RESUMEN
Ciliopathies are genetic disorders arising from dysfunction of microtubule-based cellular appendages called cilia. Different cilia types possess distinct stereotypic microtubule doublet arrangements with non-motile or 'primary' cilia having a 9+0 and motile cilia have a 9+2 array of microtubule doublets. Primary cilia are critical sensory and signaling centers needed for normal mammalian development. Defects in their structure/function result in a spectrum of clinical and developmental pathologies including abnormal neural tube and limb patterning. Altered patterning phenotypes in the limb and neural tube are due to perturbations in the hedgehog (Hh) signaling pathway. Motile cilia are important in fluid movement and defects in motility result in chronic respiratory infections, altered left-right asymmetry, and infertility. These features are the hallmarks of Primary Ciliary Dyskinesia (PCD, OMIM 244400). While mutations in several genes are associated with PCD in patients and animal models, the genetic lesion in many cases is unknown. We assessed the in vivo functions of Growth Arrest Specific 8 (GAS8). GAS8 shares strong sequence similarity with the Chlamydomonas Nexin-Dynein Regulatory Complex (NDRC) protein 4 (DRC4) where it is needed for proper flagella motility. In mammalian cells, the GAS8 protein localizes not only to the microtubule axoneme of motile cilia, but also to the base of non-motile cilia. Gas8 was recently implicated in the Hh signaling pathway as a regulator of Smoothened trafficking into the cilium. Here, we generate the first mouse with a Gas8 mutation and show that it causes severe PCD phenotypes; however, there were no overt Hh pathway phenotypes. In addition, we identified two human patients with missense variants in Gas8. Rescue experiments in Chlamydomonas revealed a subtle defect in swim velocity compared to controls. Further experiments using CRISPR/Cas9 homology driven repair (HDR) to generate one of these human missense variants in mice demonstrated that this allele is likely pathogenic.
Asunto(s)
Tipificación del Cuerpo/genética , Cilios/genética , Síndrome de Kartagener/genética , Proteínas/genética , Animales , Movimiento Celular/genética , Chlamydomonas/genética , Cilios/patología , Proteínas del Citoesqueleto , Citoesqueleto/genética , Modelos Animales de Enfermedad , Extremidades/crecimiento & desarrollo , Extremidades/patología , Predisposición Genética a la Enfermedad , Humanos , Síndrome de Kartagener/patología , Ratones , Microtúbulos/genética , Mutación , Tubo Neural/crecimiento & desarrollo , Tubo Neural/patología , Transducción de Señal/genéticaRESUMEN
Nephronophthisis-related ciliopathies (NPHP-RC) are recessive diseases characterized by renal dysplasia or degeneration. We here identify mutations of DCDC2 as causing a renal-hepatic ciliopathy. DCDC2 localizes to the ciliary axoneme and to mitotic spindle fibers in a cell-cycle-dependent manner. Knockdown of Dcdc2 in IMCD3 cells disrupts ciliogenesis, which is rescued by wild-type (WT) human DCDC2, but not by constructs that reflect human mutations. We show that DCDC2 interacts with DVL and DCDC2 overexpression inhibits ß-catenin-dependent Wnt signaling in an effect additive to Wnt inhibitors. Mutations detected in human NPHP-RC lack these effects. A Wnt inhibitor likewise restores ciliogenesis in 3D IMCD3 cultures, emphasizing the importance of Wnt signaling for renal tubulogenesis. Knockdown of dcdc2 in zebrafish recapitulates NPHP-RC phenotypes, including renal cysts and hydrocephalus, which is rescued by a Wnt inhibitor and by WT, but not by mutant, DCDC2. We thus demonstrate a central role of Wnt signaling in the pathogenesis of NPHP-RC, suggesting an avenue for potential treatment of NPHP-RC.
Asunto(s)
Enfermedades Renales Quísticas/genética , Proteínas Asociadas a Microtúbulos/genética , Vía de Señalización Wnt/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Cilios/genética , Cilios/patología , Biología Computacional , Proteínas Dishevelled , Exones , Células HEK293 , Humanos , Riñón/patología , Ratones , Microscopía Electrónica de Transmisión , Proteínas Asociadas a Microtúbulos/metabolismo , Mutación , Células 3T3 NIH , Fenotipo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Pez Cebra/genética , beta Catenina/antagonistas & inhibidores , beta Catenina/metabolismoRESUMEN
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.
