RESUMO
Motile and non-motile cilia play critical roles in mammalian development and health. These organelles are composed of a 1000 or more unique proteins, but their assembly depends entirely on proteins synthesized in the cell body and transported into the cilium by intraflagellar transport (IFT). In mammals, malfunction of non-motile cilia due to IFT dysfunction results in complex developmental phenotypes that affect most organs. In contrast, disruption of motile cilia function causes subfertility, disruption of the left-right body axis, and recurrent airway infections with progressive lung damage. In this work, we characterize allele specific phenotypes resulting from IFT74 dysfunction in human and mice. We identified two families carrying a deletion encompassing IFT74 exon 2, the first coding exon, resulting in a protein lacking the first 40 amino acids and two individuals carrying biallelic splice site mutations. Homozygous exon 2 deletion cases presented a ciliary chondrodysplasia with narrow thorax and progressive growth retardation along with a mucociliary clearance disorder phenotype with severely shorted cilia. Splice site variants resulted in a lethal skeletal chondrodysplasia phenotype. In mice, removal of the first 40 amino acids likewise results in a motile cilia phenotype but with little effect on primary cilia structure. Mice carrying this allele are born alive but are growth restricted and developed hydrocephaly in the first month of life. In contrast, a strong, likely null, allele of Ift74 in mouse completely blocks ciliary assembly and causes severe heart defects and midgestational lethality. In vitro studies suggest that the first 40 amino acids of IFT74 are dispensable for binding of other IFT subunits but are important for tubulin binding. Higher demands on tubulin transport in motile cilia compared to primary cilia resulting from increased mechanical stress and repair needs could account for the motile cilia phenotype observed in human and mice.
Assuntos
Cílios , Ciliopatias , Humanos , Animais , Camundongos , Cílios/genética , Cílios/metabolismo , Tubulina (Proteína)/metabolismo , Proteínas/genética , Aminoácidos/metabolismo , Mamíferos/metabolismo , Proteínas do Citoesqueleto/genéticaRESUMO
Bardet-Biedl syndrome (BBS), a ciliopathy, is a rare genetic condition characterised by retinal degeneration, obesity, kidney failure, and cognitive impairment. In spite of progress made in our general understanding of BBS aetiology, the molecular and cellular mechanisms underlying cognitive impairment in BBS remain elusive. Here, we report that the loss of BBS proteins causes synaptic dysfunction in principal neurons, providing a possible explanation for the cognitive impairment phenotype observed in BBS patients. Using synaptosomal proteomics and immunocytochemistry, we demonstrate the presence of Bbs proteins in the postsynaptic density (PSD) of hippocampal neurons. Loss of Bbs results in a significant reduction of dendritic spines in principal neurons of Bbs mouse models. Furthermore, we show that spine deficiency correlates with events that destabilise spine architecture, such as impaired spine membrane receptor signalling, known to be involved in the maintenance of dendritic spines. Our findings suggest a role for BBS proteins in dendritic spine homeostasis that may be linked to the cognitive phenotype observed in BBS.
Assuntos
Síndrome de Bardet-Biedl/patologia , Proteínas do Citoesqueleto/metabolismo , Espinhas Dendríticas/patologia , Animais , Ansiedade , Síndrome de Bardet-Biedl/metabolismo , Síndrome de Bardet-Biedl/fisiopatologia , Síndrome de Bardet-Biedl/psicologia , Giro Denteado/fisiopatologia , Modelos Animais de Doenças , Potenciais Pós-Sinápticos Excitadores , Feminino , Masculino , Memória , Camundongos , Receptor IGF Tipo 1/metabolismo , Sinaptossomos/metabolismoRESUMO
[This corrects the article DOI: 10.1371/journal.pbio.3000414.].
RESUMO
BACKGROUND: Development of therapeutic approaches for rare respiratory diseases is hampered by the lack of systems that allow medium-to-high-throughput screening of fully differentiated respiratory epithelium from affected patients. This is a particular problem for primary ciliary dyskinesia (PCD), a rare genetic disease caused by mutations in genes that adversely affect ciliary movement and consequently mucociliary transport. Primary cell culture of basal epithelial cells from nasal brush biopsies followed by ciliated differentiation at the air-liquid interface (ALI) has proven to be a useful tool in PCD diagnostics but the technique's broader utility, including in pre-clinical PCD research, has been restricted by the limited number of basal cells that can be expanded from such biopsies. METHODS: We describe an immunofluorescence screening method, enabled by extensive expansion of basal cells from PCD patients and the directed differentiation of these cells into ciliated epithelium in miniaturised 96-well transwell format ALI cultures. As proof-of-principle, we performed a personalised investigation in a patient with a rare and severe form of PCD (reduced generation of motile cilia), in this case caused by a homozygous nonsense mutation in the MCIDAS gene. RESULTS: Initial analyses of ciliary ultrastructure, beat pattern and beat frequency in the 96-well transwell format ALI cultures indicate that a range of different PCD defects can be retained in these cultures. The screening system in our proof-of-principal investigation allowed drugs that induce translational readthrough to be evaluated alone or in combination with nonsense-mediated decay inhibitors. We observed restoration of basal body formation but not the generation of cilia in the patient's nasal epithelial cells in vitro. CONCLUSION: Our study provides a platform for higher throughput analyses of airway epithelia that is applicable in a range of settings and suggests novel avenues for drug evaluation and development in PCD caused by nonsense mutations.
