RESUMO
TTC12 is a cytoplasmic and centromere-localized protein that plays a role in the proper assembly of dynein arm complexes in motile cilia in both respiratory cells and sperm flagella. This finding underscores its significance in cellular motility and function. However, the wide role of TTC12 in human spermatogenesis-associated primary ciliary dyskinesia (PCD) still needs to be elucidated. Whole-exome sequencing (WES) and Sanger sequencing were performed to identify potentially pathogenic variants causing PCD and multiple morphological abnormalities of sperm flagella (MMAF) in an infertile Pakistani man. Diagnostic imaging techniques were used for PCD screening in the patient. Real-time polymerase chain reaction (RTâPCR) was performed to detect the effect of mutations on the mRNA abundance of the affected genes. Papanicolaou staining and scanning electron microscopy (SEM) were carried out to examine sperm morphology. Transmission electron microscopy (TEM) was performed to examine the ultrastructure of the sperm flagella, and the results were confirmed by immunofluorescence staining. Using WES and Sanger sequencing, a novel homozygous missense variant (c.C1069T; p.Arg357Trp) in TTC12 was identified in a patient from a consanguineous family. A computed tomography scan of the paranasal sinuses confirmed the symptoms of the PCD. RT-PCR showed a decrease in TTC12 mRNA in the patient's sperm sample. Papanicolaou staining, SEM, and TEM analysis revealed a significant change in shape and a disorganized axonemal structure in the sperm flagella of the patient. Immunostaining assays revealed that TTC12 is distributed throughout the flagella and is predominantly concentrated in the midpiece in normal spermatozoa. In contrast, spermatozoa from patient deficient in TTC12 showed minimal staining intensity for TTC12 or DNAH17 (outer dynein arms components). This could lead to MMAF and result in male infertility. This novel TTC12 variant not only illuminates the underlying genetic causes of male infertility but also paves the way for potential treatments targeting these genetic factors. This study represents a significant advancement in understanding the genetic basis of PCD-related infertility.
Assuntos
Homozigoto , Infertilidade Masculina , Mutação de Sentido Incorreto , Cauda do Espermatozoide , Humanos , Masculino , Mutação de Sentido Incorreto/genética , Paquistão , Infertilidade Masculina/genética , Infertilidade Masculina/patologia , Cauda do Espermatozoide/patologia , Cauda do Espermatozoide/ultraestrutura , Cauda do Espermatozoide/metabolismo , Adulto , Linhagem , Astenozoospermia/genética , Astenozoospermia/patologia , Transtornos da Motilidade Ciliar/genética , Transtornos da Motilidade Ciliar/patologia , Sequenciamento do Exoma , Oligospermia/genética , Oligospermia/patologia , Síndrome de Kartagener/genética , Síndrome de Kartagener/patologiaRESUMO
Primary ciliary dyskinesia (PCD) is a rare genetic disorder with a variable clinical phenotype that is accompanied by reduced motility of the cilia in the respiratory tract and numerous other organs. This leads to various characteristic symptoms and disease manifestations, primarily affecting the lungs (chronic persistent productive cough, bronchiectasis), the nose and paranasal sinuses (chronic persistent rhinitis or rhinosinusitis) as well as the middle ear (chronic otitis media, middle ear effusion). Moreover, PCD is associated with impaired fertility or lateralization defects (situs anomalies, congenital heart defects). The diagnostics of PCD are complex and require a combination of several sophisticated instrument-based diagnostic procedures. Through thorough history taking and evaluation, suspected cases can be comparatively well identified based on typical clinical features and referred to further diagnostics. In recent years, molecular genetic analysis through panel diagnostics or whole exome and whole genome sequencing, has gained in importance as this enables affected individuals to participate in disease-specific and genotype-specific clinical trials. Although the current treatment is purely symptomatic, the earliest possible diagnosis is crucial for connecting patients to specialized PCD centers, which can have a significant impact on the clinical course of the affected individuals.