Asunto(s)
Discapacidad Intelectual/genética , Enfermedades Renales Quísticas/genética , Proteínas Asociadas a Microtúbulos/genética , Mutación , Alelos , Sistema Nervioso Central/anomalías , Centriolos/genética , Centriolos/metabolismo , Preescolar , Cilios/metabolismo , Exones , Femenino , Humanos , Lactante , Masculino , Proteínas Asociadas a Microtúbulos/metabolismo , Síndromes Orofaciodigitales/genéticaRESUMEN
BACKGROUND: The term nephronophthisis-related ciliopathies (NPHP-RC) describes a group of rare autosomal-recessive cystic kidney diseases, characterised by broad genetic and clinical heterogeneity. NPHP-RC is frequently associated with extrarenal manifestations and accounts for the majority of genetically caused chronic kidney disease (CKD) during childhood and adolescence. Generation of a molecular diagnosis has been impaired by this broad genetic heterogeneity. However, recently developed high-throughput exon sequencing techniques represent powerful and efficient tools to screen large cohorts for dozens of causative genes. METHODS: Therefore, we performed massively multiplexed targeted sequencing using the modified molecular inversion probe strategy (MIPs) in an international cohort of 384 patients diagnosed with NPHP-RC. RESULTS: As a result, we established the molecular diagnoses in 81/384 unrelated individuals (21.1%). We detected 127 likely disease-causing mutations in 18 of 34 evaluated NPHP-RC genes, 22 of which were novel. We further compared a subgroup of current findings to the results of a previous study in which we used an array-based microfluidic PCR technology in the same cohort. While 78 likely disease-causing mutations were previously detected by the array-based microfluidic PCR, the MIPs approach identified 94 likely pathogenic mutations. Compared with the previous approach, MIPs redetected 66 out of 78 variants and 28 previously unidentified variants, for a total of 94 variants. CONCLUSIONS: In summary, we demonstrate that the modified MIPs technology is a useful approach to screen large cohorts for a multitude of established NPHP genes in order to identify the underlying molecular cause. Combined application of two independent library preparation and sequencing techniques, however, may still be indicated for Mendelian diseases with extensive genetic heterogeneity in order to further increase diagnostic sensitivity.
Asunto(s)
Secuenciación de Nucleótidos de Alto Rendimiento , Enfermedades Renales Quísticas/genética , Técnicas de Diagnóstico Molecular , Heterogeneidad Genética , Humanos , Sensibilidad y EspecificidadRESUMEN
Karyomegalic interstitial nephritis (KIN) is a chronic interstitial nephropathy characterized by tubulointerstitial nephritis and formation of enlarged nuclei in the kidneys and other tissues. We recently reported that recessive mutations in the gene encoding FANCD2/FANCI-associated nuclease 1 (FAN1) cause KIN in humans. FAN1 is a major component of the Fanconi anemia-related pathway of DNA damage response (DDR) signaling. To study the pathogenesis of KIN, we generated a Fan1 knockout mouse model, with abrogation of Fan1 expression confirmed by quantitative RT-PCR. Challenging Fan1-/- and wild-type mice with 20 mg/kg cisplatin caused AKI in both genotypes. In contrast, chronic injection of cisplatin at 2 mg/kg induced KIN that led to renal failure within 5 weeks in Fan1-/- mice but not in wild-type mice. Cell culture studies showed decreased survival and reduced colony formation of Fan1-/- mouse embryonic fibroblasts and bone marrow mesenchymal stem cells compared with wild-type counterparts in response to treatment with genotoxic agents, suggesting that FAN1 mutations cause chemosensitivity and bone marrow failure. Our data show that Fan1 is involved in the physiologic response of kidney tubular cells to DNA damage, which contributes to the pathogenesis of CKD. Moreover, Fan1-/- mice provide a new model with which to study the pathomechanisms of CKD.
