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Rationale: Bronchiectasis is a pathological dilatation of the bronchi in the respiratory airways associated with environmental or genetic causes (e.g., cystic fibrosis, primary ciliary dyskinesia, and primary immunodeficiency disorders), but most cases remain idiopathic. Objectives: To identify novel genetic defects in unsolved cases of bronchiectasis presenting with severe rhinosinusitis, nasal polyposis, and pulmonary Pseudomonas aeruginosa infection. Methods: DNA was analyzed by next-generation or targeted Sanger sequencing. RNA was analyzed by quantitative PCR and single-cell RNA sequencing. Patient-derived cells, cell cultures, and secretions (mucus, saliva, seminal fluid) were analyzed by Western blotting and immunofluorescence microscopy, and mucociliary activity was measured. Blood serum was analyzed by electrochemiluminescence immunoassay. Protein structure and proteomic analyses were used to assess the impact of a disease-causing founder variant. Measurements and Main Results: We identified biallelic pathogenic variants in WAP four-disulfide core domain 2 (WFDC2) in 11 individuals from 10 unrelated families originating from the United States, Europe, Asia, and Africa. Expression of WFDC2 was detected predominantly in secretory cells of control airway epithelium and also in submucosal glands. We demonstrate that WFDC2 is below the limit of detection in blood serum and hardly detectable in samples of saliva, seminal fluid, and airway surface liquid from WFDC2-deficient individuals. Computer simulations and deglycosylation assays indicate that the disease-causing founder variant p.Cys49Arg structurally hampers glycosylation and, thus, secretion of mature WFDC2. Conclusions: WFDC2 dysfunction defines a novel molecular etiology of bronchiectasis characterized by the deficiency of a secreted component of the airways. A commercially available blood test combined with genetic testing allows its diagnosis.
Assuntos
Bronquiectasia , Pólipos Nasais , Adolescente , Adulto , Criança , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto Jovem , Bronquiectasia/genética , Bronquiectasia/fisiopatologia , Pólipos Nasais/genética , Proteína 2 do Domínio Central WAP de Quatro DissulfetosRESUMO
BACKGROUND: Primary ciliary dyskinesia (PCD) is a genetic disorder caused by aberrant motile cilia function that results in defective ciliary airway clearance and subsequently to recurrent airway infections and bronchiectasis. QUESTION: How many functional multiciliated airway cells are sufficient to maintain ciliary airway clearance? METHODS: To answer this question we exploited the molecular defects of the X-linked recessive PCD variant caused by pathogenic variants in DNAAF6 (PIH1D3), characterized by immotile cilia in the affected males. We carefully analyzed the clinical phenotype, molecular defect (immunofluorescence and transmission-electron microscopy) and performed in vitro (particle tracking in air-liquid interface cultures) and in vivo (radiolabeled tracer studies) studies to assess ciliary clearance of respiratory cells from females with heterozygous and males with hemizygous pathogenic DNAAF6 variants. RESULTS: PCD males with hemizygous pathogenic DNAAF6 variants displayed exclusively immotile cilia, absence of ciliary clearance and severe PCD symptoms. Due to random or skewed X-chromosome inactivation in six females with heterozygous pathogenic DNAAF6 variants, 54.3%±10 (range 38%-70%) of multiciliated cells were defective. Nevertheless, in vitro and in vivo assessment of the ciliary airway clearance was normal or slightly abnormal. Consistently, heterozygous female individuals showed no or only mild respiratory symptoms. CONCLUSIONS: Our findings indicate that 30%-62% of functioning multiciliated respiratory cells are able to generate either normal or slightly reduced ciliary clearance. Because heterozygous females displayed either no or subtle respiratory symptoms, complete correction of 30% of cells by precision medicine might be able to improve ciliary airway clearance in PCD individuals as well as clinical symptoms.
