HY-DIN' in the Cilia: Discovery of Central Pair-related Mutations in Primary Ciliary Dyskinesia.
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ABSTRACT:
SUBMITTER: Dutcher SK
PROVIDER: S-EPMC7055694 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Primary ciliary dyskinesia (PCD) is a genetically heterogeneous inherited disorder arising from dysmotility of motile cilia and sperm. This is associated with a variety of ultrastructural defects of the cilia and sperm axoneme that affect movement, leading to clinical consequences on respiratory-tract mucociliary clearance and lung function, fertility, and left-right body-axis determination. We performed whole-genome SNP-based linkage analysis in seven consanguineous families with PCD and central-microtubular-pair abnormalities. This identified two loci, in two families with intermittent absence of the central-pair structure (chromosome 6p21.1, Zmax 6.7) and in five families with complete absence of the central pair (chromosome 6q22.1, Zmax 7.0). Mutations were subsequently identified in two positional candidate genes, RSPH9 on chromosome 6p21.1 and RSPH4A on chromosome 6q22.1. Haplotype analysis identified a common ancestral founder effect RSPH4A mutation present in UK-Pakistani pedigrees. Both RSPH9 and RSPH4A encode protein components of the axonemal radial spoke head. In situ hybridization of murine Rsph9 shows gene expression restricted to regions containing motile cilia. Investigation of the effect of knockdown or mutations of RSPH9 orthologs in zebrafish and Chlamydomonas indicate that radial spoke head proteins are important in maintaining normal movement in motile, "9+2"-structure cilia and flagella. This effect is rescued by reintroduction of gene expression for restoration of a normal beat pattern in zebrafish. Disturbance in function of these genes was not associated with defects in left-right axis determination in humans or zebrafish.
| S-EPMC2668031 | biostudies-literature
Project description:Zebrafish is a vertebrate teleost widely used in many areas of research. As embryos, they develop quickly and provide unique opportunities for research studies owing to their transparency for at least 48 h post fertilization. Zebrafish have many ciliated organs that include primary cilia as well as motile cilia. Using zebrafish as an animal model helps to better understand human diseases such as Primary Ciliary Dyskinesia (PCD), an autosomal recessive disorder that affects cilia motility, currently associated with more than 50 genes. The aim of this study was to validate zebrafish motile cilia, both in mono and multiciliated cells, as organelles for PCD research. For this purpose, we obtained systematic high-resolution data in both the olfactory pit (OP) and the left-right organizer (LRO), a superficial organ and a deep organ embedded in the tail of the embryo, respectively. For the analysis of their axonemal ciliary structure, we used conventional transmission electron microscopy (TEM) and electron tomography (ET). We characterised the wild-type OP cilia and showed, for the first time in zebrafish, the presence of motile cilia (9 + 2) in the periphery of the pit and the presence of immotile cilia (still 9 + 2), with absent outer dynein arms, in the centre of the pit. In addition, we reported that a central pair of microtubules in the LRO motile cilia is common in zebrafish, contrary to mouse embryos, but it is not observed in all LRO cilia from the same embryo. We further showed that the outer dynein arms of the microtubular doublet of both the OP and LRO cilia are structurally similar in dimensions to the human respiratory cilia at the resolution of TEM and ET. We conclude that zebrafish is a good model organism for PCD research but investigators need to be aware of the specific physical differences to correctly interpret their results.