Assuntos
Transtornos da Motilidade Ciliar , Síndrome de Kartagener , Cílios , Transtornos da Motilidade Ciliar/diagnóstico , Transtornos da Motilidade Ciliar/tratamento farmacológico , Transtornos da Motilidade Ciliar/genética , Avaliação Pré-Clínica de Medicamentos , Ensaios de Triagem em Larga Escala , Humanos , Síndrome de Kartagener/diagnóstico , Síndrome de Kartagener/tratamento farmacológico , Síndrome de Kartagener/genética , Depuração MucociliarRESUMO
3MC syndrome is an autosomal recessive heterogeneous disorder with features linked to developmental abnormalities. The main features include facial dysmorphism, craniosynostosis and cleft lip/palate; skeletal structures derived from cranial neural crest cells (cNCC). We previously reported that lectin complement pathway genes COLEC11 and MASP1/3 are mutated in 3MC syndrome patients. Here we define a new gene, COLEC10, also mutated in 3MC families and present novel mutations in COLEC11 and MASP1/3 genes in a further five families. The protein products of COLEC11 and COLEC10, CL-K1 and CL-L1 respectively, form heteromeric complexes. We show COLEC10 is expressed in the base membrane of the palate during murine embryo development. We demonstrate how mutations in COLEC10 (c.25C>T; p.Arg9Ter, c.226delA; p.Gly77Glufs*66 and c.528C>G p.Cys176Trp) impair the expression and/or secretion of CL-L1 highlighting their pathogenicity. Together, these findings provide further evidence linking the lectin complement pathway and complement factors COLEC11 and COLEC10 to morphogenesis of craniofacial structures and 3MC etiology.
Assuntos
Anormalidades Múltiplas/genética , Fissura Palatina/genética , Colectinas/genética , Anormalidades Craniofaciais/genética , Craniossinostoses/genética , Mutação , Anormalidades Múltiplas/metabolismo , Anormalidades Múltiplas/patologia , Animais , Sequência de Bases , Western Blotting , Linhagem Celular , Fissura Palatina/metabolismo , Colectinas/metabolismo , Anormalidades Craniofaciais/metabolismo , Craniossinostoses/metabolismo , Exoma/genética , Saúde da Família , Feminino , Predisposição Genética para Doença/genética , Células HEK293 , Células HeLa , Humanos , Masculino , Camundongos , Análise de Sequência de DNA/métodos , SíndromeRESUMO
BACKGROUND: Rare genetic conditions are frequent risk factors for, or direct causes of, paediatric intensive care unit (PICU) admission. Such conditions are frequently suspected but unidentified at PICU admission. Compassionate and effective care is greatly assisted by definitive diagnostic information. There is therefore a need to provide a rapid genetic diagnosis to inform clinical management.To date, whole genome sequencing (WGS) approaches have proved successful in diagnosing a proportion of children with rare diseases, but results may take months to report. Our aim was to develop an end-to-end workflow for the use of rapid WGS for diagnosis in critically ill children in a UK National Health Service (NHS) diagnostic setting. METHODS: We sought to establish a multidisciplinary Rapid Paediatric Sequencing team for case selection, trio WGS, rapid bioinformatics sequence analysis and a phased analysis and reporting system to prioritise genes with a high likelihood of being causal. RESULTS: Trio WGS in 24 critically ill children led to a molecular diagnosis in 10 (42%) through the identification of causative genetic variants. In 3 of these 10 individuals (30%), the diagnostic result had an immediate impact on the individual's clinical management. For the last 14 trios, the shortest time taken to reach a provisional diagnosis was 4 days (median 8.5 days). CONCLUSION: Rapid WGS can be used to diagnose and inform management of critically ill children within the constraints of an NHS clinical diagnostic setting. We provide a robust workflow that will inform and facilitate the rollout of rapid genome sequencing in the NHS and other healthcare systems globally.