Asunto(s)
Endodesoxirribonucleasas/genética , Mutación , Nefritis Intersticial/enzimología , Nefritis Intersticial/genética , Animales , Modelos Animales de Enfermedad , Exodesoxirribonucleasas , Ratones , Ratones Noqueados , Enzimas Multifuncionales , Insuficiencia Renal Crónica/etiologíaRESUMEN
Chronically increased echogenicity on renal ultrasound is a sensitive early finding of chronic kidney disease that can be detected before manifestation of other symptoms. Increased echogenicity, however, is not specific for a certain etiology of chronic kidney disease. Here, we performed whole exome sequencing in 79 consanguineous or familial cases of suspected nephronophthisis in order to determine the underlying molecular disease cause. In 50 cases, there was a causative mutation in a known monogenic disease gene. In 32 of these cases whole exome sequencing confirmed the diagnosis of a nephronophthisis-related ciliopathy. In 8 cases it revealed the diagnosis of a renal tubulopathy. The remaining 10 cases were identified as Alport syndrome (4), autosomal-recessive polycystic kidney disease (2), congenital anomalies of the kidney and urinary tract (3), and APECED syndrome (1). In 5 families, in whom mutations in known monogenic genes were excluded, we applied homozygosity mapping for variant filtering and identified 5 novel candidate genes (RBM48, FAM186B, PIAS1, INCENP, and RCOR1) for renal ciliopathies. Thus, whole exome sequencing allows the detection of the causative mutation in 2/3 of affected individuals, thereby presenting the etiologic diagnosis, and allows identification of novel candidate genes.
Asunto(s)
Insuficiencia Renal Crónica/genética , Edad de Inicio , Estudios de Cohortes , Análisis Mutacional de ADN , Exoma , Humanos , Enfermedades Renales Quísticas/congénito , Enfermedades Renales Quísticas/genética , Insuficiencia Renal Crónica/diagnóstico por imagenRESUMEN
Primary ciliary dyskinesia (PCD) is a genetically heterogeneous, autosomal-recessive disorder, characterized by oto-sino-pulmonary disease and situs abnormalities. PCD-causing mutations have been identified in 20 genes, but collectively they account for only â¼65% of all PCDs. To identify mutations in additional genes that cause PCD, we performed exome sequencing on three unrelated probands with ciliary outer and inner dynein arm (ODA+IDA) defects. Mutations in SPAG1 were identified in one family with three affected siblings. Further screening of SPAG1 in 98 unrelated affected individuals (62 with ODA+IDA defects, 35 with ODA defects, 1 without available ciliary ultrastructure) revealed biallelic loss-of-function mutations in 11 additional individuals (including one sib-pair). All 14 affected individuals with SPAG1 mutations had a characteristic PCD phenotype, including 8 with situs abnormalities. Additionally, all individuals with mutations who had defined ciliary ultrastructure had ODA+IDA defects. SPAG1 was present in human airway epithelial cell lysates but was not present in isolated axonemes, and immunofluorescence staining showed an absence of ODA and IDA proteins in cilia from an affected individual, thus indicating that SPAG1 probably plays a role in the cytoplasmic assembly and/or trafficking of the axonemal dynein arms. Zebrafish morpholino studies of spag1 produced cilia-related phenotypes previously reported for PCD-causing mutations in genes encoding cytoplasmic proteins. Together, these results demonstrate that mutations in SPAG1 cause PCD with ciliary ODA+IDA defects and that exome sequencing is useful to identify genetic causes of heterogeneous recessive disorders.
Asunto(s)
Antígenos de Superficie/genética , Cilios/genética , Trastornos de la Motilidad Ciliar/genética , Dineínas/genética , Proteínas de Unión al GTP/genética , Síndrome de Kartagener/genética , Mutación/genética , Adolescente , Adulto , Animales , Axonema/genética , Niño , Preescolar , Citoplasma/genética , Células Epiteliales/metabolismo , Exoma , Femenino , Humanos , Lactante , Masculino , Linaje , Fenotipo , Adulto Joven , Pez CebraRESUMEN
Defects of motile cilia cause primary ciliary dyskinesia (PCD), characterized by recurrent respiratory infections and male infertility. Using whole-exome resequencing and high-throughput mutation analysis, we identified recessive biallelic mutations in ZMYND10 in 14 families and mutations in the recently identified LRRC6 in 13 families. We show that ZMYND10 and LRRC6 interact and that certain ZMYND10 and LRRC6 mutations abrogate the interaction between the LRRC6 CS domain and the ZMYND10 C-terminal domain. Additionally, ZMYND10 and LRRC6 colocalize with the centriole markers SAS6 and PCM1. Mutations in ZMYND10 result in the absence of the axonemal protein components DNAH5 and DNALI1 from respiratory cilia. Animal models support the association between ZMYND10 and human PCD, given that zmynd10 knockdown in zebrafish caused ciliary paralysis leading to cystic kidneys and otolith defects and that knockdown in Xenopus interfered with ciliogenesis. Our findings suggest that a cytoplasmic protein complex containing ZMYND10 and LRRC6 is necessary for motile ciliary function.
