoding a transcription factor not previously implicated in CAKUT in humans: a frameshift in the Arabic and a missense variant each in the Turkish and the Israeli family with segregation patterns consistent with autosomal recessive inheritance. CRISPR/Cas9-derived Foxd2 knockout mice presented with a bilateral dilated kidney pelvis accompanied by atrophy of the kidney papilla and mandibular, ophthalmologic, and behavioral anomalies, recapitulating the human phenotype. In a complementary approach to study pathomechanisms of FOXD2-dysfunction-mediated developmental kidney defects, we generated CRISPR/Cas9-mediated knockout of Foxd2 in ureteric bud-induced mouse metanephric mesenchyme cells. Transcriptomic analyses revealed enrichment of numerous differentially expressed genes important for kidney/urogenital development, including Pax2 and Wnt4 as well as gene expression changes indicating a shift toward a stromal cell identity. Histology of Foxd2 knockout mouse kidneys confirmed increased fibrosis. Further, genome-wide association studies suggest that FOXD2 could play a role for maintenance of podocyte integrity during adulthood. Thus, our studies help in genetic diagnostics of monogenic CAKUT and in understanding of monogenic and multifactorial kidney diseases.

Nephronophthisis (NPH) is an autosomal-recessive ciliopathy representing one of the most frequent causes of kidney failure in childhood characterized by a broad clinical and genetic heterogeneity. Applied to one of the worldwide largest cohorts of patients with NPH, genetic analysis encompassing targeted and whole exome sequencing identified disease-causing variants in 600 patients from 496 families with a detection rate of 71%. Of 788 pathogenic variants, 40 known ciliopathy genes were identified. However, the majority of patients (53%) bore biallelic pathogenic variants in NPHP1. NPH-causing gene alterations affected all ciliary modules defined by structural and/or functional subdomains. Seventy six percent of these patients had progressed to kidney failure, of which 18% had an infantile form (under five years) and harbored variants affecting the Inversin compartment or intraflagellar transport complex A. Forty eight percent of patients showed a juvenile (5-15 years) and 34% a late-onset disease (over 15 years), the latter mostly carrying variants belonging to the Transition Zone module. Furthermore, while more than 85% of patients with an infantile form presented with extra-kidney manifestations, it only concerned half of juvenile and late onset cases. Eye involvement represented a predominant feature, followed by cerebellar hypoplasia and other brain abnormalities, liver and skeletal defects. The phenotypic variability was in a large part associated with mutation types, genes and corresponding ciliary modules with hypomorphic variants in ciliary genes playing a role in early steps of ciliogenesis associated with juvenile-to-late onset NPH forms. Thus, our data confirm a considerable proportion of late-onset NPH suggesting an underdiagnosis in adult chronic kidney disease.

We report the screening of a large panel of genes in a series of 100 fetuses (98 families) affected with severe renal defects. Causative variants were identified in 22% of cases, greatly improving genetic counseling. The percentage of variants explaining the phenotype was different according to the type of phenotype. The highest diagnostic yield was found in cases affected with the ciliopathy-like phenotype (11/15 families and, in addition, a single heterozygous or a homozygous Class 3 variant in PKHD1 in three unrelated cases with autosomal recessive polycystic kidney disease). The lowest diagnostic yield was observed in cases with congenital anomalies of the kidney and urinary tract (9/78 families and, in addition, Class 3 variants in GREB1L in three unrelated cases with bilateral renal agenesis). Inheritance was autosomal recessive in nine genes (PKHD1, NPHP3, CEP290, TMEM67, DNAJB11, FRAS1, ACE, AGT, and AGTR1), and autosomal dominant in six genes (PKD1, PKD2, PAX2, EYA1, BICC1, and MYOCD). Finally, we developed an original approach of next-generation sequencing targeted RNA sequencing using the custom capture panel used for the sequencing of DNA, to validate one MYOCD heterozygous splicing variant identified in two male siblings with megabladder and inherited from their healthy mother.

