2d-3d-human-induced-pluripotent-stem-cell-based-models-to-dissect

2D and 3D Human Induced Pluripotent Stem Cell-Based Models to Dissect Primary Cilium Involvement during Neocortical Development

Primary cilia (PC) are non-motile dynamic microtubule-based organelles that protrude from the surface of most mammalian cells. They emerge from the older ...

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. 

Mutations of CEP83 cause infantile nephronophthisis and intellectual disability.

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.

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

Oral-facial-digital syndromes (OFDS) gather rare genetic disorders characterised by facial, oral and digital abnormalities associated with a wide range of additional features (polycystic kidney disease, cerebral malformations and several others) to delineate a growing list of OFDS subtypes. The most frequent, OFD type I, is caused by a heterozygous mutation in the  gene encoding a centrosomal protein. The wide clinical heterogeneity of OFDS suggests the involvement of other ciliary genes. For 15 years, we have aimed to identify the molecular bases of OFDS. This effort has been greatly helped by the recent development of whole-exome sequencing (WES). Here, we present all our published and unpublished results for WES in 24 cases with OFDS. We identified causal variants in five new genes (, , ,  and ) and related the clinical spectrum of four genes in other ciliopathies (, ,  and ) to OFDS. Mutations were also detected in two genes previously implicated in OFDS. Functional studies revealed the involvement of centriole elongation, transition zone and intraflagellar transport defects in OFDS, thus characterising three ciliary protein modules: the complex KIAA0753-FOPNL-OFD1, a regulator of centriole elongation; the Meckel-Gruber syndrome module, a major component of the transition zone; and the CPLANE complex necessary for IFT-A assembly. OFDS now appear to be a distinct subgroup of ciliopathies with wide heterogeneity, which makes the initial classification obsolete. A clinical classification restricted to the three frequent/well-delineated subtypes could be proposed, and for patients who do not fit one of these three main subtypes, a further classification could be based on the genotype.

Fifteen years of research on oral-facial-digital syndromes: from 1 to 16 causal genes.

Genetic and phenotypic heterogeneity and the lack of sufficiently large patient cohorts pose a significant challenge to understanding genetic associations in rare disease. Here we identify  (alias ) as a genetic modifier of cystic kidney disease in Joubert syndrome, using a -deficient mouse model to recapitulate the phenotypic variability observed in patients by mixing genetic backgrounds in a controlled manner and performing genome-wide analysis of these mice. Experimental down-regulation of  in the parental mouse strain phenocopied the severe cystic kidney phenotype. A common polymorphism within human  significantly associates with kidney disease severity in a patient cohort with  mutations. The striking phenotypic modifications we describe are a timely reminder of the value of mouse models and highlight the significant contribution of genetic background. Furthermore, if appropriately managed, this can be exploited as a powerful tool to elucidate mechanisms underlying human disease heterogeneity.

Mouse genetics reveals Barttin as a genetic modifier of Joubert syndrome.

Rare diseases affect approximately 350 million people worldwide. Delayed diagnosis is frequent due to lack of knowledge of most clinicians and a small number of expert centers. Consequently, computerized diagnosis support systems have been developed to address these issues, with many relying on rare disease expertise and taking advantage of the increasing volume of generated and accessible health-related data. Our objective is to perform a review of all initiatives aiming to support the diagnosis of rare diseases.

Diagnosis support systems for rare diseases: a scoping review.

Biallelic mutations in MAPKBP1 were recently associated with late-onset cilia-independent nephronophthisis. MAPKBP1 was found at mitotic spindle poles but could not be detected at primary cilia or centrosomes. Here, by identification and characterization of novel MAPKBP1 variants, we aimed at further investigating its role in health and disease. Genetic analysis was done by exome sequencing, homozygosity mapping, and a targeted kidney gene panel while coimmunoprecipitation was used to explore wild-type and mutant protein-protein interactions. Expression of MAPKBP1 in non-ciliated HeLa and ciliated inner medullary collecting duct cells enabled co-localization studies by fluorescence microscopy. By next generation sequencing, we identified two novel homozygous MAPKBP1 splice-site variants in patients with nephronophthisis-related chronic kidney disease. Splice-site analyses revealed truncation of C-terminal coiled-coil domains and patient-derived deletion constructs lost their ability to homodimerize and heterodimerize with paralogous WDR62. While wild-type MAPKBP1 exhibited centrosomal, basal body, and microtubule association, mutant proteins lost the latter and showed reduced recruitment to cell cycle dependent centriolar structures. Wild-type and mutant proteins had no reciprocal influence upon co-expression excluding dominant negative effects. Thus, MAPKBP1 appears to be a novel microtubule-binding protein with cell cycle dependent centriolar localization. Truncation of its coiled-coil domain is enough to abrogate its dimerization and results in severely disturbed intracellular localizations. Delineating the impact of impaired dimerization on cell cycle regulation and intracellular kidney signaling may provide new insights into common mechanisms of kidney degeneration. Thus, due to milder clinical presentation, MAPKBP1-associated nephronophthisis should be considered in adult patients with otherwise unexplained chronic kidney disease.

Novel nephronophthisis-associated variants reveal functional importance of MAPKBP1 dimerization for centriolar recruitment.