Asunto(s)
Cilios/genética , Síndrome de Kartagener/genética , Proteínas/genética , Sistema Respiratorio/metabolismo , Proteínas Supresoras de Tumor/genética , Animales , Autoantígenos/genética , Autoantígenos/metabolismo , Dineínas Axonemales/genética , Dineínas Axonemales/metabolismo , Biomarcadores/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cilios/metabolismo , Cilios/patología , Proteínas del Citoesqueleto , Exoma , Regulación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Síndrome de Kartagener/metabolismo , Síndrome de Kartagener/patología , Masculino , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Mutación , Linaje , Unión Proteica , Estructura Terciaria de Proteína , Proteínas/metabolismo , Ratas , Sistema Respiratorio/patología , Proteínas Supresoras de Tumor/metabolismo , Xenopus laevis/genética , Xenopus laevis/metabolismo , Pez Cebra/genética , Pez Cebra/metabolismoRESUMEN
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.
Asunto(s)
Ataxia Cerebelosa/genética , Síndrome de Ellis-Van Creveld/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Retinitis Pigmentosa/genética , Alelos , Secuencia de Aminoácidos , Animales , Pueblo Asiatico/genética , Huesos/anomalías , Huesos/metabolismo , Huesos/patología , Ataxia Cerebelosa/patología , Craneosinostosis/genética , Craneosinostosis/patología , Dineínas Citoplasmáticas/genética , Dineínas Citoplasmáticas/metabolismo , Dineínas/genética , Dineínas/metabolismo , Displasia Ectodérmica/genética , Displasia Ectodérmica/patología , Síndrome de Ellis-Van Creveld/patología , Epistasis Genética , Femenino , Fibroblastos/patología , Técnicas de Silenciamiento del Gen , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Enfermedades Renales Quísticas/genética , Enfermedades Renales Quísticas/patología , Masculino , Datos de Secuencia Molecular , Mutación , Fenotipo , Retinitis Pigmentosa/patología , Población Blanca/genética , Pez Cebra/genéticaRESUMEN
Primary ciliary dyskinesia (PCD) is caused when defects of motile cilia lead to chronic airway infections, male infertility, and situs abnormalities. Multiple causative PCD mutations account for only 65% of cases, suggesting that many genes essential for cilia function remain to be discovered. By using zebrafish morpholino knockdown of PCD candidate genes as an in vivo screening platform, we identified c21orf59, ccdc65, and c15orf26 as critical for cilia motility. c21orf59 and c15orf26 knockdown in zebrafish and planaria blocked outer dynein arm assembly, and ccdc65 knockdown altered cilia beat pattern. Biochemical analysis in Chlamydomonas revealed that the C21orf59 ortholog FBB18 is a flagellar matrix protein that accumulates specifically when cilia motility is impaired. The Chlamydomonas ida6 mutant identifies CCDC65/FAP250 as an essential component of the nexin-dynein regulatory complex. Analysis of 295 individuals with PCD identified recessive truncating mutations of C21orf59 in four families and CCDC65 in two families. Similar to findings in zebrafish and planaria, mutations in C21orf59 caused loss of both outer and inner dynein arm components. Our results characterize two genes associated with PCD-causing mutations and elucidate two distinct mechanisms critical for motile cilia function: dynein arm assembly for C21orf59 and assembly of the nexin-dynein regulatory complex for CCDC65.
Asunto(s)
Trastornos de la Motilidad Ciliar/genética , Glicoproteínas/genética , Síndrome de Kartagener/genética , Pez Cebra/genética , Animales , Chlamydomonas/genética , Cilios/genética , Análisis Mutacional de ADN/métodos , Dineínas/genética , Femenino , Humanos , Masculino , Mutación , Sistemas de Lectura Abierta , Planarias/genética , Proteoma/genéticaRESUMEN
BACKGROUND: Bidirectional intraflagellar transport (IFT) consists of two major protein complexes, IFT-A and IFT-B. In contrast to the IFT-B complex, all components of IFT-A have recently been linked to human ciliopathies when defective. We therefore hypothesised that mutations in additional IFT-B encoding genes can be found in patients with multisystemic ciliopathies. METHODS: We screened 1628 individuals with reno-ocular ciliopathies by targeted next-generation sequencing of ciliary candidate genes, including all IFT-B encoding genes. RESULTS: Consequently, we identified a homozygous mutation in IFT81 affecting an obligatory donor splice site in an individual with nephronophthisis and polydactyly. Further, we detected a loss-of-stop mutation with extension of the deduced protein by 10 amino acids in an individual with neuronal ceroid lipofuscinosis-1. This proband presented with retinal dystrophy and brain lesions including cerebellar atrophy, a phenotype to which the IFT81 variant might contribute. Cultured fibroblasts of this latter affected individual showed a significant decrease in ciliated cell abundance compared with controls and increased expression of the transcription factor GLI2 suggesting deranged sonic hedgehog signalling. CONCLUSIONS: This work describes identification of mutations of IFT81 in individuals with symptoms consistent with the clinical spectrum of ciliopathies. It might represent the rare case of a core IFT-B complex protein found associated with human disease. Our data further suggest that defects in the IFT-B core are an exceedingly rare finding, probably due to its indispensable role for ciliary assembly in development.