RESUMO
BACKGROUND: Primary ciliary dyskinesia (PCD) represents a group of rare hereditary disorders characterised by deficient ciliary airway clearance that can be associated with laterality defects. We aimed to describe the underlying gene defects, geographical differences in genotypes and their relationship to diagnostic findings and clinical phenotypes. METHODS: Genetic variants and clinical findings (age, sex, body mass index, laterality defects, forced expiratory volume in 1â s (FEV1)) were collected from 19 countries using the European Reference Network's ERN-LUNG international PCD Registry. Genetic data were evaluated according to American College of Medical Genetics and Genomics guidelines. We assessed regional distribution of implicated genes and genetic variants as well as genotype correlations with laterality defects and FEV1. RESULTS: The study included 1236 individuals carrying 908 distinct pathogenic DNA variants in 46 PCD genes. We found considerable variation in the distribution of PCD genotypes across countries due to the presence of distinct founder variants. The prevalence of PCD genotypes associated with pathognomonic ultrastructural defects (mean 72%, range 47-100%) and laterality defects (mean 42%, range 28-69%) varied widely among countries. The prevalence of laterality defects was significantly lower in PCD individuals without pathognomonic ciliary ultrastructure defects (18%). The PCD cohort had a reduced median FEV1 z-score (-1.66). Median FEV1 z-scores were significantly lower in CCNO (-3.26), CCDC39 (-2.49) and CCDC40 (-2.96) variant groups, while the FEV1 z-score reductions were significantly milder in DNAH11 (-0.83) and ODAD1 (-0.85) variant groups compared to the whole PCD cohort. CONCLUSION: This unprecedented multinational dataset of DNA variants and information on their distribution across countries facilitates interpretation of the genetic epidemiology of PCD and indicates that the genetic variant can predict diagnostic and phenotypic features such as the course of lung function.
Assuntos
Estudos de Associação Genética , Genótipo , Fenótipo , Humanos , Masculino , Feminino , Adulto , Criança , Adolescente , Adulto Jovem , Pessoa de Meia-Idade , Europa (Continente) , Sistema de Registros , Dineínas do Axonema/genética , Volume Expiratório Forçado , Pré-Escolar , Síndrome de Kartagener/genética , Síndrome de Kartagener/fisiopatologia , Variação Genética , Mutação , Idoso , Lactente , Proteínas do Citoesqueleto , ProteínasRESUMO
Axonemal protein complexes, such as outer (ODA) and inner (IDA) dynein arms, are responsible for the generation and regulation of flagellar and ciliary beating. Studies in various ciliated model organisms have shown that axonemal dynein arms are first assembled in the cell cytoplasm and then delivered into axonemes during ciliogenesis. In humans, mutations in genes encoding for factors involved in this process cause structural and functional defects of motile cilia in various organs such as the airways and result in the hereditary disorder primary ciliary dyskinesia (PCD). Despite extensive knowledge about the cytoplasmic assembly of axonemal dynein arms in respiratory cilia, this process is still poorly understood in sperm flagella. To better define its clinical relevance on sperm structure and function, and thus male fertility, further investigations are required. Here we report the fertility status in different axonemal dynein preassembly mutant males (DNAAF2/ KTU, DNAAF4/ DYX1C1, DNAAF6/ PIH1D3, DNAAF7/ZMYND10, CFAP300/C11orf70 and LRRC6). Besides andrological examinations, we functionally and structurally analyzed sperm flagella of affected individuals by high-speed video- and transmission electron microscopy as well as systematically compared the composition of dynein arms in sperm flagella and respiratory cilia by immunofluorescence microscopy. Furthermore, we analyzed the flagellar length in dynein preassembly mutant sperm. We found that the process of axonemal dynein preassembly is also critical in sperm, by identifying defects of ODAs and IDAs in dysmotile sperm of these individuals. Interestingly, these mutant sperm consistently show a complete loss of ODAs, while some respiratory cilia from the same individual can retain ODAs in the proximal ciliary compartment. This agrees with reports of solely one distinct ODA type in sperm, compared to two different ODA types in proximal and distal respiratory ciliary axonemes. Consistent with observations in model organisms, we also determined a significant reduction of sperm flagellar length in these individuals. These findings are relevant to subsequent studies on the function and composition of sperm flagella in PCD patients and non-syndromic infertile males. Our study contributes to a better understanding of the fertility status in PCD-affected males and should help guide genetic and andrological counselling for affected males and their families.