Assuntos
Síndrome de Kartagener , Humanos , Síndrome de Kartagener/diagnóstico , Síndrome de Kartagener/genética , Síndrome de Kartagener/terapia , Síndrome de Kartagener/patologia , Síndrome de Kartagener/fisiopatologia , FenótipoRESUMO
BACKGROUND: Primary ciliary dyskinesia (PCD) is a rare airway disorder caused by defective motile cilia. Only male patients have been reported with pathogenic mutations in X-linked DNAAF6, which result in the absence of ciliary dynein arms, whereas their heterozygous mothers are supposedly healthy. Our objective was to assess the possible clinical and ciliary consequences of X-chromosome inactivation (XCI) in these mothers. METHODS: XCI patterns of six mothers of male patients with DNAAF6-related PCD were determined by DNA-methylation studies and compared with their clinical phenotype (6/6 mothers), as well as their ciliary phenotype (4/6 mothers), as assessed by immunofluorescence and high-speed videomicroscopy analyses. The mutated X chromosome was tracked to assess the percentage of cells with a normal inactivated DNAAF6 allele. RESULTS: The mothers' phenotypes ranged from absence of symptoms to mild/moderate or severe airway phenotypes, closely reflecting their XCI pattern. Analyses of the symptomatic mothers' airway ciliated cells revealed the coexistence of normal cells and cells with immotile cilia lacking dynein arms, whose ratio closely mirrored their XCI pattern. CONCLUSION: This study highlights the importance of searching for heterozygous pathogenic DNAAF6 mutations in all female relatives of male PCD patients with a DNAAF6 defect, as well as in females consulting for mild chronic respiratory symptoms. Our results also demonstrate that about one-third-ranging from 20% to 50%-normal ciliated airway cells sufficed to avoid severe PCD, a result paving the way for gene therapy.
Assuntos
Cílios , Inativação do Cromossomo X , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Cílios/patologia , Cílios/genética , Transtornos da Motilidade Ciliar/genética , Transtornos da Motilidade Ciliar/patologia , Metilação de DNA/genética , Dineínas/genética , Síndrome de Kartagener/genética , Síndrome de Kartagener/patologia , Mutação , Fenótipo , Inativação do Cromossomo X/genéticaRESUMO
BACKGROUND: Primary ciliary dyskinesia (PCD) is a rare and heterogeneous disease that is difficult to diagnose and requires complex and expensive diagnostic tools. The saccharin transit time test is a simple and inexpensive tool that may assist in screening patients with PCD. OBJECTIVES: This study aimed to compare changes in the electron microscopy findings with clinical variables and saccharin tests in individuals diagnosed with clinical PCD (cPCD) and a control group. DESIGN AND SETTING: An observational cross-sectional study was conducted in an otorhinolaryngology outpatient clinic from August 2012 to April 2021. METHOD: Patients with cPCD underwent clinical screening questionnaires, nasal endoscopy, the saccharin transit time test, and nasal biopsy for transmission electron microscopy. RESULTS: Thirty-four patients with cPCD were evaluated. The most prevalent clinical comorbidities in the cPCD group were recurrent pneumonia, bronchiectasis, and chronic rhinosinusitis. Electron microscopy confirmed the clinical diagnosis of PCD in 16 of the 34 (47.1%) patients. CONCLUSION: The saccharin test could assist in screening patients with PCD due to its association with clinical alterations related to PCD.
Assuntos
Síndrome de Kartagener , Pneumonia , Humanos , Síndrome de Kartagener/diagnóstico , Síndrome de Kartagener/patologia , Estudos Transversais , Sacarina , Microscopia Eletrônica de TransmissãoRESUMO
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) causes cellular cilia motility alterations, leading to clinical manifestations in the upper and lower respiratory tract and situs abnormalities. The PCD diagnosis was improved after the inclusion of diagnostic tools, such as transmission electron microscopy and genetic screening; however, the PCD screening is a challenge yet. In this context, we aimed to describe the clinical, genetic, and ultra-ciliary characteristics in individuals with clinical suspicion of PCD (cPCD) from a Brazilian Tertiary Hospital. An observational study was carried out with individuals during the follow-up between 2011 and 2021. The individuals were submitted to clinical questionnaires, transmission electron microscopy, and genetic screening for pathogenic variants in PCD-related genes. Those patients were classified according to the degree of suspicion for PCD. In our study, we enrolled thirty-seven cPCD individuals; 20/37 (54.1%) had chronic rhinosinusitis, 28/37 (75.6%) had bronchiectasis, and 29/37 (78.4%) had recurrent pneumonia. A total of 17/37 (45.9%) individuals had transmission electron microscopy or genetic confirmation of PCD; 10 individuals had at least one positive pathogenic genetic variant in the PCD-related genes; however, only seven patients presented a conclusive result according to the American College of Medical Genetics and Genomics and the Association for Molecular Pathology with two pathogenic variants in homozygous or compound heterozygous. The median age at diagnosis was 13 years, and the median time between suspicion and diagnosis was four years. Sixteen patients had class I electron microscopy alterations, seven had class II alterations, and 14 had normal transmission electron microscopy according to the international consensus guideline for reporting transmission electron microscopy results in the diagnosis of PCD (BEAT-PCD TEM Criteria). Genetic screening for pathogenic variants in PCD-related genes and transmission electron microscopy can help determine the PCD diagnosis; however, they are still unavailable to all individuals with clinical suspicion in Brazil. We described ultrastructural alterations found in our population along with the identification of pathogenic variants in PCD-related genes.
