RESUMO
Soon, the genetic basis of most human Mendelian diseases will be solved. The next challenge will be to leverage this information to uncover basic mechanisms of disease and develop new therapies. To understand how this transformation is already beginning to unfold, we focus on the ciliopathies, a class of multi-organ diseases caused by disruption of the primary cilium. Through a convergence of data involving mutant gene discovery, proteomics, and cell biology, more than a dozen phenotypically distinguishable conditions are now united as ciliopathies. Sitting at the interface between simple and complex genetic conditions, these diseases provide clues to the future direction of human genetics.
Assuntos
Cílios/genética , Doença/genética , Animais , Transtornos da Motilidade Ciliar/genética , Humanos , SíndromeRESUMO
Nephronophthisis (NPHP), Joubert (JBTS), and Meckel-Gruber (MKS) syndromes are autosomal-recessive ciliopathies presenting with cystic kidneys, retinal degeneration, and cerebellar/neural tube malformation. Whether defects in kidney, retinal, or neural disease primarily involve ciliary, Hedgehog, or cell polarity pathways remains unclear. Using high-confidence proteomics, we identified 850 interactors copurifying with nine NPHP/JBTS/MKS proteins and discovered three connected modules: "NPHP1-4-8" functioning at the apical surface, "NPHP5-6" at centrosomes, and "MKS" linked to Hedgehog signaling. Assays for ciliogenesis and epithelial morphogenesis in 3D renal cultures link renal cystic disease to apical organization defects, whereas ciliary and Hedgehog pathway defects lead to retinal or neural deficits. Using 38 interactors as candidates, linkage and sequencing analysis of 250 patients identified ATXN10 and TCTN2 as new NPHP-JBTS genes, and our Tctn2 mouse knockout shows neural tube and Hedgehog signaling defects. Our study further illustrates the power of linking proteomic networks and human genetics to uncover critical disease pathways.
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Doenças Renais Císticas/genética , Proteínas de Membrana/genética , Transdução de Sinais , Animais , Ataxina-10 , Centrossomo/metabolismo , Cílios/metabolismo , Transtornos da Motilidade Ciliar/genética , Encefalocele/genética , Proteínas Hedgehog/metabolismo , Humanos , Doenças Renais Císticas/metabolismo , Camundongos , Células NIH 3T3 , Proteínas do Tecido Nervoso/genética , Doenças Renais Policísticas/genética , Retinose Pigmentar , Peixe-ZebraRESUMO
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.
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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
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
BACKGROUND: Primary ciliary dyskinesia (PCD) is an autosomal recessive hereditary disease characterized by recurrent respiratory infections. In clinical manifestations, DNAH5 (NM_001361.3) is one of the recessive pathogenic genes. Primary familial brain calcification (PFBC) is a neurodegenerative disease characterized by bilateral calcification in the basal ganglia and other brain regions. PFBC can be inherited in an autosomal dominant or recessive manner. A family with PCD caused by a DNAH5 compound heterozygous variant and PFBC caused by a MYORG homozygous variant was analyzed. METHODS: In this study, we recruited three generations of Han families with primary ciliary dyskinesia combined with primary familial brain calcification. Their clinical phenotype data were collected, next-generation sequencing was performed to screen suspected pathogenic mutations in the proband and segregation analysis of families was carried out by Sanger sequencing. The mutant and wild-type plasmids were constructed and transfected into HEK293T cells instantaneously, and splicing patterns were detected by Minigene splicing assay. The structure and function of mutations were analyzed by bioinformatics analysis. RESULTS: The clinical phenotypes of the proband (II10) and his sister (II8) were bronchiectasis, recurrent pulmonary infection, multiple symmetric calcifications of bilateral globus pallidus and cerebellar dentate nucleus, paranasal sinusitis in the whole group, and electron microscopy of bronchial mucosa showed that the ciliary axoneme was defective. There was also total visceral inversion in II10 but not in II8. A novel splice variant C.13,338 + 5G > C and a frameshift variant C.4314delT (p. Asn1438lysfs *10) were found in the DNAH5 gene in proband (II10) and II8. c.347_348dupCTGGCCTTCCGC homozygous insertion variation was found in the MYORG of the proband. The two pathogenic genes were co-segregated in the family. Minigene showed that DNAH5 c.13,338 + 5G > C has two abnormal splicing modes: One is that part of the intron bases where the mutation site located is translated, resulting in early translation termination of DNAH5; The other is the mutation resulting in the deletion of exon76. CONCLUSIONS: The newly identified DNAH5 splicing mutation c.13,338 + 5G > C is involved in the pathogenesis of PCD in the family, and forms a compound heterozygote with the pathogenic variant DNAH5 c.4314delT lead to the pathogenesis of PCD.