Assuntos
Dineínas do Axonema/metabolismo , Axonema/metabolismo , Cílios/metabolismo , Flagelos/metabolismo , Infertilidade Masculina/metabolismo , Espermatozoides/metabolismo , Dineínas do Axonema/genética , Dineínas do Axonema/ultraestrutura , Axonema/genética , Axonema/ultraestrutura , Cílios/genética , Estudos de Coortes , Citoplasma/metabolismo , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Flagelos/genética , Flagelos/ultraestrutura , Humanos , Infertilidade Masculina/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Microscopia Eletrônica de Transmissão , Mutação , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Espermatozoides/ultraestruturaRESUMO
PURPOSE: Primary ciliary dyskinesia (PCD) is a heterogeneous disorder that includes respiratory symptoms, laterality defects, and infertility caused by dysfunction of motile cilia. Most PCD-causing variants result in abnormal outer dynein arms (ODAs), which provide the generative force for respiratory ciliary beating and proper mucociliary clearance. METHODS: In addition to studies in mouse and planaria, clinical exome sequencing and functional analyses in human were performed. RESULTS: In this study, we identified homozygous pathogenic variants in CLXN (EFCAB1/ODAD5) in 3 individuals with laterality defects and respiratory symptoms. Consistently, we found that Clxn is expressed in mice left-right organizer. Transmission electron microscopy depicted ODA defects in distal ciliary axonemes. Immunofluorescence microscopy revealed absence of CLXN from the ciliary axonemes, absence of the ODA components DNAH5, DNAI1, and DNAI2 from the distal axonemes, and mislocalization or absence of DNAH9. In addition, CLXN was undetectable in ciliary axonemes of individuals with defects in the ODA-docking machinery: ODAD1, ODAD2, ODAD3, and ODAD4. Furthermore, SMED-EFCAB1-deficient planaria displayed ciliary dysmotility. CONCLUSION: Our results revealed that pathogenic variants in CLXN cause PCD with defects in the assembly of distal ODAs in the respiratory cilia. CLXN should be referred to as ODA-docking complex-associated protein ODAD5.
Assuntos
Cílios , Síndrome de Kartagener , Humanos , Animais , Camundongos , Cílios/genética , Síndrome de Kartagener/genética , Síndrome de Kartagener/metabolismo , Síndrome de Kartagener/patologia , Proteínas de Ligação ao Cálcio , Axonema/genética , Axonema/metabolismo , Axonema/patologia , Mutação , Dineínas do Axonema/genética , Dineínas do Axonema/metabolismoRESUMO
Primary ciliary dyskinesia (PCD) is characterized by chronic airway disease, male infertility, and randomization of the left/right body axis as a result of defects of motile cilia and sperm flagella. We identified loss-of-function mutations in the open-reading frame C11orf70 in PCD individuals from five distinct families. Transmission electron microscopy analyses and high-resolution immunofluorescence microscopy demonstrate that loss-of-function mutations in C11orf70 cause immotility of respiratory cilia and sperm flagella, respectively, as a result of the loss of axonemal outer (ODAs) and inner dynein arms (IDAs), indicating that C11orf70 is involved in cytoplasmic assembly of dynein arms. Expression analyses of C11orf70 showed that C11orf70 is expressed in ciliated respiratory cells and that the expression of C11orf70 is upregulated during ciliogenesis, similar to other previously described cytoplasmic dynein-arm assembly factors. Furthermore, C11orf70 shows an interaction with cytoplasmic ODA/IDA assembly factor DNAAF2, supporting our hypothesis that C11orf70 is a preassembly factor involved in the pathogenesis of PCD. The identification of additional genetic defects that cause PCD and male infertility is of great importance for the clinic as well as for genetic counselling.
Assuntos
Padronização Corporal , Dineínas/genética , Síndrome de Kartagener/genética , Mutação/genética , Proteínas Nucleares/genética , Cílios/metabolismo , Cílios/ultraestrutura , Dineínas/ultraestrutura , Feminino , Genes Recessivos , Humanos , Mutação com Perda de Função/genética , Masculino , Cauda do Espermatozoide/metabolismoRESUMO
Motile cilia line the efferent ducts of the mammalian male reproductive tract. Several recent mouse studies have demonstrated that a reduced generation of multiple motile cilia in efferent ducts is associated with obstructive oligozoospermia and fertility issues. However, the sole impact of efferent duct cilia dysmotility on male infertility has not been studied so far either in mice or human. Using video microscopy, histological- and ultrastructural analyses, we examined male reproductive tracts of mice deficient for the axonemal motor protein DNAH5: this defect exclusively disrupts the outer dynein arm (ODA) composition of motile cilia but not the ODA composition and motility of sperm flagella. These mice have immotile efferent duct cilia that lack ODAs, which are essential for ciliary beat generation. Furthermore, they show accumulation of sperm in the efferent duct. Notably, the ultrastructure and motility of sperm from these males are unaffected. Likewise, human individuals with loss-of-function DNAH5 mutations present with reduced sperm count in the ejaculate (oligozoospermia) and dilatations of the epididymal head but normal sperm motility, similar to DNAH5 deficient mice. The findings of this translational study demonstrate, in both mice and men, that efferent duct ciliary motility is important for male reproductive fitness and uncovers a novel pathomechanism distinct from primary defects of sperm motility (asthenozoospermia). If future work can identify environmental factors or defects in genes other than DNAH5 that cause efferent duct cilia dysmotility, this will help unravel other causes of oligozoospermia and may influence future practices in genetic and fertility counseling as well as ART.