Assuntos
Síndrome de Kartagener , Adolescente , Brasil/epidemiologia , Cílios , Humanos , Síndrome de Kartagener/diagnóstico , Síndrome de Kartagener/genética , Síndrome de Kartagener/patologia , Microscopia Eletrônica de Transmissão , Centros de Atenção TerciáriaRESUMO
Background: Primary ciliary dyskinesia (PCD) is a clinical syndrome characterized by cilia with an abnormal structure or function. Its main clinical manifestations comprise chronic bronchitis, cough, recurrent respiratory infections, situs inversus, and male infertility. Single-gene variants are widely assumed to be the main cause of this rare disease, and more than 40 genes have been described to be associated with its onset. CCDC39 is essential for assembling the inner dynein arms and dynein regulatory complex and is important in cilia motility. CCDC39 variants were reported as a monogenic etiology of PCD. Methods: This study investigated two unrelated Chinese patients diagnosed as PCD. The chest computed tomography scan was performed to identify PCD phenotypes of the two probands. Considering the effect of PCD on male fertility, routine semen analysis, sperm morphology examination, and scanning electron microscopy were performed to assess the semen characteristics of male proband in family 2 (F2 II-1), who had a history of infertility. Subsequently, the peripheral blood samples of probands were collected to perform whole-exome sequencing (WES) to explore the possible genetic causes of this disease. Results: Whole-exome sequencing revealed a homozygous CCDC39 variant in the female proband of family 1 (F1 II-1: c.286C>T:p.Arg96Ter) and two compound heterozygous CCDC39 variants in the male proband of family 2 (F2 II-1: c.732_733del: p.Ala245PhefsTer18; c.2800_2802dup:p.Val934dup). The two probands showed the typical PCD phenotypes, including chronic bronchitis, recurrent respiratory infections, and situs inversus. The male proband also showed oligoasthenoteratospermia with multiple morphological abnormalities of the sperm flagella. Additionally, CCDC39 protein level was significantly lower in the sperm of male proband than in the sperm from normal controls. Conclusion: We identified a homozygous variant reported previously and two compound heterozygous variants of CCDC39 possibly responsible for PCD pathogenesis, expanding the variant spectrum of Chinese PCD, Kartagener syndrome, and morphological abnormalities of the sperm flagella involving CCDC39.
Assuntos
Anormalidades Múltiplas , Bronquite Crônica , Proteínas do Citoesqueleto , Síndrome de Kartagener , Anormalidades Múltiplas/patologia , Bronquite Crônica/patologia , Cílios/genética , Cílios/patologia , Proteínas do Citoesqueleto/genética , Dineínas/genética , Feminino , Humanos , Síndrome de Kartagener/genética , Síndrome de Kartagener/patologia , Masculino , SêmenRESUMO
Primary cilia dyskinesia (PCD) is a rare genetic disease caused by ciliary structural or functional defects. It causes severe outcomes in patients, including recurrent upper and lower airway infections, progressive lung failure, and randomization of heterotaxy. To date, although 50 genes have been shown to be responsible for PCD, the etiology remains elusive. Meanwhile, owing to the lack of a model mimicking the pathogenesis that can be used as a drug screening platform, thereby slowing the development of related therapies. In the current study, we identified compound mutation of DNAH9 in a patient with PCD with the following clinical features: recurrent respiratory tract infections, low lung function, and ultrastructural defects of the outer dynein arms (ODAs). Bioinformatic analysis, structure simulation assay, and western blot analysis showed that the mutations affected the structure and expression of DNAH9 protein. Dnah9 knock-down (KD) mice recapitulated the patient phenotypes, including low lung function, mucin accumulation, and increased immune cell infiltration. Immunostaining, western blot, and co-immunoprecipitation analyses were performed to clarify that DNAH9 interacted with CCDC114/GAS8 and diminished their protein levels. Furthermore, we constructed an airway organoid of Dnah9 KD mice and discovered that it could mimic the key features of the PCD phenotypes. We then used organoid as a drug screening model to identify mitochondrial-targeting drugs that can partially elevate cilia beating in Dnah9 KD organoid. Collectively, our results demonstrated that Dnah9 KD mice and an organoid model can recapture the clinical features of patients with PCD and provide an excellent drug screening platform for human ciliopathies.