Asunto(s)
Cilios/genética , Cilios/patología , Ojo/patología , Riñón/patología , Proteínas Musculares/genética , Humanos , Mutación , Análisis de Secuencia de ADNRESUMEN
Nephrolithiasis is a prevalent condition with a high morbidity. Although dozens of monogenic causes have been identified, the fraction of single-gene disease has not been well studied. To determine the percentage of cases that can be molecularly explained by mutations in 1 of 30 known kidney stone genes, we conducted a high-throughput mutation analysis in a cohort of consecutively recruited patients from typical kidney stone clinics. The cohort comprised 272 genetically unresolved individuals (106 children and 166 adults) from 268 families with nephrolithiasis (n=256) or isolated nephrocalcinosis (n=16). We detected 50 likely causative mutations in 14 of 30 analyzed genes, leading to a molecular diagnosis in 14.9% (40 of 268) of all cases; 20 of 50 detected mutations were novel (40%). The cystinuria gene SLC7A9 (n=19) was most frequently mutated. The percentage of monogenic cases was notably high in both the adult (11.4%) and pediatric cohorts (20.8%). Recessive causes were more frequent among children, whereas dominant disease occurred more abundantly in adults. Our study provides an in-depth analysis of monogenic causes of kidney stone disease. We suggest that knowledge of the molecular cause of nephrolithiasis and nephrocalcinosis may have practical implications and might facilitate personalized treatment.
Asunto(s)
Nefrocalcinosis/genética , Nefrolitiasis/genética , Adulto , Niño , Estudios de Cohortes , Análisis Mutacional de ADN , Humanos , Mutación MissenseRESUMEN
The VATER/VACTERL association describes the combination of congenital anomalies including vertebral defects, anorectal malformations, cardiac defects, tracheoesophageal fistula with or without esophageal atresia, renal malformations, and limb defects. As mutations in ciliary genes were observed in diseases related to VATER/VACTERL, we performed targeted resequencing of 25 ciliary candidate genes as well as disease-associated genes (FOXF1, HOXD13, PTEN, ZIC3) in 123 patients with VATER/VACTERL or VATER/VACTERL-like phenotype. We detected no biallelic mutation in any of the 25 ciliary candidate genes; however, identified an identical, probably disease-causing ZIC3 missense mutation (p.Gly17Cys) in four patients and a FOXF1 de novo mutation (p.Gly220Cys) in a further patient. In situ hybridization analyses in mouse embryos between E9.5 and E14.5 revealed Zic3 expression in limb and prevertebral structures, and Foxf1 expression in esophageal, tracheal, vertebral, anal, and genital tubercle tissues, hence VATER/VACTERL organ systems. These data provide strong evidence that mutations in ZIC3 or FOXF1 contribute to VATER/VACTERL.