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Calcinose , Mutação , Linhagem , Humanos , Masculino , Calcinose/genética , Calcinose/patologia , Feminino , Dineínas do Axonema/genética , Adulto , Transtornos da Motilidade Ciliar/genética , Encefalopatias/genética , Fenótipo , Células HEK293 , China , Splicing de RNA/genética , Pessoa de Meia-Idade , Glicosídeo HidrolasesRESUMO
INTRODUCTION: Meckel-Gruber Syndrome (MKS) is an autosomal recessive genetic disorder, notable for its triad of occipital encephalocele, polycystic renal dysplasia, and postaxial polydactyly. Identified by Johann Friederich Meckel in 1822, MKS is categorized as a ciliopathy due to gene mutations. Diagnosis is confirmed by the presence of at least two key features. The condition is incompatible with life, leading to death in the womb or shortly after birth. Recent studies have largely focused on the genetic aspects of MKS, with limited information regarding the impact of neurosurgical approaches, particularly in treating encephaloceles. METHODS: A systematic review was performed according to the PRISMA statement. The PubMed, Embase, and Web of Science databases were consulted for data screening and extraction, which was conducted by two independent reviewers. The search strategy aimed to encompass studies documenting cases of MKS with published reports of encephalocele excisions, and the search strings for all databases were: Meckel-Gruber syndrome OR Meckel Gruber syndrome OR Meckel-gruber OR Meckel Gruber. RESULTS: The study included 10 newborns with MKS associated with occipital encephalocele or meningocele, all of whom underwent surgical repair of the occipital sac. The mean gestational age at birth was 36 (± 2) weeks. The mean of birth weight was 3.14 (± 0.85) kilograms. The average head circumference at birth was 33.82 cm (± 2.17). The mean diameter of the encephalocele/meningocele was 5.91 (± 1.02) cm. Other common central nervous system abnormalities included hydrocephalus, Dandy-Walker malformation, and agenesis of the corpus callosum. 40% required shunting for hydrocephalus. Surgery to remove the occipital sac occurred at a median age of 2.5 days (1.5-6.5). The most common post-surgical complication was the need for mechanical ventilation. The most common cause of death was pneumonia and the median age at death was 6.66 (0.03-18) months. CONCLUSION: Our findings suggest that neurosurgical intervention, especially for managing encephaloceles, may offer some improvement in survival, albeit within a context of generally poor prognosis. However, these results should be interpreted with caution.
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Transtornos da Motilidade Ciliar , Encefalocele , Procedimentos Neurocirúrgicos , Doenças Renais Policísticas , Retinose Pigmentar , Humanos , Encefalocele/cirurgia , Encefalocele/diagnóstico por imagem , Retinose Pigmentar/cirurgia , Procedimentos Neurocirúrgicos/métodos , Transtornos da Motilidade Ciliar/cirurgia , Transtornos da Motilidade Ciliar/genética , Doenças Renais Policísticas/cirurgia , Doenças Renais Policísticas/genética , Anormalidades do Olho/cirurgia , Recém-NascidoRESUMO
PURPOSE: This study aimed to identify the genetic causes of male infertility and primary ciliary dyskinesia (PCD)/PCD-like phenotypes in three unrelated Han Chinese families. METHODS: We conducted whole-exome sequencing of three patients with male infertility and PCD/PCD-like phenotypes from three unrelated Chinese families. Ultrastructural and immunostaining analyses of patient spermatozoa and respiratory cilia and in vitro analyses were performed to analyze the effects of SPEF2 variants. Intracytoplasmic sperm injection (ICSI) was administered to three affected patients. RESULTS: We identified four novel SPEF2 variants, including one novel homozygous splicing site variant [NC_000005.10(NM_024867.4): c.4447 + 1G > A] of the SPEF2 gene in family 1, novel compound heterozygous nonsense variants [NC_000005.10(NM_024867.4): c.1339C > T (p.R447*) and NC_000005.10(NM_024867.4): c.1645G > T (p.E549*)] in family 2, and one novel homozygous missense variant [NC_000005.10(NM_024867.4): c.2524G > A (p.D842N)] in family 3. All the patients presented with male infertility and PCD/likely PCD. All variants were present at very low levels in public databases, predicted to be deleterious in silico prediction tools, and were further confirmed deleterious by in vitro analyses. Ultrastructural analyses of the spermatozoa of the patients revealed the absence of the central pair complex in the sperm flagella. Immunostaining of the spermatozoa and respiratory cilia of the patients validated the pathogenicity of the SPEF2 variants. All patients carrying SPEF2 variants underwent one ICSI cycle and delivered healthy infants. CONCLUSION: Our study reported four novel pathogenic variants of SPEF2 in three male patients with infertility and PCD/PCD-like phenotypes, which not only extend the spectrum of SPEF2 mutations but also provide information for genetic counseling and treatment of such conditions.