Background Co-occurrence of polycystic kidney disease and hyperinsulinemic hypoglycemia has been reported in children in a few families associated with a variant in the promotor of the PMM2 gene, at position -167 upstream of the coding sequence. PMM2 encodes phosphomannomutase 2, a key enzyme in N-glycosylation. While biallelic coding PMM2 mutations are involved in congenital disorder of glycosylation CDG1A, that particular variant in the promoter of the gene, either in the homozygous state or associated with a mutation in the coding exons of the gene, is thought to restrict the N-glycosylation defect to the kidney and the pancreas. Methods Targeted exome sequencing of a panel of genes involved in monogenic kidney diseases. Results We identified a PMM2 variant at position -167 associated with a pathogenic PMM2 variant in the coding exons in 3 families, comprising 6 cases affected with a cystic kidney disease. The spectrum of phenotypes was very broad, from extremely enlarged fetal cystic kidneys in the context of a COACH-like syndrome, to isolated cystic kidney disease with small kidneys, slowly progressing toward kidney failure in adulthood. Hypoglycemia was reported only in one case. Conclusion These data show that the PMM2 promotor variation, in trans of a PMM2 coding mutation, is associated with a wide spectrum of kidney phenotypes, and is not always associated with extra-renal symptoms. When present, extra-renal defects may include COACH-like syndrome. These data prompt screening of PMM2 in unresolved cases of fetal hyperechogenic/cystic kidneys as well as in cystic kidney disease in children and adults. Graphical Abstract

Mutations 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.

In the version of this article initially published, affiliation 38 incorrectly read “ICNU-Nephrology and Urology Department, Barcelona, Spain”; “Renal Division, Hospital Clinic, IDIBAPS, University of Barcelona, Barcelona, Spain” is the correct affiliation. The error has been corrected in the HTML and PDF versions of the article.

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.

Congenital anomalies of the kidney and urinary tract (CAKUT) are a major cause of pediatric kidney failure. We performed a genome-wide analysis of copy number variants (CNVs) in 2,824 cases and 21,498 controls. Affected individuals carried a significant burden of rare exonic (that is, affecting coding regions) CNVs and were enriched for known genomic disorders (GD). Kidney anomaly (KA) cases were most enriched for exonic CNVs, encompassing GD-CNVs and novel deletions; obstructive uropathy (OU) had a lower CNV burden and an intermediate prevalence of GD-CNVs; and vesicoureteral reflux (VUR) had the fewest GD-CNVs but was enriched for novel exonic CNVs, particularly duplications. Six loci (1q21, 4p16.1-p16.3, 16p11.2, 16p13.11, 17q12 and 22q11.2) accounted for 65% of patients with GD-CNVs. Deletions at 17q12, 4p16.1-p16.3 and 22q11.2 were specific for KA; the 16p11.2 locus showed extensive pleiotropy. Using a multidisciplinary approach, we identified TBX6 as a driver for the CAKUT subphenotypes in the 16p11.2 microdeletion syndrome.

BACKGROUNDThe DiGeorge syndrome, the most common of the microdeletion syndromes, affects multiple organs, including the heart, the nervous system, and the kidney. It is caused by deletions on chromosome 22q11.2; the genetic driver of the kidney defects is unknown.METHODSWe conducted a genomewide search for structural variants in two cohorts: 2080 patients with congenital kidney and urinary tract anomalies and 22,094 controls. We performed exome and targeted resequencing in samples obtained from 586 additional patients with congenital kidney anomalies. We also carried out functional studies using zebrafish and mice.RESULTSWe identified heterozygous deletions of 22q11.2 in 1.1% of the patients with congenital kidney anomalies and in 0.01% of population controls (odds ratio, 81.5; P = 4.5x10-14). We localized the main drivers of renal disease in the DiGeorge syndrome to a 370-kb region containing nine genes. In zebrafish embryos, an induced loss of function in snap29, aifm3, and crkl resulted in renal defects; the loss of crkl alone was sufficient to induce defects. Five of 586 patients with congenital urinary anomalies had newly identified, heterozygous protein-altering variants, including a premature termination codon, in CRKL. The inactivation of Crkl in the mouse model induced developmental defects similar to those observed in patients with congenital urinary anomalies.CONCLUSIONSWe identified a recurrent 370-kb deletion at the 22q11.2 locus as a driver of kidney defects in the DiGeorge syndrome and in sporadic congenital kidney and urinary tract anomalies. Of the nine genes at this locus, SNAP29, AIFM3, and CRKL appear to be critical to the phenotype, with haploinsufficiency of CRKL emerging as the main genetic driver.