Assuntos
Estado Terminal , Doenças Genéticas Inatas/diagnóstico , Doenças Genéticas Inatas/genética , Sequenciamento Completo do Genoma , Criança , Gerenciamento Clínico , Estudo de Associação Genômica Ampla/métodos , Estudo de Associação Genômica Ampla/normas , Humanos , Unidades de Terapia Intensiva Pediátrica , Doenças Raras , Sequenciamento Completo do Genoma/métodos , Fluxo de TrabalhoRESUMO
We identified de novo nonsense variants in KIDINS220/ARMS in three unrelated patients with spastic paraplegia, intellectual disability, nystagmus, and obesity (SINO). KIDINS220 is an essential scaffold protein coordinating neurotrophin signal pathways in neurites and is spatially and temporally regulated in the brain. Molecular analysis of patients' variants confirmed expression and translation of truncated transcripts similar to recently characterized alternative terminal exon splice isoforms of KIDINS220 KIDINS220 undergoes extensive alternative splicing in specific neuronal populations and developmental time points, reflecting its complex role in neuronal maturation. In mice and humans, KIDINS220 is alternative spliced in the middle region as well as in the last exon. These full-length and KIDINS220 splice variants occur at precise moments in cortical, hippocampal, and motor neuron development, with splice variants similar to the variants seen in our patients and lacking the last exon of KIDINS220 occurring in adult rather than in embryonic brain. We conducted tissue-specific expression studies in zebrafish that resulted in spasms, confirming a functional link with disruption of the KIDINS220 levels in developing neurites. This work reveals a crucial physiological role of KIDINS220 in development and provides insight into how perturbation of the complex interplay of KIDINS220 isoforms and their relative expression can affect neuron control and human metabolism. Altogether, we here show that de novo protein-truncating KIDINS220 variants cause a new syndrome, SINO. This is the first report of KIDINS220 variants causing a human disease.
Assuntos
Deficiência Intelectual/genética , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , Nistagmo Congênito/genética , Obesidade/genética , Paraplegia/genética , Proteínas de Peixe-Zebra/genética , Processamento Alternativo/genética , Animais , Códon sem Sentido , Modelos Animais de Doenças , Humanos , Deficiência Intelectual/fisiopatologia , Neuritos/metabolismo , Neuritos/patologia , Neurogênese/genética , Neurônios/metabolismo , Neurônios/patologia , Nistagmo Congênito/fisiopatologia , Obesidade/patologia , Células PC12 , Paraplegia/fisiopatologia , Ligação Proteica/genética , Ratos , Transdução de SinaisRESUMO
BACKGROUND: Nephronophthisis is an autosomal recessive ciliopathy and important cause of end-stage renal disease (ESRD) in children and young adults. Diagnostic delay is frequent. This study investigates clinical characteristics, initial symptoms, and genetic defects in a cohort with nephronophthisis-related ciliopathy, to improve early detection and genetic counseling. METHODS: Forty patients from 36 families with nephronophthisis-related ciliopathy were recruited at university medical centers and online. Comprehensive clinical and genotypic data were recorded. Patients without molecular diagnosis were offered genetic analysis. RESULTS: Of 40 patients, 45% had isolated nephronophthisis, 48% syndromic diagnosis, and 7% nephronophthisis with extrarenal features not constituting a recognizable syndrome. Patients developed ESRD at median 13 years (range 5-47). Median age of symptom onset was 9 years in both isolated and syndromic forms (range 5-26 vs. 5-33). Common presenting symptoms were fatigue (42%), polydipsia/polyuria (33%), and hypertension (21%). Renal ultrasound showed small-to-normal-sized kidneys, increased echogenicity (65%), cysts (43%), and abnormal corticomedullary differentiation (32%). Renal biopsies in eight patients showed nonspecific signs of chronic kidney disease (CKD). Twenty-three patients (58%) had genetic diagnosis upon inclusion. Thirteen of those without a genetic diagnosis gave consent for genetic testing, and a cause was identified in five (38%). CONCLUSIONS: Nephronophthisis is genetically and phenotypically heterogeneous and should be considered in children and young adults presenting with persistent fatigue and polyuria, and in all patients with unexplained CKD. As symptom onset can occur into adulthood, presymptomatic monitoring of kidney function in syndromic ciliopathy patients should continue until at least age 30.