Assuntos
Infertilidade Masculina , Linhagem , Injeções de Esperma Intracitoplásmicas , Espermatozoides , Adulto , Humanos , Masculino , China , Cílios/genética , Cílios/patologia , Cílios/ultraestrutura , Transtornos da Motilidade Ciliar/genética , Transtornos da Motilidade Ciliar/patologia , Sequenciamento do Exoma , Homozigoto , Infertilidade Masculina/genética , Infertilidade Masculina/patologia , Mutação/genética , Fenótipo , Espermatozoides/patologia , Espermatozoides/ultraestrutura , Espermatozoides/metabolismoRESUMO
OBJECTIVE: To carry out prenatal diagnosis for a fetus with Meckel syndrome (MKS) and explore its genetic basis. METHODS: A pregnant woman presented at Suzhou Municipal Hospital in February 2018 was selected as the study subject. Clinical data was collected. Muscle tissue sample from the abortus and peripheral blood samples from the couple were collected. Genomic DNA was extracted and subjected to chromosomal microarray analysis (CMA) and whole exome sequencing. Candidate variant was verified by Sanger sequencing. RESULTS: The fetus was found to have microcephaly, oligohydramnios, polycystic kidneys and banana-shaped cerebellum at 18 weeks of gestation. After induction of labor, it was found to have encephalocele, renal cysts and polydactyly. CMA has found no abnormality. Whole exome sequencing revealed novel compound heterozygous variants c.296delA (p.Lys99SerfsTer6) and c.1243G>A (p.Val415Met) in the TMEM67 gene. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), the c.296delA variant was predicted to be pathogenic (PVS1+PM2_Supporting+PP4), whilst the c.1243G>A variant was predicted to be likely pathogenic (PM2_Supporting+PM3+PP3_Moderate+PP4). CONCLUSION: The c.296delA and c.1243G>A compound heterozygous variants of the TMEM67 gene probably underlay the MKS in this fetus.
Assuntos
Transtornos da Motilidade Ciliar , Doenças Renais Policísticas , Retinose Pigmentar , Feminino , Gravidez , Humanos , Encefalocele/genética , Doenças Renais Policísticas/genética , Feto , Transtornos da Motilidade Ciliar/genética , Mutação , Proteínas de Membrana/genéticaRESUMO
OBJECTIVE: To investigate the clinical and genetic characteristics of a case of primary ciliary dyskinesia (PCD). METHODS: We collected the clinical data on a case of PCD treated in the Department of Reproductive Medicine of Linyi People's Hospital in July 2020, detected the genes of the patient by whole-exome sequencing (WES), verified the candidate mutations by Sanger sequencing, and predicted the protein structure of the mutant gene by SWISS-MODEL. RESULTS: The proband was found with the clinical phenotypes of chronic rhinitis, bronchiectasis, visceral transposition and male infertility. WES revealed a homozygous frameshift variation of c.12890dup (p.N4297Kfs*13) in exon 74 of the DNAH5 gene, which led to the premature termination of polypeptide chain synthesis and affected the gene function. SWISS-MODEL prediction showed that some of the amino acid residues were deleted after mutation, resulting in a 3D conformational change of the protein. This variation was not recorded in the ClinVar, gnomAD and OMIM databases and, according to the relevant guidelines of the American College of Genetics and Genomics, was classified as a pathogenic variation (PVS1+PM2_P+PM3_P). CONCLUSION: The homozygous variation of the DNAH5 gene c.12890dup (p.N4297Kfs*13) may be the cause of the clinical phenotype of this case of PCD, and the above findings have enriched the variation spectrum of the DNAH5 gene.
