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1.
Kidney Int ; 105(4): 844-864, 2024 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-38154558

RESUMEN

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.


Asunto(s)
Estructuras Embrionarias , Factores de Transcripción Forkhead , Enfermedades Renales , Riñón , Nefronas , Sistema Urinario , Anomalías Urogenitales , Reflujo Vesicoureteral , Adulto , Animales , Humanos , Ratones , Estudio de Asociación del Genoma Completo , Riñón/anomalías , Riñón/embriología , Enfermedades Renales/genética , Ratones Noqueados , Nefronas/embriología , Factores de Transcripción/genética , Anomalías Urogenitales/genética , Reflujo Vesicoureteral/genética , Factores de Transcripción Forkhead/deficiencia , Factores de Transcripción Forkhead/metabolismo
2.
J Med Genet ; 54(9): 624-632, 2017 09.
Artículo en Inglés | MEDLINE | ID: mdl-28442542

RESUMEN

BACKGROUND: Recent findings suggesting that Abelson helper integration site 1 (AHI1) is involved in non-syndromic retinal disease have been debated, as the functional significance of identified missense variants was uncertain. We assessed whether AHI1 variants cause non-syndromic retinitis pigmentosa (RP). METHODS: Exome sequencing was performed in three probands with RP. The effects of the identified missense variants in AHI1 were predicted by three-dimensional structure homology modelling. Ciliary parameters were evaluated in patient's fibroblasts, and recombinant mutant proteins were expressed in ciliated retinal pigmented epithelium cells. RESULTS: In the three patients with RP, three sets of compound heterozygous variants were detected in AHI1 (c.2174G>A; p.Trp725* and c.2258A>T; p.Asp753Val, c.660delC; p.Ser221Glnfs*10 and c.2090C>T; p.Pro697Leu, c.2087A>G; p.His696Arg and c.2429C>T; p.Pro810Leu). All four missense variants were present in the conserved WD40 domain of Jouberin, the ciliary protein encoded by AHI1, with variable predicted implications for the domain structure. No significant changes in the percentage of ciliated cells, nor in cilium length or intraflagellar transport were detected. However, expression of mutant recombinant Jouberin in ciliated cells showed a significantly decreased enrichment at the ciliary base. CONCLUSIONS: This report confirms that mutations in AHI1 can underlie autosomal recessive RP. Moreover, it structurally and functionally validates the effect of the RP-associated AHI1 variants on protein function, thus proposing a new genotype-phenotype correlation for AHI1 mutation associated retinal ciliopathies.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Mutación Missense , Retinitis Pigmentosa/genética , Anomalías Múltiples/genética , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras del Transporte Vesicular , Adulto , Cerebelo/anomalías , Anomalías del Ojo/genética , Femenino , Humanos , Enfermedades Renales Quísticas/genética , Masculino , Persona de Mediana Edad , Linaje , Dominios Proteicos/genética , Retina/anomalías
3.
Hum Mol Genet ; 24(6): 1584-601, 2015 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-25398945

RESUMEN

Leber congenital amaurosis (LCA) and juvenile retinitis pigmentosa (RP) are severe hereditary diseases that causes visual impairment in infants and children. SPATA7 has recently been identified as the LCA3 and juvenile RP gene in humans, whose function in the retina remains elusive. Here, we show that SPATA7 localizes at the primary cilium of cells and at the connecting cilium (CC) of photoreceptor cells, indicating that SPATA7 is a ciliary protein. In addition, SPATA7 directly interacts with the retinitis pigmentosa GTPase regulator interacting protein 1 (RPGRIP1), a key connecting cilium protein that has also been linked to LCA. In the retina of Spata7 null mutant mice, a substantial reduction of RPGRIP1 levels at the CC of photoreceptor cells is observed, suggesting that SPATA7 is required for the stable assembly and localization of the ciliary RPGRIP1 protein complex. Furthermore, our results pinpoint a role of this complex in protein trafficking across the CC to the outer segments, as we identified that rhodopsin accumulates in the inner segments and around the nucleus of photoreceptors. This accumulation then likely triggers the apoptosis of rod photoreceptors that was observed. Loss of Spata7 function in mice indeed results in a juvenile RP-like phenotype, characterized by progressive degeneration of photoreceptor cells and a strongly decreased light response. Together, these results indicate that SPATA7 functions as a key member of a retinal ciliopathy-associated protein complex, and that apoptosis of rod photoreceptor cells triggered by protein mislocalization is likely the mechanism of disease progression in LCA3/ juvenile RP patients.


