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
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
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
Airway mucociliary regeneration and function are key players for airway defense and are impaired in chronic obstructive pulmonary disease (COPD). Using transcriptome analysis in COPD-derived bronchial biopsies, we observed a positive correlation between cilia-related genes and microRNA-449 (miR449). In vitro, miR449 was strongly increased during airway epithelial mucociliary differentiation. In vivo, miR449 was upregulated during recovery from chemical or infective insults. miR0449-/- mice (both alleles are deleted) showed impaired ciliated epithelial regeneration after naphthalene and Haemophilus influenzae exposure, accompanied by more intense inflammation and emphysematous manifestations of COPD. The latter occurred spontaneously in aged miR449-/- mice. We identified Aurora kinase A and its effector target HDAC6 as key mediators in miR449-regulated ciliary homeostasis and epithelial regeneration. Aurora kinase A is downregulated upon miR449 overexpression in vitro and upregulated in miR449-/- mouse lungs. Accordingly, imaging studies showed profoundly altered cilia length and morphology accompanied by reduced mucociliary clearance. Pharmacological inhibition of HDAC6 rescued cilia length and coverage in miR449-/- cells, consistent with its tubulin-deacetylating function. Altogether, our study establishes a link between miR449, ciliary dysfunction, and COPD pathogenesis.
Assuntos
Aurora Quinase A/metabolismo , Desacetilase 6 de Histona/metabolismo , MicroRNAs , Doença Pulmonar Obstrutiva Crônica , Animais , Aurora Quinase A/genética , Cílios/genética , Células Epiteliais , Camundongos , MicroRNAs/genética , Doença Pulmonar Obstrutiva Crônica/genética , Tubulina (Proteína)/genéticaRESUMO
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
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
Axonemal dynein ATPases direct ciliary and flagellar beating via adenosine triphosphate (ATP) hydrolysis. The modulatory effect of adenosine monophosphate (AMP) and adenosine diphosphate (ADP) on flagellar beating is not fully understood. Here, we describe a deficiency of cilia and flagella associated protein 45 (CFAP45) in humans and mice that presents a motile ciliopathy featuring situs inversus totalis and asthenospermia. CFAP45-deficient cilia and flagella show normal morphology and axonemal ultrastructure. Proteomic profiling links CFAP45 to an axonemal module including dynein ATPases and adenylate kinase as well as CFAP52, whose mutations cause a similar ciliopathy. CFAP45 binds AMP in vitro, consistent with structural modelling that identifies an AMP-binding interface between CFAP45 and AK8. Microtubule sliding of dyskinetic sperm from Cfap45-/- mice is rescued with the addition of either AMP or ADP with ATP, compared to ATP alone. We propose that CFAP45 supports mammalian ciliary and flagellar beating via an adenine nucleotide homeostasis module.
Assuntos
Nucleotídeos de Adenina/metabolismo , Astenozoospermia/genética , Proteínas do Citoesqueleto/deficiência , Situs Inversus/genética , Adolescente , Adulto , Animais , Astenozoospermia/patologia , Axonema/ultraestrutura , Sistemas CRISPR-Cas/genética , Cílios/metabolismo , Cílios/ultraestrutura , Proteínas do Citoesqueleto/genética , Análise Mutacional de DNA , Modelos Animais de Doenças , Epididimo/patologia , Feminino , Flagelos/metabolismo , Flagelos/ultraestrutura , Humanos , Mutação com Perda de Função , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Planárias/citologia , Planárias/genética , Planárias/metabolismo , Mucosa Respiratória/citologia , Mucosa Respiratória/patologia , Situs Inversus/diagnóstico por imagem , Situs Inversus/patologia , Motilidade dos Espermatozoides/genética , Tomografia Computadorizada por Raios X , Sequenciamento do ExomaRESUMO
Hydrocephalus is one of the most prevalent form of developmental central nervous system (CNS) malformations. Cerebrospinal fluid (CSF) flow depends on both heartbeat and body movement. Furthermore, it has been shown that CSF flow within and across brain ventricles depends on cilia motility of the ependymal cells lining the brain ventricles, which play a crucial role to maintain patency of the narrow sites of CSF passage during brain formation in mice. Using whole-exome and whole-genome sequencing, we identified an autosomal-dominant cause of a distinct motile ciliopathy related to defective ciliogenesis of the ependymal cilia in six individuals. Heterozygous de novo mutations in FOXJ1, which encodes a well-known member of the forkhead transcription factors important for ciliogenesis of motile cilia, cause a motile ciliopathy that is characterized by hydrocephalus internus, chronic destructive airway disease, and randomization of left/right body asymmetry. Mutant respiratory epithelial cells are unable to generate a fluid flow and exhibit a reduced number of cilia per cell, as documented by high-speed video microscopy (HVMA), transmission electron microscopy (TEM), and immunofluorescence analysis (IF). TEM and IF demonstrate mislocalized basal bodies. In line with this finding, the focal adhesion protein PTK2 displays aberrant localization in the cytoplasm of the mutant respiratory epithelial cells.