Congenital anomalies of the kidney and urinary tract (CAKUT) constitute a major cause of chronic kidney disease in children and 20% of prenatally detected anomalies. CAKUT encompass a spectrum of developmental kidney defects, including renal agenesis, hypoplasia, and cystic and non-cystic dysplasia. More than 50 genes have been reported as mutated in CAKUT-affected case subjects. However, the pathophysiological mechanisms leading to bilateral kidney agenesis (BKA) remain largely elusive. Whole-exome or targeted exome sequencing of 183 unrelated familial and/or severe CAKUT-affected case subjects, including 54 fetuses with BKA, led to the identification of 16 heterozygous variants in GREB1L (growth regulation by estrogen in breast cancer 1-like), a gene reported as a target of retinoic acid signaling. Four loss-of-function and 12 damaging missense variants, 14 being absent from GnomAD, were identified. Twelve of them were present in familial or simplex BKA-affected case subjects. Female BKA-affected fetuses also displayed uterus agenesis. We demonstrated a significant association between GREB1L variants and BKA. By in situ hybridization, we showed expression of Greb1l in the nephrogenic zone in developing mouse kidney. We generated a Greb1l knock-out mouse model by CRISPR-Cas9. Analysis at E13.5 revealed lack of kidneys and genital tract anomalies in male and female Greb1l(-/-) embryos and a slight decrease in ureteric bud branching in Greb1l(+/-) embryos. We showed that Greb1l invalidation in mIMCD3 cells affected tubulomorphogenesis in 3D-collagen culture, a phenotype rescued by expression of the wild-type human protein. This demonstrates that GREB1L plays a major role in early metanephros and genital development in mice and humans.

Congenital anomalies of the kidney and urinary tract (CAKUT) occur in three to six of 1000 live births, represent about 20% of the prenatally detected anomalies, and constitute the main cause of CKD in children. These disorders are phenotypically and genetically heterogeneous. Monogenic causes of CAKUT in humans and mice have been identified. However, despite high-throughput sequencing studies, the cause of the disease remains unknown in most patients, and several studies support more complex inheritance and the role of environmental factors and/or epigenetics in the pathophysiology of CAKUT. Here, we report the targeted exome sequencing of 330 genes, including genes known to be involved in CAKUT and candidate genes, in a cohort of 204 unrelated patients with CAKUT; 45% of the patients were severe fetal cases. We identified pathogenic mutations in 36 of 204 (17.6%) patients. These mutations included five de novo heterozygous loss of function mutations/deletions in the PBX homeobox 1 gene (PBX1), a gene known to have a crucial role in kidney development. In contrast, the frequency of SOX17 and DSTYK variants recently reported as pathogenic in CAKUT did not indicate causality. These findings suggest that PBX1 is involved in monogenic CAKUT in humans and call into question the role of some gene variants recently reported as pathogenic in CAKUT. Targeted exome sequencing also proved to be an efficient and cost-effective strategy to identify pathogenic mutations and deletions in known CAKUT genes.

Congenital anomalies of the kidney and urinary tract (CAKUT) are a major cause of renal failure in children. CAKUT are phenotypically and genetically heterogeneous and more than 50 genes have been reported as mutated in patients. The most frequently mutated genes are those encoding transcription factors HNF1B, PAX2, EYA1 and SIX1. Most of the other genes are only mutated in a few patients and their implication is sometimes elusive. We developped a targeted exome sequencing strategy («CAKUTOME», sureselect Agilent) focusing on 330 genes, including known validated or likely causative CAKUT genes, as well as candidate genes. A total of 215 unrelated patients were analysed, including 50 who had previously been tested for HNF1B, PAX2, EYA1 and/or SIX1 mutations by Sanger sequencing. This approach proved to be an efficient and cost-effective strategy to identify pathogenic mutations and copy number variations in known CAKUT genes. The 25/165 rate of mutation we identified in HNF1B, PAX2, EYA1 or SIX1 is similar to the one obtained by Sanger sequencing. In addition, we identified heterozygous mutations in ANOS1 (Kallmann syndrome), GATA3 (hypoparathyroidism, deafness and kidney disease), or CHD7 (Charge syndrome), and biallelic mutation in KIF14 in 2, 3, 1 and 2 cases, respectively. Our data also led to the identification of a novel CAKUT gene, the mutation/deletion of which affected 5 unrelated cases in the cohort. Moreover, we identified variants in several other genes never reported as mutated in CAKUT patients, whose pathogenicity is being tested. Finally, no relevant variant was identified in 40 % of our series. Although mutations in gene(s), non-coding regions or microRNAs not targeted in our CAKUTOME could be involved in some of these cases, complex inheritance, somatic events, and/or environmental factors or epigenetic mechanisms likely explain this large fraction of cases.