Assuntos
Ciliopatias/diagnóstico , Aconselhamento Genético , Testes Genéticos , Doenças Renais Císticas/congênito , Falência Renal Crônica/prevenção & controle , Proteínas Adaptadoras de Transdução de Sinal/genética , Adolescente , Adulto , Idade de Início , Biópsia , Criança , Ciliopatias/complicações , Ciliopatias/genética , Ciliopatias/patologia , Proteínas do Citoesqueleto , Diagnóstico Tardio/prevenção & controle , Feminino , Humanos , Rim/diagnóstico por imagem , Rim/patologia , Doenças Renais Císticas/complicações , Doenças Renais Císticas/diagnóstico , Doenças Renais Císticas/genética , Doenças Renais Císticas/patologia , Falência Renal Crônica/etiologia , Masculino , Proteínas de Membrana/genética , Pessoa de Meia-Idade , Países Baixos , Sistema de Registros/estatística & dados numéricos , Fatores de Tempo , Ultrassonografia , Sequenciamento do Exoma , Adulto JovemRESUMO
Bardet-Biedl syndrome is a rare autosomal recessive, multisystem disease characterized by retinal dystrophy, renal malformation, obesity, intellectual disability, polydactyly, and hypogonadism. Nineteen disease-causing genes (BBS1-19) have been identified, of which mutations in BBS1 are most common in North America and Europe. A hallmark of the disease, renal malformation is heterogeneous and is a cause of morbidity and mortality through the development of CKD. We studied the prevalence and severity of CKD in 350 patients with Bardet-Biedl syndrome-related renal disease attending the United Kingdom national Bardet-Biedl syndrome clinics to further elucidate the phenotype and identify risk indicators of CKD. Overall, 31% of children and 42% of adults had CKD; 6% of children and 8% of adults had stage 4-5 CKD. In children, renal disease was often detected within the first year of life. Analysis of the most commonly mutated disease-associated genes revealed that, compared with two truncating mutations, two missense mutations associated with less severe CKD in adults. Moreover, compared with mutations in BBS10, mutations in BBS1 associated with less severe CKD or lack of CKD in adults. Finally, 51% of patients with available ultrasounds had structural renal abnormalities, and 35% of adults were hypertensive. The presence of structural abnormalities or antihypertensive medication also correlated statistically with stage 3b-5 CKD. This study describes the largest reported cohort of patients with renal disease in Bardet-Biedl syndrome and identifies risk factors to be considered in genetic counseling.
Assuntos
Síndrome de Bardet-Biedl/complicações , Insuficiência Renal Crônica/epidemiologia , Insuficiência Renal Crônica/etiologia , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Mutação , Prevalência , Insuficiência Renal Crônica/genética , Estudos Retrospectivos , Fatores de Risco , Índice de Gravidade de Doença , Adulto JovemRESUMO
Jeune asphyxiating thoracic dystrophy (JATD) is a skeletal dysplasia characterized by a small thoracic cage and a range of skeletal and extra-skeletal anomalies. JATD is genetically heterogeneous with at least nine genes identified, all encoding ciliary proteins, hence the classification of JATD as a skeletal ciliopathy. Consistent with the observation that the heterogeneous molecular basis of JATD has not been fully determined yet, we have identified two consanguineous Saudi families segregating JATD who share a single identical ancestral homozygous haplotype among the affected members. Whole-exome sequencing revealed a single novel variant within the disease haplotype in CEP120, which encodes a core centriolar protein. Subsequent targeted sequencing of CEP120 in Saudi and European JATD cohorts identified two additional families with the same missense mutation. Combining the four families in linkage analysis confirmed a significant genome-wide linkage signal at the CEP120 locus. This missense change alters a highly conserved amino acid within CEP120 (p.Ala199Pro). In addition, we show marked reduction of cilia and abnormal number of centrioles in fibroblasts from one affected individual. Inhibition of the CEP120 ortholog in zebrafish produced pleiotropic phenotypes characteristic of cilia defects including abnormal body curvature, hydrocephalus, otolith defects and abnormal renal, head and craniofacial development. We also demonstrate that in CEP120 morphants, cilia are shortened in the neural tube and disorganized in the pronephros. These results are consistent with aberrant CEP120 being implicated in the pathogenesis of JATD and expand the role of centriolar proteins in skeletal ciliopathies.