Assuntos
Dineínas do Axonema , Discinesias , Síndrome de Kartagener , Animais , Dineínas do Axonema/genética , Dineínas do Axonema/metabolismo , Cílios/metabolismo , Avaliação Pré-Clínica de Medicamentos , Dineínas/metabolismo , Discinesias/metabolismo , Discinesias/patologia , Humanos , Síndrome de Kartagener/genética , Síndrome de Kartagener/metabolismo , Síndrome de Kartagener/patologia , Camundongos , Mutação/genética , Organoides/metabolismoRESUMO
Congenital hydrocephalus affects approximately one in 1000 newborn children and is fatal in approximately 50% of untreated cases. The currently known management protocols usually necessitate multiple interventions and long-term use of healthcare resources due to a relatively high incidence of complications, and many of them mostly provide a treatment of the effect rather than the cause of cerebrospinal fluid flow reduction or outflow obstruction. Future studies discussing etiology specific hydrocephalus alternative treatments are needed. We systematically reviewed the available literature on the effect of ciliary abnormality on congenital hydrocephalus pathogenesis, to open a discussion on the feasibility of factoring ciliary abnormality in future research on hydrocephalus treatment modalities. Although there are different forms of ciliopathies, we focused in this review on primary ciliary dyskinesia. There is growing evidence of association of other ciliary syndromes and hydrocephalus, such as the reduced generation of multiple motile cilia, which is distinct from primary ciliary dyskinesia. Data for this review were identified by searching PubMed using the search terms 'hydrocephalus,' 'Kartagener syndrome,' 'primary ciliary dyskinesia,' and 'immotile cilia syndrome.' Only articles published in English and reporting human patients were included. Seven studies met our inclusion criteria, reporting 12 cases of hydrocephalus associated with primary ciliary dyskinesia. The patients had variable clinical presentations, genetic backgrounds, and ciliary defects. The ependymal water propelling cilia differ in structure and function from the mucus propelling cilia, and there is a possibility of isolated non-syndromic ependymal ciliopathy causing only hydrocephalus with growing evidence in the literature for the association ependymal ciliary abnormality and hydrocephalus. Abdominal and thoracic situs in children with hydrocephalus can be evaluated, and secondary damage of ependymal cilia causing hydrocephalus in cases with generalized ciliary abnormality can be considered.
Assuntos
Hidrocefalia , Síndrome de Kartagener , Cílios/genética , Cílios/patologia , Epêndima/patologia , Humanos , Hidrocefalia/etiologia , Hidrocefalia/patologia , Recém-Nascido , Síndrome de Kartagener/complicações , Síndrome de Kartagener/genética , Síndrome de Kartagener/patologiaRESUMO
Primary ciliary dyskinesia (PCD) is a congenital disorder predominantly inherited in an autosomal recessive trait. The disorder causes disturbance in the motion of cilia, leading to severe impairment of mucociliary clearance (MCC). If undiagnosed or diagnosed too late, the condition leads to the development of bronchiectasis and serious damage to the lungs in later life. Most of the methods for diagnosing PCD are time-consuming and demand extensive economic resources to establish them. High-speed video microscopy analysis (HSVMA) is the only diagnostic tool to visualize and analyze living respiratory cells with beating cilia in vitro. It is fast, cost-effective, and, in experienced hands, very reliable as a diagnostic tool for PCD. In addition, classical diagnostic measures such as transmission electron microscopy (TEM) are not applicable for some mutations as morphological changes are absent. This paper describes the process of collecting respiratory epithelial cells, the further preparation of the specimen, and the process of HSVMA. We also describe how brushed cells can be successfully kept unharmed and beating by keeping them in a nourishing medium for storage and transport to the investigation site in cases where a clinic does not possess the equipment to perform HSVMA. Also shown are videos with pathologic beating patterns from patients with a mutation in the dynein arm heavy chain 11 gene (DNAH11), which cannot be diagnosed with TEM; the result of an inconclusive HSVMA due to infection of the upper airways, as well as an unsuccessful brushing with superimposition of red blood cells. With this article, we would like to encourage every unit dealing with pulmonology patients and rare lung diseases to perform HSVMA as part of their daily routine diagnostics for PCD or send the specimens over to a center specializing in performing HSVMA.