Assuntos
Dineínas do Axonema/metabolismo , Axonema/metabolismo , Cílios/metabolismo , Genitália Masculina/metabolismo , Motilidade dos Espermatozoides , Espermatozoides/patologia , Animais , Dineínas do Axonema/genética , Axonema/genética , Axonema/ultraestrutura , Cílios/genética , Cílios/ultraestrutura , Transtornos da Motilidade Ciliar/genética , Transtornos da Motilidade Ciliar/metabolismo , Transtornos da Motilidade Ciliar/patologia , Predisposição Genética para Doença , Genitália Masculina/ultraestrutura , Humanos , Masculino , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Movimento , Mutação , Oligospermia/genética , Oligospermia/metabolismo , Oligospermia/patologia , Fenótipo , Espermatozoides/ultraestruturaRESUMO
Primary ciliary dyskinesia (PCD) is a genetically heterogeneous chronic destructive airway disease. PCD is traditionally diagnosed by nasal nitric oxide measurement, analysis of ciliary beating, transmission electron microscopy (TEM), and/or genetic testing. In most genetic PCD variants, laterality defects can occur. However, it is difficult to establish a diagnosis in individuals with PCD and central pair (CP) defects, and alternative strategies are required because of very subtle ciliary beating abnormalities, a normal ciliary ultrastructure, and normal situs composition. Mutations in HYDIN are known to cause CP defects, but the genetic analysis of HYDIN variants is confounded by the pseudogene HYDIN2, which is almost identical in terms of intron/exon structure. We have previously shown that several types of PCD can be diagnosed via immunofluorescence (IF) microscopy analyses. Here, using IF microscopy, we demonstrated that in individuals with PCD and CP defects, the CP-associated protein SPEF2 is absent in HYDIN-mutant cells, revealing its dependence on functional HYDIN. Next, we performed IF analyses of SPEF2 in respiratory cells from 189 individuals with suspected PCD and situs solitus. Forty-one of the 189 individuals had undetectable SPEF2 and were subjected to a genetic analysis, which revealed one novel loss-of-function mutation in SPEF2 and three reported and 13 novel HYDIN mutations in 15 individuals. The remaining 25 individuals are good candidates for new, as-yet uncharacterized PCD variants that affect the CP apparatus. SPEF2 mutations have been associated with male infertility but have not previously been identified to cause PCD. We identified a mutation of SPEF2 that is causative for PCD with a CP defect. We conclude that SPEF2 IF analyses can facilitate the detection of CP defects and evaluation of the pathogenicity of HYDIN variants, thus aiding the molecular diagnosis of CP defects.
Assuntos
Proteínas de Ciclo Celular/deficiência , Cílios/química , Transtornos da Motilidade Ciliar/genética , Proteínas dos Microfilamentos/genética , Axonema/química , Axonema/ultraestrutura , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/fisiologia , Transtornos da Motilidade Ciliar/diagnóstico , Transtornos da Motilidade Ciliar/patologia , Códon sem Sentido , Estudos de Coortes , Análise Mutacional de DNA , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Feminino , Heterogeneidade Genética , Homozigoto , Humanos , Mutação com Perda de Função , Masculino , Proteínas dos Microfilamentos/fisiologia , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Depuração Mucociliar/genética , Mutação , Mutação de Sentido Incorreto , Linhagem , Cultura Primária de Células , Situs Inversus/diagnóstico , Situs Inversus/genética , Situs Inversus/patologiaRESUMO
Multiprotein complexes referred to as outer dynein arms (ODAs) develop the main mechanical force to generate the ciliary and flagellar beat. ODA defects are the most common cause of primary ciliary dyskinesia (PCD), a congenital disorder of ciliary beating, characterized by recurrent infections of the upper and lower airways, as well as by progressive lung failure and randomization of left-right body asymmetry. Using a whole-exome sequencing approach, we identified recessive loss-of-function mutations within TTC25 in three individuals from two unrelated families affected by PCD. Mice generated by CRISPR/Cas9 technology and carrying a deletion of exons 2 and 3 in Ttc25 presented with laterality defects. Consistently, we observed immotile nodal cilia and missing leftward flow via particle image velocimetry. Furthermore, transmission electron microscopy (TEM) analysis in TTC25-deficient mice revealed an absence of ODAs. Consistent with our findings in mice, we were able to show loss of the ciliary ODAs in humans via TEM and immunofluorescence (IF) analyses. Additionally, IF analyses revealed an absence of the ODA docking complex (ODA-DC), along with its known components CCDC114, CCDC151, and ARMC4. Co-immunoprecipitation revealed interaction between the ODA-DC component CCDC114 and TTC25. Thus, here we report TTC25 as a new member of the ODA-DC machinery in humans and mice.