Assuntos
Osso e Ossos/anormalidades , Proteínas de Ciclo Celular/genética , Centríolos/genética , Síndrome de Ellis-Van Creveld/genética , Mutação de Sentido Incorreto , Sequência de Aminoácidos , Animais , Osso e Ossos/metabolismo , Proteínas de Ciclo Celular/metabolismo , Centríolos/metabolismo , Mapeamento Cromossômico , Cromossomos Humanos Par 5/genética , Cromossomos Humanos Par 5/metabolismo , Cílios/patologia , Estudos de Coortes , Modelos Animais de Doenças , Europa (Continente) , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Loci Gênicos , Estudo de Associação Genômica Ampla , Humanos , Lactente , Imageamento por Ressonância Magnética , Masculino , Dados de Sequência Molecular , Linhagem , Fenótipo , Arábia Saudita , Peixe-ZebraRESUMO
Bardet-Biedl syndrome (BBS) and autosomal dominant polycystic kidney disease (ADPKD) are two genetically distinct ciliopathies but share common phenotypes such as renal cysts. Seven BBS proteins form a complex called the BBSome which is localized at the basal body or ciliary axoneme and regulates the ciliary entry or flagellar exit of several signaling molecules. Here, we demonstrate that, unlike the seven-span somatostatin receptor 3 or the leptin receptor that interacts with all subunits of the BBSome, the ADPKD protein polycystin-1 (PC1) interacts with BBS1, BBS4, BBS5 and BBS8, four of the seven components of the BBSome. Only depletion or mutation of BBS1, but not depletion of BBS5 and BBS8, or knockout of BBS4, impairs ciliary trafficking of PC1 in kidney epithelial cells. Depletion of these BBS proteins affects neither the ciliary length nor the plasma membrane targeting of PC1. Expression of a pathogenic BBS3/Arl6 mutant (T31R) that locks Arl6 in the GDP form leads to stunted cilia and inhibition of PC1 on primary cilia. We propose that the 11-span membrane protein PC1 is a BBSome cargo and that the components of the BBSome may possess subunit-specific functions. Moreover, physical interactions between the BBS and ADPKD proteins may underline the overlapping renal phenotypes in these two diseases.
Assuntos
Fatores de Ribosilação do ADP/metabolismo , Rim/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Canais de Cátion TRPP/metabolismo , Fatores de Ribosilação do ADP/genética , Animais , Linhagem Celular , Cílios/metabolismo , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Transporte Proteico , Canais de Cátion TRPP/genéticaRESUMO
Ocular coloboma is a congenital defect resulting from failure of normal closure of the optic fissure during embryonic eye development. This birth defect causes childhood blindness worldwide, yet the genetic etiology is poorly understood. Here, we identified a novel homozygous mutation in the SALL2 gene in members of a consanguineous family affected with non-syndromic ocular coloboma variably affecting the iris and retina. This mutation, c.85G>T, introduces a premature termination codon (p.Glu29*) predicted to truncate the SALL2 protein so that it lacks three clusters of zinc-finger motifs that are essential for DNA-binding activity. This discovery identifies SALL2 as the third member of the Drosophila homeotic Spalt-like family of developmental transcription factor genes implicated in human disease. SALL2 is expressed in the developing human retina at the time of, and subsequent to, optic fissure closure. Analysis of Sall2-deficient mouse embryos revealed delayed apposition of the optic fissure margins and the persistence of an anterior retinal coloboma phenotype after birth. Sall2-deficient embryos displayed correct posterior closure toward the optic nerve head, and upon contact of the fissure margins, dissolution of the basal lamina occurred and PAX2, known to be critical for this process, was expressed normally. Anterior closure was disrupted with the fissure margins failing to meet, or in some cases misaligning leading to a retinal lesion. These observations demonstrate, for the first time, a role for SALL2 in eye morphogenesis and that loss of function of the gene causes ocular coloboma in humans and mice.