Le séquençage d’exome permet une analyse génétique précise des pathologies rares du développement humain. Toutefois, cette technique aboutit à l’identification d’un grand nombre de variants, dont la pathogénie doit ensuite être démontrée. Nous proposons ici une nouvelle stratégie, rapide et peu onéreuse, pour déterminer le rôle fonctionnel et biologique de gènes candidats, obtenus par séquençage d’exome, dans l’hypodysplasie rénale. Cette approche consiste à utiliser un vivo-morpholino en culture de reins murins ex vivo, afin d’inhiber l’expression d’un gène candidat au cours du développement rénal et ainsi, récapituler le phénotype observé chez le patient. La présence d’un phénotype rénal est analysée après culture rénale par acquisition multiphotonique après immunofluorescence en whole-mount. Six gènes candidats ont ainsi été testés, potentiellement responsables d’hypodysplasie rénale dans 7 familles différentes. Un gène candidat a été identifié par la présence d’un variant hétérozygote avec décalage du cadre de lecture, ségrégeant avec un phénotype de dysplasie kystique dans une famille. Son expression était diminuée de 65 % en culture (p < 0,001). Le phénotype observé récapitulait le phénotype rénal du patient en terme de kystes et de diminution de branchements du bourgeon urétéral. Un autre gène étudié était un gène candidat dans une famille avec hypodysplasie multikystique, en raison d’un variant homozygote faux-sens supposé délétère repéré dans 3 fœtus. Son expression en culture de reins était diminuée de 60 % (p < 0,01). L’explant rénal présentait des kystes ; le branchement du bourgeon urétéral était diminué de 45 %. Le volume rénal était également diminué. L’épaisseur de la paroi tubulaire était augmentée de 28 %. Le phénotype obtenu après culture récapitulait précisément celui des patients. Parmi les gènes candidats testés, 3 n’ont pas récapitulé le phénotype observé chez les patients. Cette nouvelle approche a permis de récapituler ex vivo le phénotype observé dans 2 familles différentes, confirmant le rôle fonctionnel et biologique de 2 gènes candidats obtenus par séquençage d’exome. Cette nouvelle stratégie est rapide, fiable, et peu onéreuse, particulièrement comparée à la génération de lignées de souris transgéniques. Elle est notamment pertinente pour l’analyse de mutations avec décalage du cadre de lecture ou de mutations faux-sens, résultant en une perte de fonction de la protéine cible.

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.