Assuntos
Síndrome de Kartagener , Cílios/metabolismo , Dineínas/metabolismo , Humanos , Síndrome de Kartagener/diagnóstico , Síndrome de Kartagener/genética , Síndrome de Kartagener/patologia , Microscopia de Vídeo , FenótipoRESUMO
Although ubiquitously present, the relevance of cilia for vertebrate development and health has long been underrated. However, the aberration or dysfunction of ciliary structures or components results in a large heterogeneous group of disorders in mammals, termed ciliopathies. The majority of human ciliopathy cases are caused by malfunction of the ciliary dynein motor activity, powering retrograde intraflagellar transport (enabled by the cytoplasmic dynein-2 complex) or axonemal movement (axonemal dynein complexes). Despite a partially shared evolutionary developmental path and shared ciliary localization, the cytoplasmic dynein-2 and axonemal dynein functions are markedly different: while cytoplasmic dynein-2 complex dysfunction results in an ultra-rare syndromal skeleto-renal phenotype with a high lethality, axonemal dynein dysfunction is associated with a motile cilia dysfunction disorder, primary ciliary dyskinesia (PCD) or Kartagener syndrome, causing recurrent airway infection, degenerative lung disease, laterality defects, and infertility. In this review, we provide an overview of ciliary dynein complex compositions, their functions, clinical disease hallmarks of ciliary dynein disorders, presumed underlying pathomechanisms, and novel developments in the field.
Assuntos
Dineínas do Axonema/metabolismo , Ciliopatias/patologia , Dineínas do Citoplasma/metabolismo , Animais , Dineínas do Axonema/química , Dineínas do Axonema/genética , Cílios/metabolismo , Ciliopatias/genética , Ciliopatias/metabolismo , Dineínas do Citoplasma/química , Dineínas do Citoplasma/genética , Humanos , Síndrome de Kartagener/genética , Síndrome de Kartagener/metabolismo , Síndrome de Kartagener/patologia , Polimorfismo Genético , Síndrome de Costela Curta e Polidactilia/genética , Síndrome de Costela Curta e Polidactilia/metabolismo , Síndrome de Costela Curta e Polidactilia/patologiaRESUMO
Primary ciliary dyskinesia (PCD) is a genetically heterogeneous disorder of motile cilia. With few exceptions, PCD is an autosomal recessive condition, and there are over 40 genes associated with the condition. We present a case of a newborn female with clinical features of PCD, specifically the Kartagener syndrome phenotype, due to variants in TTC25. This gene has been previously associated with PCD in three families. Two multi-gene panels performed as a neonate and at two years of age were uninformative. Exome sequencing was performed by the Care4Rare Canada Consortium on a research basis, and an apparent homozygous intronic variant (TTC25:c.1145+1G > A) was identified that was predicted to abolish the canonical splice donor activity of exon 8. The child's mother was a heterozygous carrier of the variant. The paternal sample did not show the splice variant, and homozygosity was observed across the paternal locus. Microarray analysis showed a 50 kb heterozygous deletion spanning the genes TTC25 and CNP. This is the first example of a pathogenic gross deletion in trans with a splice variant, resulting in TTC25-related PCD.
Assuntos
Proteínas de Transporte/genética , Deleção de Genes , Síndrome de Kartagener/genética , Proteínas de Transporte/metabolismo , Variações do Número de Cópias de DNA , Feminino , Humanos , Recém-Nascido , Síndrome de Kartagener/patologia , Sítios de Splice de RNARESUMO
BACKGROUND: Kartagener syndrome is an autosomal recessive inherited disorder of primary ciliary dyskinesia. Moyamoya syndrome refers to a moyamoya angiopathy associated with other neurological and/or extra-neurological symptoms, or due to a well identified acquired or inherited cause. We herein reported a case of a 48-year-old woman who was favored the diagnosis of Kartagener syndrome and moyamoya syndrome. The whole genome sequencing and bioinformatics analysis showed a homozygotic nonsense mutation in the dynein, axonemal, heavy chain (DNAH) 5 gene, and heterozygotic missense mutation in the DNAH11 gene. This is the first report of the co-occurrence of the two rare diseases. CASE PRESENTATION: A case of a 48-year-old woman was presented with hemiplegia and slurred speech. The magnetic resonance imaging of the brain confirmed acute cerebral infarction in the right basal ganglia region, semi-oval center, insular lobe, and frontal parietal lobe. The electrocardiogram showed inverted "P" waves in L1 and AVL on left-sided chest leads and computed tomography scan of the chest showed bronchiectasis changes, cardiac shadow and apex on the right side, and situs inversus of aortic arch position. The digital subtraction angiography showed inversion of the aortic arch, and bilateral internal carotid arteries are occluded from the ophthalmic segment. The clinical, radiological, and laboratory findings made the diagnosis of Kartagener syndrome and moyamoya syndrome. The whole genome sequencing and bioinformatics analysis showed a homozygotic nonsense mutation in DNAH5 gene, and heterozygotic missense mutation in the DNAH11 gene. CONCLUSION: The combined mutation of DNAH5 and DNAH11 may lead to the overlapping dysfunction of motile and nonmotile cilia, which contribute to the co-occurrence of Kartagener syndrome and moyamoya syndrome. Our report deserves further confirm by more case reports.