Asunto(s)
Proteínas de Unión al ADN/metabolismo , Cilio Conector de los Fotorreceptores/patología , Proteínas/metabolismo , Células Fotorreceptoras Retinianas Bastones/patología , Animales , Apoptosis , Bovinos , Proteínas del Citoesqueleto , Proteínas de Unión al ADN/genética , Eliminación de Gen , Humanos , Ratones , Ratones Mutantes , Cilio Conector de los Fotorreceptores/metabolismo , Transporte de Proteínas , Células Fotorreceptoras Retinianas Conos/patología , Células Fotorreceptoras Retinianas Bastones/metabolismo , Rodopsina/metabolismo
4.
medRxiv ; 2023 Mar 22.
Artículo en Inglés | MEDLINE | ID: mdl-36993625

RESUMEN

Background: Congenital anomalies of the kidney and urinary tract (CAKUT) are the predominant cause for chronic kidney disease below 30 years of age. Many monogenic forms have been discovered mainly due to comprehensive genetic testing like exome sequencing (ES). However, disease-causing variants in known disease-associated genes still only explain a proportion of cases. Aim of this study was to unravel the underlying molecular mechanism of syndromic CAKUT in two multiplex families with presumed autosomal recessive inheritance. Methods and Results: ES in the index individuals revealed two different rare homozygous variants in FOXD2, a transcription factor not previously implicated in CAKUT in humans: a frameshift in family 1 and a missense variant in family 2 with family segregation patterns consistent with autosomal-recessive inheritance. CRISPR/Cas9-derived Foxd2 knock-out (KO) mice presented with bilateral dilated renal pelvis accompanied by renal papilla atrophy while extrarenal features included mandibular, ophthalmologic, and behavioral anomalies, recapitulating the phenotype of humans with FOXD2 dysfunction. To study the pathomechanism of FOXD2-dysfunction-mediated developmental renal defects, in a complementary approach, we generated CRISPR/Cas9-mediated KO of Foxd2 in ureteric-bud-induced mouse metanephric mesenchyme cells. Transcriptomic analyses revealed enrichment of numerous differentially expressed genes important in renal/urogenital development, including Pax2 and Wnt4 as well as gene expression changes indicating a cell identity shift towards a stromal cell identity. Histology of Foxd2 KO mouse kidneys confirmed increased fibrosis. Further, GWAS data (genome-wide association studies) suggests that FOXD2 could play a role for maintenance of podocyte integrity during adulthood. Conclusions: In summary, our data implicate that FOXD2 dysfunction is a very rare cause of autosomal recessive syndromic CAKUT and suggest disturbances of the PAX2-WNT4 cell signaling axis contribute to this phenotype.

5.
PLoS One ; 14(5): e0216705, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31095607

RESUMEN

The cilium is an essential organelle at the surface of mammalian cells whose dysfunction causes a wide range of genetic diseases collectively called ciliopathies. The current rate at which new ciliopathy genes are identified suggests that many ciliary components remain undiscovered. We generated and rigorously analyzed genomic, proteomic, transcriptomic and evolutionary data and systematically integrated these using Bayesian statistics into a predictive score for ciliary function. This resulted in 285 candidate ciliary genes. We generated independent experimental evidence of ciliary associations for 24 out of 36 analyzed candidate proteins using multiple cell and animal model systems (mouse, zebrafish and nematode) and techniques. For example, we show that OSCP1, which has previously been implicated in two distinct non-ciliary processes, causes ciliogenic and ciliopathy-associated tissue phenotypes when depleted in zebrafish. The candidate list forms the basis of CiliaCarta, a comprehensive ciliary compendium covering 956 genes. The resource can be used to objectively prioritize candidate genes in whole exome or genome sequencing of ciliopathy patients and can be accessed at http://bioinformatics.bio.uu.nl/john/syscilia/ciliacarta/.