Asunto(s)
Canal Anal/anomalías , Ano Imperforado/genética , Esófago/anomalías , Factores de Transcripción Forkhead/genética , Estudios de Asociación Genética , Cardiopatías Congénitas/genética , Proteínas de Homeodominio/genética , Riñón/anomalías , Deformidades Congénitas de las Extremidades/genética , Radio (Anatomía)/anomalías , Columna Vertebral/anomalías , Tráquea/anomalías , Factores de Transcripción/genética , Alelos , Animales , Ano Imperforado/diagnóstico , Cilios/genética , Biología Computacional/métodos , Análisis Mutacional de ADN , Femenino , Factores de Transcripción Forkhead/metabolismo , Genotipo , Cardiopatías Congénitas/diagnóstico , Secuenciación de Nucleótidos de Alto Rendimiento , Proteínas de Homeodominio/metabolismo , Humanos , Inmunohistoquímica , Deformidades Congénitas de las Extremidades/diagnóstico , Masculino , Ratones , Mutación , Fenotipo , Factores de Transcripción/metabolismoRESUMEN
Nephronophthisis-related ciliopathies (NPHP-RC) are autosomal-recessive cystic kidney diseases. More than 13 genes are implicated in its pathogenesis to date, accounting for only 40 % of all cases. High-throughput mutation screenings of large patient cohorts represent a powerful tool for diagnostics and identification of novel NPHP genes. We here performed a new high-throughput mutation analysis method to study 13 established NPHP genes (NPHP1-NPHP13) in a worldwide cohort of 1,056 patients diagnosed with NPHP-RC. We first applied multiplexed PCR-based amplification using Fluidigm Access-Array™ technology followed by barcoding and next-generation resequencing on an Illumina platform. As a result, we established the molecular diagnosis in 127/1,056 independent individuals (12.0 %) and identified a single heterozygous truncating mutation in an additional 31 individuals (2.9 %). Altogether, we detected 159 different mutations in 11 out of 13 different NPHP genes, 99 of which were novel. Phenotypically most remarkable were two patients with truncating mutations in INVS/NPHP2 who did not present as infants and did not exhibit extrarenal manifestations. In addition, we present the first case of Caroli disease due to mutations in WDR19/NPHP13 and the second case ever with a recessive mutation in GLIS2/NPHP7. This study represents the most comprehensive mutation analysis in NPHP-RC patients, identifying the largest number of novel mutations in a single study worldwide.
Asunto(s)
Enfermedad de Caroli/genética , Cilios/genética , Cilios/patología , Genes Recesivos/genética , Enfermedades Renales Quísticas/genética , Proteínas de la Membrana/genética , Mutación/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Enfermedad de Caroli/patología , Estudios de Cohortes , Proteínas del Citoesqueleto , Análisis Mutacional de ADN , Femenino , Salud Global , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Enfermedades Renales Quísticas/patología , Masculino , Reacción en Cadena de la Polimerasa Multiplex , Linaje , Proyectos PilotoRESUMEN
OBJECTIVE: To identify disease-causing mutations within coding regions of 11 known NPHP genes (NPHP1-NPHP11) in a cohort of 192 patients diagnosed with a nephronophthisis-associated ciliopathy, at low cost. METHODS: Mutation analysis was carried out using PCR-based 48.48 Access Array microfluidic technology (Fluidigm) with consecutive next-generation sequencing. We applied a 10-fold primer multiplexing approach allowing PCR-based amplification of 475 amplicons (251 exons) for 48 DNA samples simultaneously. After four rounds of amplification followed by indexing all of 192 patient-derived products with different barcodes in a subsequent PCR, 2 × 100 paired-end sequencing was performed on one lane of a HiSeq2000 instrument (Illumina). Bioinformatics analysis was performed using 'CLC Genomics Workbench' software. Potential mutations were confirmed by Sanger sequencing and shown to segregate. RESULTS: Bioinformatics analysis revealed sufficient coverage of 30 × for 168/192 (87.5%) DNA samples (median 449 ×) and of 234 out of 251 targeted coding exons (sensitivity: 93.2%). For proof-of-principle, we analysed 20 known mutations and identified 18 of them in the correct zygosity state (90%). Likewise, we identified pathogenic mutations in 34/192 patients (18%) and discovered 23 novel mutations in the genes NPHP3 (7), NPHP4 (3), IQCB1 (4), CEP290 (7), RPGRIP1L (1), and TMEM67 (1). Additionally, we found 40 different single heterozygous missense variants of unknown significance. CONCLUSIONS: We conclude that the combined approach of array-based multiplexed PCR-amplification on a Fluidigm Access Array platform followed by next-generation sequencing is highly cost-efficient and strongly facilitates diagnostic mutation analysis in broadly heterogeneous Mendelian disorders.