Assuntos
Axonema/genética , Axonema/metabolismo , Proteínas de Transporte/genética , Cílios/patologia , Dineínas/química , Dineínas/metabolismo , Síndrome de Kartagener/genética , Síndrome de Kartagener/patologia , Mutação , Animais , Axonema/patologia , Axonema/ultraestrutura , Cílios/metabolismo , Cílios/ultraestrutura , Dineínas/genética , Dineínas/ultraestrutura , Exoma/genética , Éxons/genética , Imunofluorescência , Genes Recessivos , Humanos , Camundongos , Microscopia Eletrônica de Transmissão , Ligação Proteica , Xenopus , Proteínas de Xenopus/deficiência , Proteínas de Xenopus/genéticaRESUMO
BACKGROUND: Cough is a key symptom in patients with cystic fibrosis (CF) and primary ciliary dyskinesia (PCD). OBJECTIVE: The study objectives were to test whether cough is related to parameters reflecting their disease severity and whether CF and PCD differ in cough frequency. METHODS: In this prospective observational study, we used a microphone-based monitoring system (LEOSound® Monitor) to count the coughs in healthy subjects (HS) and in stable patients with CF and PCD (25 subjects per group) on 2 consecutive nights. RESULTS: The median number of coughs/h in the HS, CF, and PCD groups was 0.0, 1.3, and 0.5 on the first night and 0.0, 2.3, and 0.2 on the second night, respectively. Patients with CF and PCD coughed more than HS (p < 0.001 and p = 0.009, respectively) and CF patients coughed more than PCD patients (p = 0.023). A multivariable mixed model analysis revealed forced expiratory volume in 1 s as an independent risk factor for increased cough frequency in patients. The reliability for repeated measurements was higher for cough epochs/h than for coughs/h (intraclass correlation coefficient: 0.75 and 0.49, respectively). CONCLUSIONS: Patients with CF cough more than patients with PCD. The cough frequency in CF and PCD is associated with parameters reflecting disease severity. Cough frequency is a possible endpoint in clinical trials and cough epochs/h may be more useful than coughs/h.
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Transtornos da Motilidade Ciliar/diagnóstico , Ritmo Circadiano/fisiologia , Tosse/diagnóstico , Fibrose Cística/diagnóstico , Monitorização Fisiológica/métodos , Adolescente , Adulto , Criança , Transtornos da Motilidade Ciliar/complicações , Tosse/etiologia , Fibrose Cística/complicações , Feminino , Seguimentos , Volume Expiratório Forçado/fisiologia , Voluntários Saudáveis , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Testes de Função Respiratória , Índice de Gravidade de Doença , Adulto JovemRESUMO
Primary ciliary dyskinesia (PCD) is a recessively inherited disease that leads to chronic respiratory disorders owing to impaired mucociliary clearance. Conventional transmission electron microscopy (TEM) is a diagnostic standard to identify ultrastructural defects in respiratory cilia but is not useful in approximately 30% of PCD cases, which have normal ciliary ultrastructure. DNAH11 mutations are a common cause of PCD with normal ciliary ultrastructure and hyperkinetic ciliary beating, but its pathophysiology remains poorly understood. We therefore characterized DNAH11 in human respiratory cilia by immunofluorescence microscopy (IFM) in the context of PCD. We used whole-exome and targeted next-generation sequence analysis as well as Sanger sequencing to identify and confirm eight novel loss-of-function DNAH11 mutations. We designed and validated a monoclonal antibody specific to DNAH11 and performed high-resolution IFM of both control and PCD-affected human respiratory cells, as well as samples from green fluorescent protein (GFP)-left-right dynein mice, to determine the ciliary localization of DNAH11. IFM analysis demonstrated native DNAH11 localization in only the proximal region of wild-type human respiratory cilia and loss of DNAH11 in individuals with PCD with certain loss-of-function DNAH11 mutations. GFP-left-right dynein mice confirmed proximal DNAH11 localization in tracheal cilia. DNAH11 retained proximal localization in respiratory cilia of individuals with PCD with distinct ultrastructural defects, such as the absence of outer dynein arms (ODAs). TEM tomography detected a partial reduction of ODAs in DNAH11-deficient cilia. DNAH11 mutations result in a subtle ODA defect in only the proximal region of respiratory cilia, which is detectable by IFM and TEM tomography.