| S-EPMC8393663 | biostudies-literature
Project description:Primary ciliary dyskinesia (PCD) is an inherited chronic respiratory obstructive disease with randomized body laterality and infertility, resulting from cilia and sperm dysmotility. PCD is characterized by clinical variability and extensive genetic heterogeneity, associated with different cilia ultrastructural defects and mutations identified in >20 genes. Next generation sequencing (NGS) technologies therefore present a promising approach for genetic diagnosis which is not yet in routine use. We developed a targeted panel-based NGS pipeline to identify mutations by sequencing of selected candidate genes in 70 genetically undefined PCD patients. This detected loss-of-function RSPH1 mutations in four individuals with isolated central pair (CP) agenesis and normal body laterality, from two unrelated families. Ultrastructural analysis in RSPH1-mutated cilia revealed transposition of peripheral outer microtubules into the 'empty' CP space, accompanied by a distinctive intermittent loss of the central pair microtubules. We find that mutations in RSPH1, RSPH4A and RSPH9, which all encode homologs of components of the 'head' structure of ciliary radial spoke complexes identified in Chlamydomonas, cause clinical phenotypes that appear to be indistinguishable except at the gene level. By high-resolution immunofluorescence we identified a loss of RSPH4A and RSPH9 along with RSPH1 from RSPH1-mutated cilia, suggesting RSPH1 mutations may result in loss of the entire spoke head structure. CP loss is seen in up to 28% of PCD cases, in whom laterality determination specified by CP-less embryonic node cilia remains undisturbed. We propose this defect could arise from instability or agenesis of the ciliary central microtubules due to loss of their normal radial spoke head tethering.
| S-EPMC4049301 | biostudies-literature
Project description:BackgroundPrimary ciliary dyskinesia (PCD) is a genetic disorder characterized by impaired ciliary function, leading to chronic sinopulmonary disease. The genetic causes of PCD are still evolving, while the diagnosis is often dependent on finding a ciliary ultrastructural abnormality and immotile cilia. Here we report a novel gene associated with PCD but without ciliary ultrastructural abnormalities evident by transmission electron microscopy, but with dyskinetic cilia beating.MethodsGenetic linkage analysis was performed in a family with a PCD subject. Gene expression was studied in Chlamydomonas reinhardtii and human airway epithelial cells, using RNA assays and immunostaining. The phenotypic effects of candidate gene mutations were determined in primary culture human tracheobronchial epithelial cells transduced with gene targeted shRNA sequences. Video-microscopy was used to evaluate cilia motion.ResultsA single novel mutation in CCDC65, which created a termination codon at position 293, was identified in a subject with typical clinical features of PCD. CCDC65, an orthologue of the Chlamydomonas nexin-dynein regulatory complex protein DRC2, was localized to the cilia of normal nasal epithelial cells but was absent in those from the proband. CCDC65 expression was up-regulated during ciliogenesis in cultured airway epithelial cells, as was DRC2 in C. reinhardtii following deflagellation. Nasal epithelial cells from the affected individual and CCDC65-specific shRNA transduced normal airway epithelial cells had stiff and dyskinetic cilia beating patterns compared to control cells. Moreover, Gas8, a nexin-dynein regulatory complex component previously identified to associate with CCDC65, was absent in airway cells from the PCD subject and CCDC65-silenced cells.ConclusionMutation in CCDC65, a nexin-dynein regulatory complex member, resulted in a frameshift mutation and PCD. The affected individual had altered cilia beating patterns, and no detectable ultrastructural defects of the ciliary axoneme, emphasizing the role of the nexin-dynein regulatory complex and the limitations of certain methods for PCD diagnosis.
| S-EPMC3753302 | biostudies-literature
Project description: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.
| S-EPMC7378221 | biostudies-literature
Project description:Primary ciliary dyskinesia (PCD) is a disorder that affects motile cilia in the airway that are required for the removal of mucus, debris, and pathogens. It is important to diagnose PCD in early childhood to preserve lung function. The confirmation of a diagnosis relies on the assessment of ciliary ultrastructure by transmission electron microscopy (TEM). TEM involves the quantitative assessment of the ciliary ultrastructure to identify PCD defects as well as abnormalities resulting from infection. Many specialist diagnostic centres still rely on physical counters to tally results and paper notes to summarise findings before transferring the results to computer databases/records. To speed up the diagnostic data collection and increase the protection of patient information, we have developed digital ciliary feature counters that conform to the PCD reporting international consensus guideline. These counters can be used on a computer or tablet, and automatically generate notes regarding sample observations. We show that the digital counters are easy to use and can generate TEM diagnostic reports that will be useful for many PCD diagnostic centres.