Assuntos
Sequenciamento do Exoma , Mutação da Fase de Leitura , Humanos , Masculino , Dineínas do Axonema/genética , Fenótipo , Homozigoto , Transtornos da Motilidade Ciliar/genética , Éxons , Infertilidade Masculina/genéticaRESUMO
A finely regulated system of airway epithelial development governs the differentiation of motile ciliated cells of the human respiratory tract, conferring the body's mucociliary clearance defence system. Human cilia dysfunction can arise through genetic mutations and this is a cause of debilitating disease morbidities that confer a greatly reduced quality of life. The inherited human motile ciliopathy disorder, primary ciliary dyskinesia (PCD), can arise from mutations in genes affecting various aspects of motile cilia structure and function through deficient production, transport and assembly of cilia motility components or through defective multiciliogenesis. Our understanding about the development of the respiratory epithelium, motile cilia biology and the implications for human pathology has expanded greatly over the past 20 years since isolation of the first PCD gene, rising to now nearly 50 genes. Systems level insights about cilia motility in health and disease have been made possible through intensive molecular and omics (genomics, transcriptomics, proteomics) research, applied in ciliate organisms and in animal and human disease modelling. Here, we review ciliated airway development and the genetic stratification that underlies PCD, for which the underlying genotype can increasingly be connected to biological mechanism and disease prognostics. Progress in this field can facilitate clinical translation of research advances, with potential for great medical impact, e.g. through improvements in ciliopathy disease diagnosis, management, family counselling and by enhancing the potential for future genetically tailored approaches to disease therapeutics.
Assuntos
Dineínas do Axonema/genética , Cílios/metabolismo , Transtornos da Motilidade Ciliar/genética , Proteínas do Olho/genética , Mutação , Mucosa Respiratória/metabolismo , Animais , Dineínas do Axonema/metabolismo , Cílios/patologia , Cílios/ultraestrutura , Transtornos da Motilidade Ciliar/metabolismo , Transtornos da Motilidade Ciliar/patologia , Proteínas do Olho/metabolismo , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica , Genótipo , Humanos , Padrões de Herança , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas/genética , Proteínas/metabolismo , Qualidade de Vida , Mucosa Respiratória/patologia , Mucosa Respiratória/ultraestrutura , Transdução de SinaisRESUMO
More and more attention is paid to diseases such as internal transfer and brain malformation which are caused by the abnormal morphogenesis of cilia. These cilia-related diseases are divided into two categories: ciliopathy resulting from defects of primary cilia and primary ciliary dyskinesia (PCD) caused by functional dysregulation of motile cilia. Cilia are widely distributed, and their related diseases can cover many human organs and tissues. Recent studies prove that primary cilia play a key role in maintaining homeostasis in the cardiovascular system. However, molecular mechanisms of cilia-related diseases remain elusive. Here, we reviewed recent research progresses on characteristics, molecular mechanisms and treatment methods of ciliopathy and PCD. Our review is beneficial to the further research on the pathogenesis and treatment strategies of cilia-related diseases.
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Transtornos da Motilidade Ciliar , Ciliopatias , Humanos , Cílios/patologia , Transtornos da Motilidade Ciliar/genética , Ciliopatias/genética , Ciliopatias/patologia , MutaçãoRESUMO
We report 4 cases of primary ciliary dyskinesia in unrelated indigenous North American children caused by identical, homozygous, likely pathogenic deletions in the DNAL1 gene. These shared DNAL1 deletions among dispersed indigenous populations suggest that primary ciliary dyskinesia accounts for more lung disease with bronchiectasis than previously recognized in indigenous North Americans.