Asunto(s)
Cilios/genética , Genómica , Animales , Teorema de Bayes , Caenorhabditis elegans/citología , Caenorhabditis elegans/genética , Anotación de Secuencia Molecular , Fenotipo , Reproducibilidad de los Resultados , Células Receptoras Sensoriales/metabolismo , Pez Cebra/genética
6.
J Cell Biol ; 217(8): 2851-2865, 2018 08 06.
Artículo en Inglés | MEDLINE | ID: mdl-29899041

RESUMEN

Photoreceptor-specific ciliopathies often affect a structure that is considered functionally homologous to the ciliary transition zone (TZ) called the connecting cilium (CC). However, it is unclear how mutations in certain ciliary genes disrupt the photoreceptor CC without impacting the primary cilia systemically. By applying stochastic optical reconstruction microscopy technology in different genetic models, we show that the CC can be partitioned into two regions: the proximal CC (PCC), which is homologous to the TZ of primary cilia, and the distal CC (DCC), a photoreceptor-specific extension of the ciliary TZ. This specialized distal zone of the CC in photoreceptors is maintained by SPATA7, which interacts with other photoreceptor-specific ciliary proteins such as RPGR and RPGRIP1. The absence of Spata7 results in the mislocalization of DCC proteins without affecting the PCC protein complexes. This collapse results in destabilization of the axonemal microtubules, which consequently results in photoreceptor degeneration. These data provide a novel mechanism to explain how genetic disruption of ubiquitously present ciliary proteins exerts tissue-specific ciliopathy phenotypes.


Asunto(s)
Proteínas de Unión al ADN/fisiología , Cilio Conector de los Fotorreceptores/metabolismo , Proteínas Adaptadoras Transductoras de Señales/análisis , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Antígenos de Neoplasias , Proteínas Portadoras/análisis , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular , Proteínas del Citoesqueleto , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas del Ojo/análisis , Proteínas del Ojo/genética , Proteínas del Ojo/metabolismo , Ratones , Ratones Endogámicos C57BL , Proteínas Nucleares/metabolismo , Proteínas Nucleares/fisiología , Cilio Conector de los Fotorreceptores/ultraestructura , Transporte de Proteínas/genética
7.
Nat Commun ; 7: 11491, 2016 05 13.
Artículo en Inglés | MEDLINE | ID: mdl-27173435

RESUMEN

Cellular organelles provide opportunities to relate biological mechanisms to disease. Here we use affinity proteomics, genetics and cell biology to interrogate cilia: poorly understood organelles, where defects cause genetic diseases. Two hundred and seventeen tagged human ciliary proteins create a final landscape of 1,319 proteins, 4,905 interactions and 52 complexes. Reverse tagging, repetition of purifications and statistical analyses, produce a high-resolution network that reveals organelle-specific interactions and complexes not apparent in larger studies, and links vesicle transport, the cytoskeleton, signalling and ubiquitination to ciliary signalling and proteostasis. We observe sub-complexes in exocyst and intraflagellar transport complexes, which we validate biochemically, and by probing structurally predicted, disruptive, genetic variants from ciliary disease patients. The landscape suggests other genetic diseases could be ciliary including 3M syndrome. We show that 3M genes are involved in ciliogenesis, and that patient fibroblasts lack cilia. Overall, this organelle-specific targeting strategy shows considerable promise for Systems Medicine.