DNAJB11 (DnaJ Heat Shock Protein Family (Hsp40) Member B11) heterozygous loss of function variations have been reported in autosomal dominant cystic kidney disease with extensive fibrosis, associated with maturation and trafficking defect involving both the autosomal dominant polycystic kidney disease protein polycystin-1 and the autosomal dominant tubulointerstitial kidney disease protein uromodulin. Here we show that biallelic pathogenic variations in DNAJB11 lead to a severe fetal disease including enlarged cystic kidneys, dilation and proliferation of pancreatic duct cells, and liver ductal plate malformation, an association known as Ivemark II syndrome. Cysts of the kidney were developed exclusively from uromodulin negative tubular segments. In addition, tubular cells from the affected kidneys had elongated primary cilia, a finding previously reported in ciliopathies. Thus, our data show that the recessive disease associated with DNAJB11 variations is a ciliopathy rather than a disease of the autosomal dominant tubulointerstitial kidney disease spectrum, and prompt screening of DNAJB11 in fetal hyperechogenic/cystic kidneys.

Bi-allelic pathogenic variations in DNAJB11 cause Ivemark II syndrome, a renal-hepatic-pancreatic dysplasia.

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.

Cystic kidney diseases associated with mutations in phosphomannomutase 2 promotor: a large spectrum of phenotypes.

To identify patients with similar clinical profiles and derive insights from the records and outcomes of similar patients can help fast and precise diagnosis and other clinical decisions for rare diseases. Similarity methods are required to take into account the semantic relations between medical concepts and also the different relevance of all medical concepts presented in patients' medical records. In this paper, we introduce the methods developed in the context of rare disease screening/diagnosis from clinical data warehouse using medical concept embedding and adjusted aggregations. Our methods provided better preliminary results than baseline methods, with a significant improvement of precision among the top ranked similar patients, which is encouraging for further fine-tuning and application on a large-scale dataset for new/candidate patient identification.

Identification of Similar Patients Through Medical Concept Embedding from Electronic Health Records: A Feasibility Study for Rare Disease Diagnosis.

Nephronophthisis (NPH) is an autosomal recessive ciliopathy and a major cause of end-stage renal disease in children. The main forms, juvenile and adult NPH, are characterized by tubulointerstitial fibrosis whereas the infantile form is more severe and characterized by cysts. NPH is caused by mutations in over 20 different genes, most of which encode components of the primary cilium, an organelle in which important cellular signaling pathways converge. Ciliary signal transduction plays a critical role in kidney development and tissue homeostasis, and disruption of ciliary signaling has been associated with cyst formation, epithelial cell dedifferentiation and kidney function decline. Drugs have been identified that target specific signaling pathways (for example cAMP/PKA, Hedgehog, and mTOR pathways) and rescue NPH phenotypes in  and/or  models. Despite identification of numerous candidate drugs in rodent models, there has been a lack of clinical trials and there is currently no therapy that halts disease progression in NPH patients. This review covers the most important findings of therapeutic approaches in NPH model systems to date, including hypothesis-driven therapies and untargeted drug screens, approached from the pathophysiology of NPH. Importantly, most animal models used in these studies represent the cystic infantile form of NPH, which is less prevalent than the juvenile form. It appears therefore important to develop new models relevant for juvenile/adult NPH. Alternative non-orthologous animal models and developments in patient-based  model systems are discussed, as well as future directions in personalized therapy for NPH.

Renal Ciliopathies: Sorting Out Therapeutic Approaches for Nephronophthisis.

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.

Article	Year
Mutations of CEP83 cause infantile nephronophthisis and intellectual disability. American journal of human genetics\| PubMed ID: 24882706	2014
Mutations in MAPKBP1 Cause Juvenile or Late-Onset Cilia-Independent Nephronophthisis. American journal of human genetics\| PubMed ID: 28089251	2017
Fifteen years of research on oral-facial-digital syndromes: from 1 to 16 causal genes. Journal of medical genetics\| PubMed ID: 28289185	2017
Mouse genetics reveals Barttin as a genetic modifier of Joubert syndrome. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 31879347	2020
Diagnosis support systems for rare diseases: a scoping review. Orphanet journal of rare diseases\| PubMed ID: 32299466	2020
Novel nephronophthisis-associated variants reveal functional importance of MAPKBP1 dimerization for centriolar recruitment. Kidney international\| PubMed ID: 32505465	2020
Bi-allelic pathogenic variations in DNAJB11 cause Ivemark II syndrome, a renal-hepatic-pancreatic dysplasia. Kidney international\| PubMed ID: 33129895	2021
Cystic kidney diseases associated with mutations in phosphomannomutase 2 promotor: a large spectrum of phenotypes. Pediatric nephrology (Berlin, Germany)\| PubMed ID: 33580824	2021
Identification of Similar Patients Through Medical Concept Embedding from Electronic Health Records: A Feasibility Study for Rare Disease Diagnosis. Studies in health technology and informatics\| PubMed ID: 34042646	2021
Renal Ciliopathies: Sorting Out Therapeutic Approaches for Nephronophthisis. Frontiers in cell and developmental biology\| PubMed ID: 34055783	2021
Targeted next-generation sequencing in a large series of fetuses with severe renal diseases. Human mutation\| PubMed ID: 35005812	2022

Sophie Saunier

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