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Bronquiectasia , Transtornos da Motilidade Ciliar , Criança , Humanos , Transtornos da Motilidade Ciliar/genética , América do Norte , Grupos RaciaisRESUMO
Primary ciliary dyskinesia (PCD) is a hereditary disease caused by pathogenic variants in genes associated with motile cilia. Some variants responsible for PCD are reported to be ethnic-specific or geographical-specific. To identify the responsible PCD variants of Japanese PCD patients, we performed next-generation sequencing of a panel of 32 PCD genes or whole-exome sequencing in 26 newly identified Japanese PCD families. We then combined their genetic data with those from 40 Japanese PCD families reported previously, for an overall analysis of 66 unrelated Japanese PCD families. We conducted Genome Aggregation Database and TogoVar database analyses to reveal the PCD genetic spectrum of the Japanese population and compare with other ethnic groups worldwide. We identified 22 unreported variants among the 31 patients in the 26 newly identified PCD families, including 17 deleterious variants estimated to cause lack of transcription or nonsense-mediated mRNA decay and 5 missense mutations. In all 76 PCD patients from the 66 Japanese families, we identified 53 variants on 141 alleles in total. Copy number variation in DRC1 is the most frequent variant in Japanese PCD patients, followed by DNAH5 c.9018C>T. We found 30 variants specific to the Japanese population, of which 22 are novel. Furthermore, 11 responsible variants in the Japanese PCD patients are common in East Asian populations, while some variants are more frequent in other ethnic groups. In conclusion, PCD is genetically heterogeneous between different ethnicities, and Japanese PCD patients have a characteristic genetic spectrum.
Assuntos
Transtornos da Motilidade Ciliar , Variações do Número de Cópias de DNA , População do Leste Asiático , Humanos , Transtornos da Motilidade Ciliar/genética , Transtornos da Motilidade Ciliar/patologia , Variações do Número de Cópias de DNA/genética , Genômica , MutaçãoRESUMO
Primary ciliary dyskinesia (PCD) is a genetically heterogeneous disorder affecting ciliary structure and function. PCD exhibiting dynein regulatory complex subunit 1 (DRC1) exon 1-4 deletion has been reported in several Japanese PCD patients; however, no large scale studies have been performed. Here, we aimed to determine the prevalence and founder effect of this variant in the Korean population. Using an in-house copy number variation tool, we screened for DRC1 exon 1-4 deletion in 20 patients with PCD and exome data of 1435 patients in the Seoul National University Hospital repository. In cases of suspected DRC1 deletion, confirmatory gap-PCR was performed. In a PCD cohort, three of 20 (15%) patients were positive for DRC1 exon 1-4 deletion (NM_145038.5(DRC1): c.1-3952_540 + 1331del27748-bp) while pathogenic variants were found in CCDC39 (N = 1), DNAAF6 (N = 1), DNAH9 (N = 1). In the 1,435-sample exome data, seven patients (0.49%) were confirmed to have DRC1 exon 1-4 deletion. A chimeric sequence including the junction was searched from the 1000 Genomes Project data repository. One Japanese patient (0.96%) was found to have the same DRC1 exon 1-4 deletion, which was absent in other populations. This study demonstrated that the DRC1 exon 1-4 deletion is a founder mutation based on haplotype analysis. In summary, the prevalence of PCD based on DRC1 exon 1-4 deletion is particularly high in Korean and Japanese populations, which is attributed to the founder effect. Genetic testing for DRC1 exon 1-4 deletion should be considered as an initial screening tool for Korean and Japanese patients with PCD.
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Transtornos da Motilidade Ciliar , Humanos , Transtornos da Motilidade Ciliar/epidemiologia , Transtornos da Motilidade Ciliar/genética , Prevalência , Efeito Fundador , Variações do Número de Cópias de DNA , Éxons/genética , República da Coreia/epidemiologia , Mutação , Dineínas do Axonema/genética , Proteínas Associadas aos Microtúbulos/genéticaRESUMO
Patient-derived human organoids can be used to model a variety of diseases. Recently, we described conditions for long-term expansion of human airway organoids (AOs) directly from healthy individuals and patients. Here, we first optimize differentiation of AOs towards ciliated cells. After differentiation of the AOs towards ciliated cells, these can be studied for weeks. When returned to expansion conditions, the organoids readily resume their growth. We apply this condition to AOs established from nasal inferior turbinate brush samples of patients suffering from primary ciliary dyskinesia (PCD), a pulmonary disease caused by dysfunction of the motile cilia in the airways. Patient-specific differences in ciliary beating are observed and are in agreement with the patients' genetic mutations. More detailed organoid ciliary phenotypes can thus be documented in addition to the standard diagnostic procedure. Additionally, using genetic editing tools, we show that a patient-specific mutation can be repaired. This study demonstrates the utility of organoid technology for investigating hereditary airway diseases such as PCD.