Assuntos
Códon sem Sentido , Coloboma/genética , Fatores de Transcrição/genética , Adolescente , Animais , Criança , Consanguinidade , Análise Mutacional de DNA , Proteínas de Ligação a DNA , Olho/embriologia , Olho/patologia , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Feminino , Expressão Gênica , Genes Recessivos , Estudos de Associação Genética , Predisposição Genética para Doença , Células HEK293 , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Homozigoto , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator de Transcrição PAX2/genética , Fator de Transcrição PAX2/metabolismo , Fator de Transcrição PAX6 , Fatores de Transcrição Box Pareados/genética , Fatores de Transcrição Box Pareados/metabolismo , Linhagem , Polimorfismo de Nucleotídeo Único , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Primary ciliary dyskinesia (PCD) is an inherited chronic respiratory obstructive disease with randomized body laterality and infertility, resulting from cilia and sperm dysmotility. PCD is characterized by clinical variability and extensive genetic heterogeneity, associated with different cilia ultrastructural defects and mutations identified in >20 genes. Next generation sequencing (NGS) technologies therefore present a promising approach for genetic diagnosis which is not yet in routine use. We developed a targeted panel-based NGS pipeline to identify mutations by sequencing of selected candidate genes in 70 genetically undefined PCD patients. This detected loss-of-function RSPH1 mutations in four individuals with isolated central pair (CP) agenesis and normal body laterality, from two unrelated families. Ultrastructural analysis in RSPH1-mutated cilia revealed transposition of peripheral outer microtubules into the 'empty' CP space, accompanied by a distinctive intermittent loss of the central pair microtubules. We find that mutations in RSPH1, RSPH4A and RSPH9, which all encode homologs of components of the 'head' structure of ciliary radial spoke complexes identified in Chlamydomonas, cause clinical phenotypes that appear to be indistinguishable except at the gene level. By high-resolution immunofluorescence we identified a loss of RSPH4A and RSPH9 along with RSPH1 from RSPH1-mutated cilia, suggesting RSPH1 mutations may result in loss of the entire spoke head structure. CP loss is seen in up to 28% of PCD cases, in whom laterality determination specified by CP-less embryonic node cilia remains undisturbed. We propose this defect could arise from instability or agenesis of the ciliary central microtubules due to loss of their normal radial spoke head tethering.
Assuntos
Proteínas de Ligação a DNA/genética , Síndrome de Kartagener/genética , Axonema/metabolismo , Axonema/fisiologia , Proteínas do Citoesqueleto/genética , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Síndrome de Kartagener/fisiopatologia , Microscopia Eletrônica , Microscopia de Fluorescência , Mutação , Proteínas/genéticaRESUMO
Short-rib polydactyly syndromes (SRPS I-V) are a group of lethal congenital disorders characterized by shortening of the ribs and long bones, polydactyly, and a range of extraskeletal phenotypes. A number of other disorders in this grouping, including Jeune and Ellis-van Creveld syndromes, have an overlapping but generally milder phenotype. Collectively, these short-rib dysplasias (with or without polydactyly) share a common underlying defect in primary cilium function and form a subset of the ciliopathy disease spectrum. By using whole-exome capture and massive parallel sequencing of DNA from an affected Australian individual with SRPS type III, we detected two novel heterozygous mutations in WDR60, a relatively uncharacterized gene. These mutations segregated appropriately in the unaffected parents and another affected family member, confirming compound heterozygosity, and both were predicted to have a damaging effect on the protein. Analysis of an additional 54 skeletal ciliopathy exomes identified compound heterozygous mutations in WDR60 in a Spanish individual with Jeune syndrome of relatively mild presentation. Of note, these two families share one novel WDR60 missense mutation, although haplotype analysis suggested no shared ancestry. We further show that WDR60 localizes at the base of the primary cilium in wild-type human chondrocytes, and analysis of fibroblasts from affected individuals revealed a defect in ciliogenesis and aberrant accumulation of the GLI2 transcription factor at the centrosome or basal body in the absence of an obvious axoneme. These findings show that WDR60 mutations can cause skeletal ciliopathies and suggest a role for WDR60 in ciliogenesis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Síndrome de Ellis-Van Creveld/genética , Mutação/genética , Síndrome de Costela Curta e Polidactilia/genética , Proteínas Adaptadoras de Transdução de Sinal/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Pré-Escolar , Condrócitos/metabolismo , Condrócitos/patologia , Segregação de Cromossomos/genética , Cílios/metabolismo , Síndrome de Ellis-Van Creveld/diagnóstico por imagem , Evolução Fatal , Feminino , Feto/diagnóstico por imagem , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Lactente , Recém-Nascido , Masculino , Camundongos , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/genética , Linhagem , Gravidez , Radiografia , Síndrome de Costela Curta e Polidactilia/diagnóstico por imagemRESUMO
Bidirectional (anterograde and retrograde) motor-based intraflagellar transport (IFT) governs cargo transport and delivery processes that are essential for primary cilia growth and maintenance and for hedgehog signaling functions. The IFT dynein-2 motor complex that regulates ciliary retrograde protein transport contains a heavy chain dynein ATPase/motor subunit, DYNC2H1, along with other less well functionally defined subunits. Deficiency of IFT proteins, including DYNC2H1, underlies a spectrum of skeletal ciliopathies. Here, by using exome sequencing and a targeted next-generation sequencing panel, we identified a total of 11 mutations in WDR34 in 9 families with the clinical diagnosis of Jeune syndrome (asphyxiating thoracic dystrophy). WDR34 encodes a WD40 repeat-containing protein orthologous to Chlamydomonas FAP133, a dynein intermediate chain associated with the retrograde intraflagellar transport motor. Three-dimensional protein modeling suggests that the identified mutations all affect residues critical for WDR34 protein-protein interactions. We find that WDR34 concentrates around the centrioles and basal bodies in mammalian cells, also showing axonemal staining. WDR34 coimmunoprecipitates with the dynein-1 light chain DYNLL1 in vitro, and mining of proteomics data suggests that WDR34 could represent a previously unrecognized link between the cytoplasmic dynein-1 and IFT dynein-2 motors. Together, these data show that WDR34 is critical for ciliary functions essential to normal development and survival, most probably as a previously unrecognized component of the mammalian dynein-IFT machinery.