Assuntos
Dineínas do Axonema/metabolismo , Cílios/metabolismo , Dineínas/metabolismo , Pulmão/metabolismo , Sequência de Bases , Cílios/ultraestrutura , Dineínas/ultraestrutura , Homozigoto , Humanos , Síndrome de Kartagener/genética , Mutação/genética , Transporte ProteicoRESUMO
The motive forces for ciliary movement are generated by large multiprotein complexes referred to as outer dynein arms (ODAs), which are preassembled in the cytoplasm prior to transport to the ciliary axonemal compartment. In humans, defects in structural components, docking complexes, or cytoplasmic assembly factors can cause primary ciliary dyskinesia (PCD), a disorder characterized by chronic airway disease and defects in laterality. By using combined high resolution copy-number variant and mutation analysis, we identified ARMC4 mutations in twelve PCD individuals whose cells showed reduced numbers of ODAs and severely impaired ciliary beating. Transient suppression in zebrafish and analysis of an ENU mouse mutant confirmed in both model organisms that ARMC4 is critical for left-right patterning. We demonstrate that ARMC4 is an axonemal protein that is necessary for proper targeting and anchoring of ODAs.
Assuntos
Proteínas do Domínio Armadillo/genética , Padronização Corporal/genética , Cílios/genética , Dineínas/genética , Síndrome de Kartagener/genética , Sistema Respiratório/metabolismo , Sequência de Aminoácidos , Animais , Proteínas do Domínio Armadillo/metabolismo , Axonema/genética , Axonema/metabolismo , Axonema/patologia , Cílios/metabolismo , Cílios/patologia , Variações do Número de Cópias de DNA , Análise Mutacional de DNA , Dineínas/metabolismo , Regulação da Expressão Gênica , Humanos , Síndrome de Kartagener/metabolismo , Síndrome de Kartagener/patologia , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Dados de Sequência Molecular , Mutação , Sistema Respiratório/patologia , Peixe-Zebra/genética , Peixe-Zebra/metabolismoRESUMO
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.
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Antígenos de Superfície/genética , Cílios/genética , Transtornos da Motilidade Ciliar/genética , Dineínas/genética , Proteínas de Ligação ao GTP/genética , Síndrome de Kartagener/genética , Mutação/genética , Adolescente , Adulto , Animais , Axonema/genética , Criança , Pré-Escolar , Citoplasma/genética , Células Epiteliais/metabolismo , Exoma , Feminino , Humanos , Lactente , Masculino , Linhagem , Fenótipo , Adulto Jovem , Peixe-ZebraRESUMO
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.
Assuntos
Cílios/genética , Síndrome de Kartagener/genética , Proteínas/genética , Sistema Respiratório/metabolismo , Proteínas Supressoras de Tumor/genética , Animais , Autoantígenos/genética , Autoantígenos/metabolismo , Dineínas do Axonema/genética , Dineínas do Axonema/metabolismo , Biomarcadores/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cílios/metabolismo , Cílios/patologia , Proteínas do Citoesqueleto , Exoma , Regulação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Síndrome de Kartagener/metabolismo , Síndrome de Kartagener/patologia , Masculino , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Mutação , Linhagem , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas/metabolismo , Ratos , Sistema Respiratório/patologia , Proteínas Supressoras de Tumor/metabolismo , Xenopus laevis/genética , Xenopus laevis/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismoRESUMO
Primary ciliary dyskinesia (PCD) is a rare autosomal recessive disorder leading to chronic upper and lower airway disease. Fundamental data on epidemiology, clinical presentation, course and treatment strategies are lacking in PCD. We have established an international PCD registry to realise an unmet need for an international platform to systematically collect data on incidence, clinical presentation, treatment and disease course.The registry was launched in January 2014. We used internet technology to ensure easy online access using a web browser under www.pcdregistry.eu. Data from 201 patients have been collected so far. The database is comprised of a basic data form including demographic and diagnostic information, and visit forms designed to monitor the disease course.To establish a definite PCD diagnosis, we used strict diagnostic criteria, which required two to three diagnostic methods in addition to classical clinical symptoms. Preliminary analysis of lung function data demonstrated a mean annual decline of percentage predicted forced expiratory volume in 1â s of 0.59% (95% CI 0.98-0.22).Here, we present the development of an international PCD registry as a new promising tool to advance the understanding of this rare disorder, to recruit candidates for research studies and ultimately to improve PCD care.