Asunto(s)
Cilios/metabolismo , Ciliopatías/genética , Enanismo/genética , Hipotonía Muscular/genética , Mapas de Interacción de Proteínas , Proteínas/metabolismo , Columna Vertebral/anomalías , Transporte Biológico/fisiología , Cromatografía de Afinidad/métodos , Ciliopatías/patología , Ciliopatías/terapia , Análisis Mutacional de ADN , Conjuntos de Datos como Asunto , Enanismo/patología , Enanismo/terapia , Fibroblastos , Células HEK293 , Humanos , Espectrometría de Masas , Terapia Molecular Dirigida/métodos , Hipotonía Muscular/patología , Hipotonía Muscular/terapia , Mapeo de Interacción de Proteínas/métodos , Proteínas/genética , Proteínas/aislamiento & purificación , Proteómica/métodos , Columna Vertebral/patología , Análisis de Sistemas
8.
Nat Cell Biol ; 17(8): 1074-1087, 2015 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-26167768

RESUMEN

Defects in primary cilium biogenesis underlie the ciliopathies, a growing group of genetic disorders. We describe a whole-genome siRNA-based reverse genetics screen for defects in biogenesis and/or maintenance of the primary cilium, obtaining a global resource. We identify 112 candidate ciliogenesis and ciliopathy genes, including 44 components of the ubiquitin-proteasome system, 12 G-protein-coupled receptors, and 3 pre-mRNA processing factors (PRPF6, PRPF8 and PRPF31) mutated in autosomal dominant retinitis pigmentosa. The PRPFs localize to the connecting cilium, and PRPF8- and PRPF31-mutated cells have ciliary defects. Combining the screen with exome sequencing data identified recessive mutations in PIBF1, also known as CEP90, and C21orf2, also known as LRRC76, as causes of the ciliopathies Joubert and Jeune syndromes. Biochemical approaches place C21orf2 within key ciliopathy-associated protein modules, offering an explanation for the skeletal and retinal involvement observed in individuals with C21orf2 variants. Our global, unbiased approaches provide insights into ciliogenesis complexity and identify roles for unanticipated pathways in human genetic disease.


Asunto(s)
Cilios/genética , Trastornos de la Motilidad Ciliar/genética , Marcadores Genéticos , Pruebas Genéticas/métodos , Genómica/métodos , Células Fotorreceptoras , Interferencia de ARN , Anomalías Múltiples , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/ultraestructura , Enfermedades Cerebelosas/genética , Cerebelo/anomalías , Cilios/metabolismo , Cilios/patología , Trastornos de la Motilidad Ciliar/metabolismo , Trastornos de la Motilidad Ciliar/patología , Proteínas del Citoesqueleto , Bases de Datos Genéticas , Síndrome de Ellis-Van Creveld/genética , Anomalías del Ojo/genética , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Células HEK293 , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Enfermedades Renales Quísticas/genética , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación , Fenotipo , Células Fotorreceptoras/metabolismo , Células Fotorreceptoras/ultraestructura , Proteínas Gestacionales/genética , Proteínas Gestacionales/metabolismo , Proteínas/genética , Proteínas/metabolismo , Reproducibilidad de los Resultados , Retina/anomalías , Factores Supresores Inmunológicos/genética , Factores Supresores Inmunológicos/metabolismo , Transfección , Pez Cebra/genética , Pez Cebra/metabolismo
9.
Nat Genet ; 45(8): 951-6, 2013 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-23793029

RESUMEN

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.


Asunto(s)
Enfermedades Renales Quísticas/genética , Cinesinas/genética , Proteínas Nucleares/genética , Proteínas Quinasas/genética , Factores de Transcripción/genética , Animales , Cilios/metabolismo , Consanguinidad , Exones , Técnicas de Silenciamiento del Gen , Humanos , Intrones , Enfermedades Renales Quísticas/metabolismo , Cinesinas/metabolismo , Ratones , Mutación , Quinasas Relacionadas con NIMA , Proteínas Nucleares/metabolismo , Fenotipo , Enfermedades Renales Poliquísticas/genética , Unión Proteica , Mapas de Interacción de Proteínas , Proteínas Quinasas/metabolismo , Transporte de Proteínas , Factores de Transcripción/metabolismo , Xenopus/embriología , Xenopus/metabolismo , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo
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