Les anomalies congénitales des reins et des voies urinaires (CAKUT) sont une cause majeure d’insuffisance rénale de l’enfant. Les CAKUT sont des pathologies génétiquement et phénotypiquement hétérogènes. Des mutations de plus de 50 gènes ont été rapportées dans des formes isolées ou syndromiques. Il s’agit le plus souvent de mutations hétérozygotes et les gènes les plus fréquemment impliqués codent les facteurs de transcription HNF1B, PAX2 et EYA1/SIX1. Ces gènes, testés en routine dans le cadre du diagnostic, permettent d’expliquer 15–20 % des cas. La plupart des autres gènes ne concernent qu’une minorité de patients et leur implication n’est pas toujours réellement établie. Afin d’améliorer l’efficacité du diagnostic génétique en testant l’ensemble des gènes connus, et d’identifier de nouveaux gènes responsables, nous avons développé une stratégie de séquençage haut débit (« Cakutome », technologie Agilent SureSelect) ciblant 388 gènes : gènes de CAKUT établis ou probables, gènes invalidés dans des modèles murins d’anomalies de développement rénal, gènes impliqués dans des processus cellulaires/signalisations importants pour le développement rénal, et gènes candidats identifiés par séquençage d’éxome de cas familiaux de CAKUT. Cent soixante-neuf échantillons ont été analysés, 63 préalablement testés pour HNF1B, PAX2, EYA1 et/ou SIX1 et sans mutation identifiée, et 106 nouveaux cas. Nous avons identifié des mutations/délétions dans HNF1B, PAX2, EYA1 ou SIX1 chez 21 des 106 patients. Ce taux est globalement similaire à celui obtenu par séquençage Sanger. Cependant, l’approche Cakutome a permis d’identifier deux délétions dans PAX2 qui n’auraient pas été trouvées par analyse Sanger. L’identification de mutations dans les gènes KAL1 (syndrome de Kallmann), GATA3 (syndrome HDR : hypoparathyroidie, surdité, rein) ou SALL1 (syndrome de Townes-Brocks) chez des 4 fœtus/patient avec CAKUT isolé, ainsi que d’une mutation d’EYA1 chez un patient chez lequel seuls HNF1B et PAX2 avaient été testés, nous ont permis de faire le diagnostic chez ces patients. Cette étude a également mis en évidence des variations potentiellement pathogènes (non-sens, délétions intragéniques, faux-sens prédits délétères) dans plusieurs gènes, dont certains non encore rapportés chez les patients CAKUT. Dans 4 familles, l’association de variations hétérozygotes dans deux gènes suggère un possible mécanisme épistatique. Enfin, l’étude de 38 fœtus avec agénésie rénale bilatérale (ARB) n’a pas permis d’identifier de nouvelles mutations d’ITGA8 et FGF20, deux gènes identifiés par notre équipe dans des ARB récessives. Cette étude souligne la complexité de la génétique des CAKUT. Une difficulté est liée à l’interprétation des variants rares dans le contexte de mutations principalement dominantes à expressivité variable. Ces résultats suggèrent l’existence, au moins dans certains cas, d’une hérédité complexe et/ou le rôle de facteurs environnementaux et épigénétiques, contribuant à la survenue et à l’hétérogénéité des CAKUT.

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.

NEK8/NPHP9 encodes a NIMA (Never-In-Mitosis A) protein essential for cell cycle control. NEK8 is composed of kinase and RCC1 domains, the latter involved in centrosomal localization. It localizes into the nucleus and at the inversin compartment in the primary cilium. Using ciliary gene-enriched exome sequencing, we identified recessive NEK8 mutations in 3 cases with severe overlapping phenotypes including renal cystic (hypo)dysplasia, situs inversus, cardiopathy and paucity of bile ducts. Two patients who died early after birth carried missense mutations in the kinase and/or RCC1 domains. A homozygous splice mutation was identified in a fetus with Meckel-like phenotype. Analyses of patient fibroblasts and IMCD3 cells expressing mutated NEK8-GFP revealed that the mutations affect NEK8 nuclear and ciliary localization. The number of ciliated cells was reduced and ciliary localization of NEK8 partner ANKS6/NPHP16 was lost, demonstrating the key role of NEK8 in cilia function. Surprisingly, in patient fibroblasts, NEK8 accumulates at the Golgi that appeared dispersed into the cytoplasm suggesting a role in vesicular trafficking. Cell cycle defects associated with abnormal nuclear accumulation of YAP, a transcriptional co-activator of the Hippo pathway was also observed, together with dysregulation of several Hippo effector/target genes. Finally, injection of nek8 morpholinos in zebrafish embryos led to ciliopathy-related phenotype (curly body axis, laterality defects, pronephric cysts) that could be rescued by RNA expression of WT NEK8 but not by the mutated forms, further demonstrating pathogenicity of the mutations. Altogether, we demonstrate that human NEK8 mutations alter developmental ciliary and non-ciliary processes, thus leading to multisystemic defects.