Assuntos
Ventrículos Cerebrais/patologia , Ciliopatias/genética , Fatores de Transcrição Forkhead/genética , Hidrocefalia/genética , Mutação/genética , Corpos Basais/patologia , Cílios/genética , Cílios/patologia , Ciliopatias/patologia , Epêndima/patologia , Células Epiteliais/patologia , Humanos , Hidrocefalia/patologiaRESUMO
Background - Nearly one in 100 live births presents with congenital heart defects (CHD). CHD are frequently associated with laterality defects, such as situs inversus totalis (SIT), a mirrored positioning of internal organs. Body laterality is established by a complex process: monocilia at the embryonic left-right organizer (LRO) facilitate both the generation and sensing of a leftward fluid flow. This induces the conserved left-sided Nodal signaling cascade to initiate asymmetric organogenesis. Primary ciliary dyskinesia (PCD) originates from dysfunction of motile cilia, causing symptoms such as chronic sinusitis, bronchiectasis and frequently SIT. The most frequently mutated gene in PCD, DNAH5 is associated with randomization of body asymmetry resulting in SIT in half of the patients; however, its relation to CHD occurrence in humans has not been investigated in detail so far. Methods - We performed genotype / phenotype correlations in 132 PCD patients carrying disease-causing DNAH5 mutations, focusing on situs defects and CHD. Using high speed video microscopy-, immunofluorescence-, and in situ hybridization analyses, we investigated the initial steps of left-right axis establishment in embryos of a Dnah5 mutant mouse model. Results - 65.9% (87 / 132) of the PCD patients carrying disease-causing DNAH5 mutations had laterality defects: 88.5% (77 / 87) presented with SIT, 11.5% (10 / 87) presented with situs ambiguus; and 6.1% (8 / 132) presented with CHD. In Dnah5mut/mut mice, embryonic LRO monocilia lack outer dynein arms resulting in immotile cilia, impaired flow at the LRO, and randomization of Nodal signaling with normal, reversed or bilateral expression of key molecules. Conclusions - For the first time, we directly demonstrate the disease-mechanism of laterality defects linked to DNAH5 deficiency at the molecular level during embryogenesis. We highlight that mutations in DNAH5 are not only associated with classical randomization of left-right body asymmetry but also with severe laterality defects including CHD.
RESUMO
Dysfunction of motile monocilia, altering the leftward flow at the embryonic node essential for determination of left-right body asymmetry, is a major cause of laterality defects. Laterality defects are also often associated with reduced mucociliary clearance caused by defective multiple motile cilia of the airway and are responsible for destructive airway disease. Outer dynein arms (ODAs) are essential for ciliary beat generation, and human respiratory cilia contain different ODA heavy chains (HCs): the panaxonemally distributed γ-HC DNAH5, proximally located ß-HC DNAH11 (defining ODA type 1), and the distally localized ß-HC DNAH9 (defining ODA type 2). Here we report loss-of-function mutations in DNAH9 in five independent families causing situs abnormalities associated with subtle respiratory ciliary dysfunction. Consistent with the observed subtle respiratory phenotype, high-speed video microscopy demonstrates distally impaired ciliary bending in DNAH9 mutant respiratory cilia. DNAH9-deficient cilia also lack other ODA components such as DNAH5, DNAI1, and DNAI2 from the distal axonemal compartment, demonstrating an essential role of DNAH9 for distal axonemal assembly of ODAs type 2. Yeast two-hybrid and co-immunoprecipitation analyses indicate interaction of DNAH9 with the ODA components DNAH5 and DNAI2 as well as the ODA-docking complex component CCDC114. We further show that during ciliogenesis of respiratory cilia, first proximally located DNAH11 and then distally located DNAH9 is assembled in the axoneme. We propose that the ß-HC paralogs DNAH9 and DNAH11 achieved specific functional roles for the distinct axonemal compartments during evolution with human DNAH9 function matching that of ancient ß-HCs such as that of the unicellular Chlamydomonas reinhardtii.
Assuntos
Dineínas do Axonema/genética , Cílios/genética , Dineínas/genética , Mutação/genética , Axonema/genética , Transtornos da Motilidade Ciliar/genética , Humanos , Síndrome de Kartagener/genética , FenótipoRESUMO
The clinical spectrum of ciliopathies affecting motile cilia spans impaired mucociliary clearance in the respiratory system, laterality defects including heart malformations, infertility and hydrocephalus. Using linkage analysis and whole exome sequencing, we identified two recessive loss-of-function MNS1 mutations in five individuals from four consanguineous families: 1) a homozygous nonsense mutation p.Arg242* in four males with laterality defects and infertility and 2) a homozygous nonsense mutation p.Gln203* in one female with laterality defects and recurrent respiratory infections additionally carrying homozygous mutations in DNAH5. Consistent with the laterality defects observed in these individuals, we found Mns1 to be expressed in mouse embryonic ventral node. Immunofluorescence analysis further revealed that MNS1 localizes to the axonemes of respiratory cilia as well as sperm flagella in human. In-depth ultrastructural analyses confirmed a subtle outer dynein arm (ODA) defect in the axonemes of respiratory epithelial cells resembling findings reported in Mns1-deficient mice. Ultrastructural analyses in the female carrying combined mutations in MNS1 and DNAH5 indicated a role for MNS1 in the process of ODA docking (ODA-DC) in the distal respiratory axonemes. Furthermore, co-immunoprecipitation and yeast two hybrid analyses demonstrated that MNS1 dimerizes and interacts with the ODA docking complex component CCDC114. Overall, we demonstrate that MNS1 deficiency in humans causes laterality defects (situs inversus) and likely male infertility and that MNS1 plays a role in the ODA-DC assembly.
Assuntos
Códon sem Sentido , Lateralidade Funcional/genética , Homozigoto , Infertilidade Masculina/genética , Proteínas Nucleares/metabolismo , Adolescente , Adulto , Animais , Dineínas do Axonema/genética , Dineínas do Axonema/metabolismo , Axonema/metabolismo , Proteínas de Ciclo Celular , Criança , Pré-Escolar , Cílios/ultraestrutura , Feminino , Regulação da Expressão Gênica , Ligação Genética , Humanos , Lactente , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Linhagem , Polimorfismo de Nucleotídeo Único , Cauda do Espermatozoide , Sequenciamento do Exoma , Adulto JovemRESUMO
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.