Asunto(s)
Análisis Mutacional de ADN , Secuenciación de Nucleótidos de Alto Rendimiento , Enfermedades Renales Quísticas/congénito , Reacción en Cadena de la Polimerasa Multiplex , Secuencia de Bases , Cilios/patología , Biología Computacional/métodos , Exones , Genotipo , Humanos , Enfermedades Renales Quísticas/genética , Enfermedades Renales Quísticas/patología , Mutación , Reproducibilidad de los ResultadosRESUMEN
BACKGROUND: Excessive diagnostic testing and defensive medicine contribute to billions of dollars in avoidable costs in the United States annually. Our objective was to determine the influence of financial incentives, accompanied with information regarding test risk and benefit, on patient preference for diagnostic testing. METHODS: We conducted a cross-sectional survey of patients at the University of Michigan emergency department (ED). Each participant was presented with a hypothetical scenario involving an ED visit following minor traumatic brain injury. Participants were given information regarding potential benefit (detecting brain hemorrhage) and risk (developing cancer) of head computed tomography scan, as well as an incentive of $0 or $100 to forego testing. We used 0.1 and 1% for test benefit and risk, and values for risk, benefit, and financial incentive varied across participants. Our primary outcome was patient preference to undergo testing. We also collected demographic and numeracy information. We then used logistic regression to estimate odds ratios (ORs), which were adjusted for multiple potential confounders. Our sample size was designed to find at least 300 events (preference for testing) to allow for inclusion of up to 30 covariates in fully adjusted models. We had 85% to 90% power to detect a 10% absolute difference in testing rate across groups, assuming a 95% significance level. RESULTS: We surveyed 913 patients. Increasing test benefit from 0.1% to 1% significantly increased test acceptance (adjusted OR [AOR] = 1.6, 95% confidence interval [CI] = 1.2 to 2.1) and increasing test risk from 0.1% to 1% significantly decreased test acceptance (AOR = 0.70, 95% CI = 0.52 to 0.93). Finally, a $100 incentive to forego low-value testing significantly reduced test acceptance (AOR = 0.6; 95% CI = 0.4 to 0.8). CONCLUSIONS: Providing financial incentives to forego testing significantly decreased patient preference for testing, even when accounting for test benefit and risk. This work is preliminary and hypothetical and requires confirmation in larger patient cohorts facing these actual decisions.
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Toma de Decisiones , Motivación , Prioridad del Paciente , Tomografía Computarizada por Rayos X/economía , Adulto , Estudios Transversales , Servicio de Urgencia en Hospital/estadística & datos numéricos , Femenino , Humanos , Modelos Logísticos , Masculino , Persona de Mediana Edad , Encuestas y Cuestionarios , Tomografía Computarizada por Rayos X/efectos adversos , Estados UnidosRESUMEN
BACKGROUND: Diagnostic testing is common during emergency department (ED) visits. Little is understood about patient preferences for such testing. We hypothesized that a patient's willingness to undergo diagnostic testing is influenced by the potential benefit, risk, and personal cost. METHODS: We conducted a cross sectional survey among ED patients for diagnostic testing in two hypothetical scenarios: chest pain (CP) and mild traumatic brain injury (mTBI). Each scenario defined specific risks, benefits, and costs of testing. The odds of a participant desiring diagnostic testing were calculated using a series of nested multivariable logistic regression models. RESULTS: Participants opted for diagnostic testing 68.2% of the time, including 69.7% of CP and 66.7% of all mTBI scenarios. In the CP scenario, 81% of participants desired free testing versus 59% when it was associated with a $100 copay (difference = 22%, 95% confidence interval [CI] = 16% to 28%). Similarly, in the mTBI scenario, 73% of adult participants desired free testing versus 56% when charged a $100 copayment (difference = 17%, 95% CI = 11% to 24%). Benefit and risk had mixed effects across the scenarios. In fully adjusted models, the association between cost and desire for testing persisted in the CP (odds ratio [OR] = 0.33, 95% CI = 0.23 to 0.47) and adult mTBI (OR = 0.47, 95% CI = 0.33 to 0.67) scenarios. CONCLUSIONS: In this ED-based study, patient preferences for diagnostic testing differed significantly across levels of risk, benefit, and cost of diagnostic testing. Cost was the strongest and most consistent factor associated with decreased desire for testing.