Assuntos
Dineínas do Axonema/genética , Síndrome de Kartagener/diagnóstico , Doença de Moyamoya/diagnóstico , Encéfalo/patologia , Feminino , Humanos , Síndrome de Kartagener/genética , Síndrome de Kartagener/patologia , Pessoa de Meia-Idade , Mutação , Mutação de Sentido Incorreto , Sequenciamento Completo do GenomaRESUMO
Primary ciliary dyskinesia (PCD) is a genetically heterogeneous syndrome that results from defects in motile cilia. The ciliary axoneme has a 9 + 2 microtubule structure consisting of nine peripheral doublets surrounding a central pair apparatus (CPA), which plays a critical role in regulating proper ciliary function. We have previously shown that mouse models with mutations in CPA genes CFAP221, CFAP54, and SPEF2 have a PCD phenotype with defects in ciliary motility. In this study, we investigated potential genetic interaction between these CPA genes by generating each combination of double heterozygous and double homozygous mutants. No detectable cilia-related phenotypes were observed in double heterozygotes, but all three double homozygous mutant lines exhibit early mortality and typically develop severe PCD-associated phenotypes of hydrocephalus, mucociliary clearance defects in the upper airway, and abnormal spermatogenesis. Double homozygous cilia are generally intact and display a normal morphology and distribution. Spermiogenesis is aborted in double homozygotes, with an absence of mature flagella on elongating spermatids and epididymal sperm. These findings identify genetic interactions between CPA genes and genetic mechanisms regulating the CPA and motile cilia function.
Assuntos
Proteínas de Ligação a Calmodulina , Cílios , Síndrome de Kartagener , Proteínas Associadas aos Microtúbulos , Proteínas , Animais , Proteínas de Ligação a Calmodulina/genética , Proteínas de Ligação a Calmodulina/metabolismo , Cílios/genética , Cílios/metabolismo , Cílios/patologia , Modelos Animais de Doenças , Síndrome de Kartagener/genética , Síndrome de Kartagener/metabolismo , Síndrome de Kartagener/patologia , Masculino , Camundongos , Camundongos Mutantes , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas/genética , Proteínas/metabolismo , Espermátides/metabolismo , Espermátides/patologia , Espermatogênese/genéticaRESUMO
PURPOSE: Primary ciliary dyskinesia (PCD) is a ciliopathy caused by dysfunction of motile cilia. As there is still no standard PCD diagnostics, the final diagnosis requires a combination of several tests. The genetic screening is a hallmark for the final diagnosis and requires high-throughput techniques, such as whole-exome sequencing (WES). Nevertheless, WES has limitations that may prevent a definitive genetic diagnosis. Here we present a case that demonstrates how the PCD genetic diagnosis may not be trivial. MATERIALS/METHODS: A child with PCD and situs inversus totalis (designated as Kartagener syndrome (KS)) was subjected to clinical assessments, ultrastructural analysis of motile cilia, extensive genetic evaluation by WES and chromosomal array analysis, bioinformatic analysis, gene expression analysis and immunofluorescence to identify the genetic etiology. His parents and sister, as well as healthy controls were also evaluated. RESULTS: Here we show that a disease-causing variant in the USP11 gene and copy number variations in CRHR1 and KRT34 genes may be involved in the patient PCD phenotype. None of these genes were previously reported in PCD patients and here we firstly show its presence and immunolocalization in respiratory cells. CONCLUSIONS: This work highlights how the genetic diagnosis can turn to be rather complex and that combining several approaches may be needed. Overall, our results contribute to increase the understanding of the genetic factors involved in the pathophysiology of PCD/KS, which is of paramount importance to assist the current diagnosis and future development of newer therapies.