Assuntos
Síndrome de Kartagener/diagnóstico , Síndrome de Kartagener/epidemiologia , Sistema de Registros , Adolescente , Adulto , Distribuição por Idade , Idoso , Criança , Pré-Escolar , Progressão da Doença , Europa (Continente) , Feminino , Volume Expiratório Forçado , Humanos , Incidência , Lactente , Internet , Colaboração Intersetorial , Masculino , Pessoa de Meia-Idade , América do Norte , Seleção de Pacientes , Adulto JovemRESUMO
Primary ciliary dyskinesia (PCD) is a genetically heterogeneous recessive disorder caused by several distinct defects in genes responsible for ciliary beating, leading to defective mucociliary clearance often associated with randomization of left/right body asymmetry. Individuals with PCD caused by defective radial spoke (RS) heads are difficult to diagnose owing to lack of gross ultrastructural defects and absence of situs inversus. Thus far, most mutations identified in human radial spoke genes (RSPH) are loss-of-function mutations, and missense variants have been rarely described. We studied the consequences of different RSPH9, RSPH4A, and RSPH1 mutations on the assembly of the RS complex to improve diagnostics in PCD. We report 21 individuals with PCD (16 families) with biallelic mutations in RSPH9, RSPH4A, and RSPH1, including seven novel mutations comprising missense variants, and performed high-resolution immunofluorescence analysis of human respiratory cilia. Missense variants are frequent genetic defects in PCD with RS defects. Absence of RSPH4A due to mutations in RSPH4A results in deficient axonemal assembly of the RS head components RSPH1 and RSPH9. RSPH1 mutant cilia, lacking RSPH1, fail to assemble RSPH9, whereas RSPH9 mutations result in axonemal absence of RSPH9, but do not affect the assembly of the other head proteins, RSPH1 and RSPH4A. Interestingly, our results were identical in individuals carrying loss-of-function mutations, missense variants, or one amino acid deletion. Immunofluorescence analysis can improve diagnosis of PCD in patients with loss-of-function mutations as well as missense variants. RSPH4A is the core protein of the RS head.
Assuntos
Proteínas do Citoesqueleto/genética , Proteínas de Ligação a DNA/genética , Síndrome de Kartagener/diagnóstico , Proteínas/genética , Adolescente , Adulto , Criança , Pré-Escolar , Proteínas do Citoesqueleto/metabolismo , Análise Mutacional de DNA , Proteínas de Ligação a DNA/metabolismo , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Síndrome de Kartagener/genética , Síndrome de Kartagener/metabolismo , Masculino , Mutação de Sentido Incorreto , Multimerização Proteica , Proteínas/metabolismo , Adulto JovemRESUMO
Primary ciliary dyskinesia (PCD) is a genetically heterogeneous recessive disorder characterized by defective cilia and flagella motility. Chronic destructive-airway disease is caused by abnormal respiratory-tract mucociliary clearance. Abnormal propulsion of sperm flagella contributes to male infertility. Genetic defects in most individuals affected by PCD cause randomization of left-right body asymmetry; approximately half show situs inversus or situs ambiguous. Almost 70 years after the hy3 mouse possessing Hydin mutations was described as a recessive hydrocephalus model, we report HYDIN mutations in PCD-affected persons without hydrocephalus. By homozygosity mapping, we identified a PCD-associated locus, chromosomal region 16q21-q23, which contains HYDIN. However, a nearly identical 360 kb paralogous segment (HYDIN2) in chromosomal region 1q21.1 complicated mutational analysis. In three affected German siblings linked to HYDIN, we identified homozygous c.3985G>T mutations that affect an evolutionary conserved splice acceptor site and that subsequently cause aberrantly spliced transcripts predicting premature protein termination in respiratory cells. Parallel whole-exome sequencing identified a homozygous nonsense HYDIN mutation, c.922A>T (p.Lys307(∗)), in six individuals from three Faroe Island PCD-affected families that all carried an 8.8 Mb shared haplotype across HYDIN, indicating an ancestral founder mutation in this isolated population. We demonstrate by electron microscopy tomography that, consistent with the effects of loss-of-function mutations, HYDIN mutant respiratory cilia lack the C2b projection of the central pair (CP) apparatus; similar findings were reported in Hydin-deficient Chlamydomonas and mice. High-speed videomicroscopy demonstrated markedly reduced beating amplitudes of respiratory cilia and stiff sperm flagella. Like the hy3 mouse model, all nine PCD-affected persons had normal body composition because nodal cilia function is apparently not dependent on the function of the CP apparatus.