Assuntos
Transtornos da Motilidade Ciliar , Organoides , Cílios , Transtornos da Motilidade Ciliar/genética , Humanos , Mutação , FenótipoRESUMO
Primary ciliary dyskinesia (PCD) represents a group of diseases characterized by impaired movement of cilia and subsequent health problems in diverse organ systems, notably the respiratory tract. Almost 50 candidate genes for PCD are known in humans. In this study, we investigated an Australian Shepherd dog with a history of recurrent respiratory infections and nasal discharge. A transmission electron microscopy investigation led to the diagnosis of PCD with central pair defect, in which the normal 9:2 arrangement of respiratory cilia was altered and reduced to a 9:0 arrangement. Whole genome sequencing data from the affected dog was obtained and searched for variants in PCD candidate genes that were not present in 918 control genomes from different breeds. This revealed a homozygous single base pair exchange at a splice site of STK36, XM_038585732.1:c.2868-1G>A. The mutant allele was absent from 281 additionally genotyped Australian Shepherd dogs. RT-PCR confirmed aberrant splicing in the affected dog with the skipping of exon 20 and the insertion of a cryptic exon, which is predicted to lead to a premature stop codon and truncation of 36% of the STK36 wild-type open reading frame, XP_038441660.1:(p.Met957Profs*11). STK36 variants were previously reported to cause PCD in humans and mice. The knowledge from other species together with the absence of the mutant allele in more than 1000 control dogs suggests STK36:c.2868-1G>A as the most likely candidate variant for PCD in the investigated case.
Assuntos
Transtornos da Motilidade Ciliar , Doenças do Cão , Animais , Cães , Transtornos da Motilidade Ciliar/genética , Transtornos da Motilidade Ciliar/veterinária , Genótipo , Homozigoto , Proteínas Serina-Treonina Quinases/genéticaRESUMO
BACKGROUND: The study describes the clinical manifestations and variant screening of two Chinese siblings with primary ciliary dyskinesia (PCD). They carry the same DNAAF2 genotype, which is an extremely rare PCD genotype in the Chinese population. In addition, the study illustrated an overview of published variants on DNAAF2 to date. METHODS: A two-child family was recruited for the study. Clinical manifestations, laboratory tests, bronchoscopic and otoscopic images, and radiographic data were collected. Whole blood was collected from siblings and their parents for whole-exome sequencing (WES) and Sanger sequencing to screen variants. RESULTS: The two siblings exhibited typical clinical manifestations of PCD. Two compound heterozygous variants in DNAAF2 were detected in both by WES. Nonsense variant c.156 C>A and frameshift variant c.177_178insA, which was a novel variant. CONCLUSION: The study identified a novel variant of DNAAF2 in Chinese children with a typical phenotype of PCD, which may enrich our knowledge of the clinical, diagnostic and genetic information of DNAAF2-induced PCD in children.
Assuntos
Transtornos da Motilidade Ciliar , Mutação da Fase de Leitura , Humanos , Transtornos da Motilidade Ciliar/diagnóstico , Transtornos da Motilidade Ciliar/genética , Genótipo , Mutação , FenótipoRESUMO
Ciliary dyneins are preassembled in the cytoplasm before being transported into cilia, and a family of proteins containing the PIH1 domain, PIH proteins, are involved in the assembly process. However, the functional differences and relationships between members of this family of proteins remain largely unknown. Using Chlamydomonas reinhardtii as a model, we isolated and characterized two novel Chlamydomonas PIH preassembly mutants, mot48-2 and twi1-1. A new allele of mot48 (ida10), mot48-2, shows large defects in ciliary dynein assembly in the axoneme and altered motility. A second mutant, twi1-1, shows comparatively smaller defects in motility and dynein assembly. A double mutant mot48-2; twi1-1 displays greater reduction in motility and in dynein assembly compared to each single mutant. Similarly, a double mutant twi1-1; pf13 also shows a significantly greater defect in motility and dynein assembly than either parent mutant. Thus, MOT48 (IDA10), TWI1 and PF13 may define different steps, and have partially overlapping functions, in a pathway required for ciliary dynein preassembly. Together, our data suggest the three PIH proteins function in preassembly steps that are both common and unique for different ciliary dyneins.