Assuntos
Síndrome de Kartagener/etiologia , Queratinas Específicas do Cabelo/genética , Queratinas Tipo I/genética , Mutação , Receptores de Hormônio Liberador da Corticotropina/genética , Tioléster Hidrolases/genética , Pré-Escolar , Cílios/metabolismo , Cílios/patologia , Variações do Número de Cópias de DNA , Feminino , Testes Genéticos , Humanos , Síndrome de Kartagener/patologia , Masculino , NADPH Oxidase 2/genética , Linhagem , FenótipoRESUMO
Primary ciliary dyskinesia (PCD) is a rare autosomal recessive disorder characterized by dysfunction of motile cilia causing ineffective mucus clearance and organ laterality defects. In this study, two unrelated Portuguese children with strong PCD suspicion underwent extensive clinical and genetic assessments by whole-exome sequencing (WES), as well as ultrastructural analysis of cilia by transmission electron microscopy (TEM) to identify their genetic etiology. These analyses confirmed the diagnostic of Kartagener syndrome (KS) (PCD with situs inversus). Patient-1 showed a predominance of the absence of the inner dynein arms with two disease-causing variants in the CCDC40 gene. Patient-2 showed the absence of both dynein arms and WES disclosed two novel high impact variants in the DNAH5 gene and two missense variants in the DNAH7 gene, all possibly deleterious. Moreover, in Patient-2, functional data revealed a reduction of gene expression and protein mislocalization in both genes' products. Our work calls the researcher's attention to the complexity of the PCD and to the possibility of gene interactions modelling the PCD phenotype. Further, it is demonstrated that even for well-known PCD genes, novel pathogenic variants could have importance for a PCD/KS diagnosis, reinforcing the difficulty of providing genetic counselling and prenatal diagnosis to families.
Assuntos
Cílios/ultraestrutura , Síndrome de Kartagener/genética , Síndrome de Kartagener/patologia , Dineínas do Axonema/genética , Criança , Dineínas/genética , Feminino , Testes Genéticos , Humanos , Masculino , Mutação , Portugal , Proteínas/genética , Sequenciamento do Exoma/métodosRESUMO
BACKGROUND: Primary ciliary dyskinesia (PCD) is a rare genetic disorder. Although the genetic tests and new diagnostic algorithms have recently been recommended, clinical signs and electron microscope (EM) findings have historically been the mainstays of diagnosis in Asia. To characterize PCD previously reported in Japan, we conducted a systematic review and meta-analysis. METHODS: A search using MEDLINE, EMBASE, and Japana Centra Revuo Medicina (in Japanese) databases was carried out to identify articles reporting PCD, Kartagener syndrome, or immotile cilia syndrome in Japanese patients and published between 1985 and 2015. RESULTS: After excluding duplication from 334 reports, we extracted 316 patients according to the criteria. Diagnosis was most frequently made in adulthood (148 patients [46.8%] ≥ 18 years old, 24 patients [7.6%] < 1 year old, 68 patients [21.5%] 1-17 years old and 76 patients [24.1%] lacking information). Of the 230 patients (72.8%) who received EM examination, there were patients with inner dynein arm (IDA) defects (n = 55; 23.9%), outer dynein arm (ODA) defects (14; 6.1%), both ODA and IDA defects (57; 24.8%), other structural abnormalities (25; 10.9%), no abnormalities (4; 1.7%), and no detailed conclusion or description (75; 32.6%). CONCLUSION: Delayed diagnosis of this congenital disease with high frequency of IDA defects and low frequency of ODA defects appear to be historical features of PCD reported in Japan, when EM was a main diagnostic tool. This review highlights problems experienced in this field, and provides basic information to establish a modernized PCD diagnosis and management system in the future.
Assuntos
Dineínas/deficiência , Síndrome de Kartagener/diagnóstico , Cílios/fisiologia , Cílios/ultraestrutura , Diagnóstico Tardio , Dineínas/ultraestrutura , Humanos , Japão , Síndrome de Kartagener/patologia , Microscopia EletrônicaRESUMO
PROPOSE: To study CCDC103 expression profiles and understand how pathogenic variants in CCDC103 affect its expression profile at mRNA and protein level. METHODS: To increase the knowledge about the CCDC103, we attempted genotype-phenotype correlations in two patients carrying novel homozygous (missense and frameshift) CCDC103 variants. Whole-exome sequencing, quantitative PCR, Western blot, electron microscopy, immunohistochemistry, immunocytochemistry, and immunogold labelling were performed to characterize CCDC103 expression profiles in reproductive and somatic cells. RESULTS: Our data demonstrate that pathogenic variants in CCDC103 gene negatively affect gene and protein expression in both patients who presented absence of DA on their axonemes. Further, we firstly report that CCDC103 is expressed at different levels in reproductive tissues and somatic cells and described that CCDC103 protein forms oligomers with tissue-specific sizes, which suggests that CCDC103 possibly undergoes post-translational modifications. Moreover, we reported that CCDC103 was restricted to the midpiece of sperm and is present at the cytoplasm of the other cells. CONCLUSIONS: Overall, our data support the CCDC103 involvement in PCD and suggest that CCDC103 may have different assemblies and roles in cilia and sperm flagella biology that are still unexplored.