Assuntos
Genes Recessivos , Síndrome de Kartagener/genética , Proteínas dos Microfilamentos/genética , Mutação , Adulto , Sequência de Bases , Cromossomos Humanos Par 1 , Cromossomos Humanos Par 16 , Cílios/genética , Análise Mutacional de DNA/métodos , Feminino , Loci Gênicos/genética , Predisposição Genética para Doença , Haplótipos , Homozigoto , Humanos , Hidrocefalia/genética , Masculino , Dados de Sequência Molecular , Linhagem , Splicing de RNA/genética , Irmãos , Situs Inversus/genéticaRESUMO
STUDY QUESTION: What is the motor protein composition and function of human fallopian tube (FT) cilia? SUMMARY ANSWER: Although the motor protein composition and function of human FT cilia resemble that of respiratory cilia, females with primary ciliary dyskinesia (PCD) are not necessarily infertile. WHAT IS KNOWN ALREADY: FTs are lined with multiple motile cilia, which show a 9 + 2 ultrastructure by transmission electron microscopy. Case reports suggest an increased incidence of subfertility and ectopic pregnancy in women with PCD, a disease characterized by dysfunction of motile cilia and flagella. STUDY DESIGN, SIZE, DURATION: This study consisted of an observational laboratory study on human FT specimens from five healthy females recruited from April 2012 to December 2013 and a descriptive observational retrospective analysis of a clinical PCD database. PARTICIPANTS/MATERIALS, SETTING, METHODS: Human FT tissue was obtained from five healthy females after tubal ligation during caesarean delivery. Motor protein composition was assessed by immunofluorescence microscopy using antibodies against dynein arms and nexin-dynein regulatory complex subunits. Ciliary motility was analysed by high-speed video microscopy. A retrospective search of our database of PCD individuals was performed for information on conception and childbirth. MAIN RESULTS AND THE ROLE OF CHANCE: The motor protein composition of human FT cilia was identical to that of respiratory cilia. FT cilia showed coordinated beating, resulting in a directed fluid flow towards the uterine cavity. We identified nine PCD individuals with severe dysfunction of respiratory cilia who gave birth to children after spontaneous conception. This suggests that ciliary beating is not the key motor of ovum transport. LIMITATIONS, REASON FOR CAUTION: FT cilia of affected PCD females were not available for analysis. Thus, it remains to be proven that FT cilia indeed show the same defects as respiratory cilia in PCD individuals. Comprehensive epidemiological studies are needed to determine the extent of female (sub-) fertility in PCD. WIDER IMPLICATIONS OF THE FINDINGS: Knowledge of the exact protein composition and function of FT cilia will contribute to a better understanding of cilia-generated fluid flow in female reproduction. These findings are important for subsequent studies of function and protein composition of FT cilia in PCD patients.
Assuntos
Dineínas do Axonema/metabolismo , Cílios/fisiologia , Tubas Uterinas/metabolismo , Síndrome de Kartagener/metabolismo , Tubulina (Proteína)/metabolismo , Adulto , Transporte Biológico , Feminino , Humanos , Estudos RetrospectivosRESUMO
Primary ciliary dyskinesia (PCD) is a rare genetic disorder leading to recurrent respiratory tract infections. High-speed video-microscopy analysis (HVMA) of ciliary beating, currently the first-line diagnostic tool for PCD in most centres, is challenging because recent studies have expanded the spectrum of HVMA findings in PCD from grossly abnormal to very subtle. The objective of this study was to describe the diversity of HVMA findings in genetically confirmed PCD individuals. HVMA was performed as part of the routine work-up of individuals with suspected PCD. Subsequent molecular analysis identified biallelic mutations in the PCD-related genes of 66 individuals. 1072 videos of these subjects were assessed for correlation with the genotype. Biallelic mutations (19 novel) were found in 17 genes: DNAI1, DNAI2, DNAH5, DNAH11, CCDC103, ARMC4, KTU/DNAAF2, LRRC50/DNAAF1, LRRC6, DYX1C1, ZMYND10, CCDC39, CCDC40, CCDC164, HYDIN, RSPH4A and RSPH1. Ciliary beat pattern variations correlated well with the genetic findings, allowing the classification of typical HVMA findings for different genetic groups. In contrast, analysis of ciliary beat frequency did not result in additional diagnostic impact. In conclusion, this study provides detailed knowledge about the diversity of HVMA findings in PCD and may therefore be seen as a guide to the improvement of PCD diagnostics.