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Pruebas Diagnósticas de Rutina/economía , Servicio de Urgencia en Hospital/economía , Prioridad del Paciente/economía , Adulto , Anciano , Lesiones Traumáticas del Encéfalo/diagnóstico , Dolor en el Pecho/diagnóstico , Estudios Transversales , Pruebas Diagnósticas de Rutina/psicología , Femenino , Humanos , Modelos Logísticos , Masculino , Persona de Mediana Edad , Oportunidad Relativa , Prioridad del Paciente/psicología , Encuestas y Cuestionarios , Adulto JovenRESUMEN
Recessive mutations in the SDCCAG8 gene cause a nephronophthisis-related ciliopathy with Bardet-Biedl syndrome-like features in humans. Our previous characterization of the orthologous Sdccag8gt/gt mouse model recapitulated the retinal-renal disease phenotypes and identified impaired DNA damage response signaling as an underlying disease mechanism in the kidney. However, several other phenotypic and mechanistic features of Sdccag8gt/gt mice remained unexplored. Here we show that Sdccag8gt/gt mice exhibit developmental and structural abnormalities of the skeleton and limbs, suggesting impaired Hedgehog (Hh) signaling. Indeed, cell culture studies demonstrate the requirement of SDCCAG8 for ciliogenesis and Hh signaling. Using an affinity proteomics approach, we demonstrate that SDCCAG8 interacts with proteins of the centriolar satellites (OFD1, AZI1), of the endosomal sorting complex (RABEP2, ERC1), and with non-muscle myosin motor proteins (MYH9, MYH10, MYH14) at the centrosome. Furthermore, we show that RABEP2 localization at the centrosome is regulated by SDCCAG8. siRNA mediated RABEP2 knockdown in hTERT-RPE1 cells leads to defective ciliogenesis, indicating a critical role for RABEP2 in this process. Together, this study identifies several centrosome-associated proteins as novel SDCCAG8 interaction partners, and provides new insights into the function of SDCCAG8 at this structure.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Autoantígenos/metabolismo , Centriolos/metabolismo , Proteínas Hedgehog/metabolismo , Proteínas de Neoplasias/metabolismo , Transducción de Señal , Proteínas de Transporte Vesicular/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Autoantígenos/genética , Centriolos/genética , Técnicas de Silenciamiento del Gen , Células HEK293 , Proteínas Hedgehog/genética , Humanos , Ratones , Ratones Transgénicos , Miosinas/genética , Miosinas/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Transporte Vesicular/genéticaRESUMEN
Steroid-resistant nephrotic syndrome (SRNS) causes 15% of chronic kidney disease (CKD). Here we show that recessive mutations in FAT1 cause a distinct renal disease entity in four families with a combination of SRNS, tubular ectasia, haematuria and facultative neurological involvement. Loss of FAT1 results in decreased cell adhesion and migration in fibroblasts and podocytes and the decreased migration is partially reversed by a RAC1/CDC42 activator. Podocyte-specific deletion of Fat1 in mice induces abnormal glomerular filtration barrier development, leading to podocyte foot process effacement. Knockdown of Fat1 in renal tubular cells reduces migration, decreases active RAC1 and CDC42, and induces defects in lumen formation. Knockdown of fat1 in zebrafish causes pronephric cysts, which is partially rescued by RAC1/CDC42 activators, confirming a role of the two small GTPases in the pathogenesis. These findings provide new insights into the pathogenesis of SRNS and tubulopathy, linking FAT1 and RAC1/CDC42 to podocyte and tubular cell function.
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Cadherinas/genética , Adhesión Celular/genética , Movimiento Celular/genética , Fibroblastos/metabolismo , Síndrome Nefrótico/congénito , Podocitos/metabolismo , Proteínas de Pez Cebra/genética , Animales , Dilatación Patológica/genética , Técnicas de Silenciamiento del Gen , Hematuria/genética , Humanos , Túbulos Renales/citología , Túbulos Renales/metabolismo , Túbulos Renales/patología , Lisencefalia/genética , Ratones , Mutación , Síndrome Nefrótico/genética , Síndrome , Pez Cebra , Proteína de Unión al GTP cdc42/metabolismo , Proteína de Unión al GTP rac1/metabolismoRESUMEN
The Meckel syndrome (MKS) complex functions at the transition zone, located between the basal body and axoneme, to regulate the localization of ciliary membrane proteins. We investigated the role of Tmem231, a two-pass transmembrane protein, in MKS complex formation and function. Consistent with a role in transition zone function, mutation of mouse Tmem231 disrupts the localization of proteins including Arl13b and Inpp5e to cilia, resulting in phenotypes characteristic of MKS such as polydactyly and kidney cysts. Tmem231 and B9d1 are essential for each other and other complex components such as Mks1 to localize to the transition zone. As in mouse, the Caenorhabditis elegans orthologue of Tmem231 localizes to and controls transition zone formation and function, suggesting an evolutionarily conserved role for Tmem231. We identified TMEM231 mutations in orofaciodigital syndrome type 3 (OFD3) and MKS patients that compromise transition zone function. Thus, Tmem231 is critical for organizing the MKS complex and controlling ciliary composition, defects in which cause OFD3 and MKS.