Assuntos
Axonema/patologia , Transtornos da Motilidade Ciliar/genética , Infertilidade Masculina/patologia , Síndrome de Kartagener/genética , Proteínas Associadas aos Microtúbulos/genética , Mutação , Cauda do Espermatozoide/patologia , Axonema/genética , Transtornos da Motilidade Ciliar/patologia , Dineínas/metabolismo , Feminino , Humanos , Infertilidade Masculina/etiologia , Síndrome de Kartagener/patologia , Masculino , Pessoa de Meia-Idade , Reprodução , Situs Inversus/genética , Situs Inversus/patologia , Cauda do Espermatozoide/metabolismoRESUMO
OBJECTIVE: To determine whether adenoid epithelium is superior to nasal mucosa for biopsy of ciliated epithelium for electron microscopy (EM) to evaluate pediatric patients with rhinosinusitis for primary ciliary dyskinesia (PCD). METHODS: A retrospective review compared electron microscopic results in children with chronic or recurrent rhinosinusitis who underwent both adenoidectomy or nasopharyngeal biopsy and nasal mucosa biopsy in the course of evaluation for PCD at a tertiary care institution. RESULTS: Forty pediatric patients met inclusion criteria for this study. Nine of these patients had a prior adenoidectomy and therefore underwent nasopharyngeal biopsy for collection of adenoid tissue. All nine of the nasopharyngeal biopsies and 25 of the 31 (80.6%) adenoid biopsies had sufficient cilia for EM evaluation of the ultrastructure. Of the 40 patients who also had a nasal biopsy, only 12 (30.0%) had sufficient cilia for EM analysis. The distribution of sufficient versus insufficient cilia for analysis between adenoid and nasal mucosa was statistically significant (Pâ¯<â¯0.05). Abnormal cilia were found in only 2.5% of our patients. CONCLUSIONS: In current practice, the nasal cavity is a common location for obtaining ciliated epithelium for EM analysis, as it is easily accessible for biopsy and the procedure itself causes relatively low patient morbidity. Chronic rhinosinusitis, however, has been associated with decreased cilia density on nasal respiratory epithelium. Given that adenoidectomies are often performed in children with chronic rhinosinusitis, our data suggest that adenoid tissue is a better source of ciliated tissue for analysis compared to turbinate epithelium.
Assuntos
Tonsila Faríngea/patologia , Síndrome de Kartagener/patologia , Mucosa Nasal/patologia , Rinite/patologia , Sinusite/patologia , Adenoidectomia , Biópsia/métodos , Criança , Pré-Escolar , Doença Crônica , Cílios/patologia , Humanos , Lactente , Microscopia Eletrônica , Recidiva , Estudos RetrospectivosRESUMO
BACKGROUND: Primary ciliary dyskinesia can result from a number of different ciliary defects that adversely affect ciliary function resulting markedly reduced or absent mucociliary clearance. Improvement in diagnostic testing is an area of current research. During diagnostic evaluation of PCD we observed ciliated conical protrusions from part of the apical surface of ciliated cells in those diagnosed with PCD. The aim of this study was to investigate if this abnormality was specific to PCD. METHODS: Epithelial edges from 67 consecutively diagnosed PCD patients, 67 patients consecutively referred for PCD diagnostic testing in whom PCD was excluded, 22 with asthma and 18 with Cystic Fibrosis (CF) were studied retrospectively in a blinded manner using light microscopy. RESULTS: Forty six out of 67 patients with PCD had ciliated conical epithelial protrusions, whereas none were seen in patients where PCD was excluded, or in patients with asthma or CF. The sensitivity, specificity, positive predictive value and negative predictive value for the presence of the ciliated conical protrusions to predict a diagnosis of PCD were 76.5, 100, 100 and 77% respectively. CONCLUSIONS: Characteristic ciliated conical protrusions from ciliated epithelial cells maybe a useful pointer to the diagnosis of PCD. However, their absence does not exclude the diagnosis of PCD.