Assuntos
Dineínas do Axonema/metabolismo , Movimento Celular/genética , Cílios/metabolismo , Transtornos da Motilidade Ciliar/genética , Proteínas de Plantas/genética , Chlamydomonas reinhardtii , Humanos , Mutação , Proteínas de Plantas/metabolismoRESUMO
Synchronized beating of cilia on multiciliated cells (MCCs) generates a directional flow of mucus across epithelia. This motility requires a "9 + 2" microtubule (MT) configuration in axonemes and the unidirectional array of basal bodies of cilia on the MCCs. However, it is not fully understood what components are needed for central MT-pair assembly as they are not continuous with basal bodies in contrast to the nine outer MT doublets. In this study, we discovered that a homozygous knockdown mouse model for MT minus-end regulator calmodulin-regulated spectrin-associated protein 3 (CAMSAP3), Camsap3tm1a/tm1a , exhibited multiple phenotypes, some of which are typical of primary ciliary dyskinesia (PCD), a condition caused by motile cilia defects. Anatomical examination of Camsap3tm1a/tm1a mice revealed severe nasal airway blockage and abnormal ciliary morphologies in nasal MCCs. MCCs from different tissues exhibited defective synchronized beating and ineffective generation of directional flow likely underlying the PCD-like phenotypes. In normal mice, CAMSAP3 localized to the base of axonemes and at the basal bodies in MCCs. However, in Camsap3tm1a/tm1a , MCCs lacked CAMSAP3 at the ciliary base. Importantly, the central MT pairs were missing in the majority of cilia, and the polarity of the basal bodies was disorganized. These phenotypes were further confirmed in MCCs of Xenopus embryos when CAMSAP3 expression was knocked down by morpholino injection. Taken together, we identified CAMSAP3 as being important for the formation of central MT pairs, proper orientation of basal bodies, and synchronized beating of motile cilia.
Assuntos
Corpos Basais/metabolismo , Cílios/metabolismo , Transtornos da Motilidade Ciliar/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Animais , Axonema/metabolismo , Polaridade Celular , Transtornos da Motilidade Ciliar/genética , Células Epiteliais/metabolismo , Humanos , Camundongos , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/genética , XenopusRESUMO
Motile cilia/flagella are essential for swimming and generating extracellular fluid flow in eukaryotes. Motile cilia harbor a 9+2 arrangement consisting of nine doublet microtubules with dynein arms at the periphery and a pair of singlet microtubules at the center (central pair). In the central system, the radial spoke has a T-shaped architecture and regulates the motility and motion pattern of cilia. Recent cryoelectron tomography data reveal three types of radial spokes (RS1, RS2, and RS3) in the 96 nm axoneme repeat unit; however, the molecular composition of the third radial spoke, RS3 is unknown. In human pathology, it is well known mutation of the radial spoke head-related genes causes primary ciliary dyskinesia (PCD) including respiratory defect and infertility. Here, we describe the role of the primary ciliary dyskinesia protein Rsph4a in the mouse motile cilia. Cryoelectron tomography reveals that the mouse trachea cilia harbor three types of radial spoke as with the other vertebrates and that all triplet spoke heads are lacking in the trachea cilia of Rsph4a-deficient mice. Furthermore, observation of ciliary movement and immunofluorescence analysis indicates that Rsph4a contributes to the generation of the planar beating of motile cilia by building the distal architecture of radial spokes in the trachea, the ependymal tissues, and the oviduct. Although detailed mechanism of RSs assembly remains unknown, our results suggest Rsph4a is a generic component of radial spoke heads, and could explain the severe phenotype of human PCD patients with RSPH4A mutation.