Assuntos
Proteínas de Transporte/genética , Dineínas do Citoplasma/genética , Síndrome de Ellis-Van Creveld/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Animais , Povo Asiático/genética , Axonema/genética , Criança , Chlamydomonas/genética , Cílios/genética , Cílios/metabolismo , Citoesqueleto/genética , Citoesqueleto/metabolismo , Síndrome de Ellis-Van Creveld/patologia , Exoma , Éxons , Humanos , Lactente , Recém-Nascido , Mutação , Conformação Proteica , Proteômica , População Branca/genéticaRESUMO
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.
Assuntos
Ataxia Cerebelar/genética , Síndrome de Ellis-Van Creveld/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Retinose Pigmentar/genética , Alelos , Sequência de Aminoácidos , Animais , Povo Asiático/genética , Osso e Ossos/anormalidades , Osso e Ossos/metabolismo , Osso e Ossos/patologia , Ataxia Cerebelar/patologia , Craniossinostoses/genética , Craniossinostoses/patologia , Dineínas do Citoplasma/genética , Dineínas do Citoplasma/metabolismo , Dineínas/genética , Dineínas/metabolismo , Displasia Ectodérmica/genética , Displasia Ectodérmica/patologia , Síndrome de Ellis-Van Creveld/patologia , Epistasia Genética , Feminino , Fibroblastos/patologia , Técnicas de Silenciamento de Genes , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Doenças Renais Císticas/genética , Doenças Renais Císticas/patologia , Masculino , Dados de Sequência Molecular , Mutação , Fenótipo , Retinose Pigmentar/patologia , População Branca/genética , Peixe-Zebra/genéticaRESUMO
BACKGROUND: Mutations in microtubule-regulating genes are associated with disorders of neuronal migration and microcephaly. Regulation of centriole length has been shown to underlie the pathogenesis of certain ciliopathy phenotypes. Using a next-generation sequencing approach, we identified mutations in a novel centriolar disease gene in a kindred with an embryonic lethal ciliopathy phenotype and in a patient with primary microcephaly. METHODS AND RESULTS: Whole exome sequencing data from a non-consanguineous Caucasian kindred exhibiting mid-gestation lethality and ciliopathic malformations revealed two novel non-synonymous variants in CENPF, a microtubule-regulating gene. All four affected fetuses showed segregation for two mutated alleles [IVS5-2A>C, predicted to abolish the consensus splice-acceptor site from exon 6; c.1744G>T, p.E582X]. In a second unrelated patient exhibiting microcephaly, we identified two CENPF mutations [c.1744G>T, p.E582X; c.8692 C>T, p.R2898X] by whole exome sequencing. We found that CENP-F colocalised with Ninein at the subdistal appendages of the mother centriole in mouse inner medullary collecting duct cells. Intraflagellar transport protein-88 (IFT-88) colocalised with CENP-F along the ciliary axonemes of renal epithelial cells in age-matched control human fetuses but did not in truncated cilia of mutant CENPF kidneys. Pairwise co-immunoprecipitation assays of mitotic and serum-starved HEKT293 cells confirmed that IFT88 precipitates with endogenous CENP-F. CONCLUSIONS: Our data identify CENPF as a new centriolar disease gene implicated in severe human ciliopathy and microcephaly related phenotypes. CENP-F has a novel putative function in ciliogenesis and cortical neurogenesis.
Assuntos
Proteínas Cromossômicas não Histona/genética , Cílios/genética , Genética Médica , Microcefalia/genética , Proteínas dos Microfilamentos/genética , Animais , Centríolos/genética , Cílios/patologia , Exoma/genética , Feminino , Feto , Células HEK293 , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Camundongos , Microcefalia/patologia , Mutação , Células NIH 3T3 , Linhagem , Gravidez , Peixe-ZebraRESUMO
Jeune asphyxiating thoracic dystrophy, an autosomal recessive chondrodysplasia, often leads to death in infancy because of a severely constricted thoracic cage and respiratory insufficiency; retinal degeneration, cystic renal disease and polydactyly may be complicating features. We show that IFT80 mutations underlie a subset of Jeune asphyxiating thoracic dystrophy cases, establishing the first association of a defective intraflagellar transport (IFT) protein with human disease. Knockdown of ift80 in zebrafish resulted in cystic kidneys, and knockdown in Tetrahymena thermophila produced shortened or absent cilia.
Assuntos
Asfixia/genética , Doenças do Desenvolvimento Ósseo/genética , Proteínas de Transporte/genética , Doenças Renais Císticas/genética , Mutação/genética , Tetrahymena thermophila/genética , Doenças Torácicas/genética , Peixe-Zebra/genética , Animais , Feminino , Humanos , Recém-Nascido , Masculino , Linhagem , Polidactilia/genética , Tetrahymena thermophila/crescimento & desenvolvimento , Peixe-Zebra/crescimento & desenvolvimentoRESUMO
Primary cilia are cellular appendages important for signal transduction and sensing the environment. Bardet-Biedl syndrome proteins form a complex that is important for several cytoskeleton-related processes such as ciliogenesis, cell migration and division. However, the mechanisms by which BBS proteins may regulate the cytoskeleton remain unclear. We discovered that Bbs4- and Bbs6-deficient renal medullary cells display a characteristic behaviour comprising poor migration, adhesion and division with an inability to form lamellipodial and filopodial extensions. Moreover, fewer mutant cells were ciliated [48% ± 6 for wild-type (WT) cells versus 23% ± 7 for Bbs4 null cells; P < 0.0001] and their cilia were shorter (2.55 µm ± 0.41 for WT cells versus 2.16 µm ± 0.23 for Bbs4 null cells; P < 0.0001). While the microtubular cytoskeleton and cortical actin were intact, actin stress fibre formation was severely disrupted, forming abnormal apical stress fibre aggregates. Furthermore, we observed over-abundant focal adhesions (FAs) in Bbs4-, Bbs6- and Bbs8-deficient cells. In view of these findings and the role of RhoA in regulation of actin filament polymerization, we showed that RhoA-GTP levels were highly upregulated in the absence of Bbs proteins. Upon treatment of Bbs4-deficient cells with chemical inhibitors of RhoA, we were able to restore the cilia length and number as well as the integrity of the actin cytoskeleton. Together these findings indicate that Bbs proteins play a central role in the regulation of the actin cytoskeleton and control the cilia length through alteration of RhoA levels.
Assuntos
Actinas/metabolismo , Síndrome de Bardet-Biedl/metabolismo , Cílios/metabolismo , Cílios/ultraestrutura , Chaperoninas do Grupo II/genética , Proteínas/genética , Proteínas de Peixe-Zebra/metabolismo , Citoesqueleto de Actina/química , Citoesqueleto de Actina/metabolismo , Actinas/química , Animais , Síndrome de Bardet-Biedl/genética , Células Cultivadas , Proteínas do Citoesqueleto , Células Epiteliais/metabolismo , Adesões Focais/metabolismo , Chaperoninas do Grupo II/metabolismo , Humanos , Medula Renal/citologia , Medula Renal/metabolismo , Camundongos , Proteínas Associadas aos Microtúbulos , Células NIH 3T3 , Fenótipo , Polimerização , Multimerização Proteica , Proteínas/metabolismo , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Proteína rhoA de Ligação ao GTP/antagonistas & inibidores , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
CCDC28B encodes a coiled coil domain-containing protein involved in ciliogenesis that was originally identified as a second site modifier of the ciliopathy Bardet-Biedl syndrome. We have previously shown that the depletion of CCDC28B leads to shortened cilia; however, the mechanism underlying how this protein controls ciliary length is unknown. Here, we show that CCDC28B interacts with SIN1, a component of the mTOR complex 2 (mTORC2), and that this interaction is important both in the context of mTOR signaling and in a hitherto unknown, mTORC-independent role of SIN1 in cilia biology. We show that CCDC28B is a positive regulator of mTORC2, participating in its assembly/stability and modulating its activity, while not affecting mTORC1 function. Further, we show that Ccdc28b regulates cilia length in vivo, at least in part, through its interaction with Sin1. Importantly, depletion of Rictor, another core component of mTORC2, does not result in shortened cilia. Taken together, our findings implicate CCDC28B in the regulation of mTORC2, and uncover a novel function of SIN1 regulating cilia length that is likely independent of mTOR signaling.