| S-EPMC7459509 | biostudies-literature
Project description:Primary ciliary dyskinesia (PCD) is an autosomal recessive disorder of cilia structure, function, and biogenesis leading to chronic infections of the respiratory tract, fertility problems, and disorders of organ laterality. The diagnosis can be challenging, using traditional tools such as characteristic clinical features, ciliary function, and ultrastructural defects and newer screening tools such as nasal nitric oxide levels and genetic testing add to the diagnostic algorithm. There are 32 known PCD-causing genes, and in the future, comprehensive genetic testing may screen young infants before developing symptoms, thus improving survival. Therapies include surveillance of pulmonary function and microbiology, in addition to airway clearance, antibiotics, and early referral to bronchiectasis centers. As with cystic fibrosis (CF), standardized care at specialized centers using a multidisciplinary approach likely improves outcomes. In conjunction with the CF foundation, the PCD foundation, with experienced investigators and clinicians, is developing a network of PCD clinical centers to coordinate the effort in North America and Europe. As the network grows, clinical care and knowledge will improve.
| S-EPMC4873960 | biostudies-literature
Project description:Primary ciliary dyskinesia (PCD) is a rare (1/20,000), multisystem disease with a complex phenotype caused by the impaired motility of cilia/flagella, usually related to ultrastructural defects of these organelles. Mutations in genes encoding radial spoke head (RSPH) proteins, elements of the ciliary ultrastructure, have been recently described. However, the relative involvement of RSPH genes in PCD pathogenesis remained unknown, due to a small number of PCD families examined for mutations in these genes. The purpose of this study was to estimate the involvement of RSPH4A and RSPH9 in PCD pathogenesis among East Europeans (West Slavs), and to shed more light on ultrastructural ciliary defects caused by mutations in these genes. The coding sequences of RSPH4A and RSPH9 were screened in PCD patients from 184 families, using single strand conformational polymorphism analysis and sequencing. Two previously described (Q109X; R490X) and two new RSPH4A mutations (W356X; IVS3_2-5del), in/around exons 1 and 3, were identified; no mutations were found in RSPH9. We estimate that mutations in RSPH4A, but not in RSPH9, are responsible for 2-3% of cases in the East European PCD population (4% in PCD families without situs inversus; 11% in families preselected for microtubular defects). Analysis of the SNP-haplotype background provided insight into the ancestry of repetitively found mutations (Q109X; R490X; IVS3_2-5del), but further studies involving other PCD cohorts are required to elucidate whether these mutations are specific for Slavic people or spread among other European populations. Ultrastructural defects associated with the mutations were analyzed in the transmission electron microscope images; almost half of the ciliary cross-sections examined in patients with RSPH4A mutations had the microtubule transposition phenotype (9+0 and 8+1 pattern). While microtubule transposition was a prevalent ultrastructural defect in cilia from patients with RSPH4A mutations, similar defects were also observed in PCD patients with mutations in other genes.
| S-EPMC3308995 | biostudies-literature
Project description:Primary ciliary dyskinesia (PCD) is an autosomal recessive, genetically heterogeneous disorder characterised by oto-sino-pulmonary disease and situs abnormalities (Kartagener syndrome) due to abnormal structure and/or function of cilia. Most patients currently recognised to have PCD have ultrastructural defects of cilia; however, some patients have clinical manifestations of PCD and low levels of nasal nitric oxide, but normal ultrastructure, including a few patients with biallelic mutations in dynein axonemal heavy chain 11 (DNAH11).To test further for mutant DNAH11 as a cause of PCD, DNAH11 was sequenced in patients with a PCD clinical phenotype, but no known genetic aetiology.82 exons and intron/exon junctions in DNAH11 were sequenced in 163 unrelated patients with a clinical phenotype of PCD, including those with normal ciliary ultrastructure (n=58), defects in outer and/or inner dynein arms (n=76), radial spoke/central pair defects (n=6), and 23 without definitive ultrastructural results, but who had situs inversus (n=17), or bronchiectasis and/or low nasal nitric oxide (n=6). Additionally, DNAH11 was sequenced in 13 subjects with isolated situs abnormalities to see if mutant DNAH11 could cause situs defects without respiratory disease.Of the 58 unrelated patients with PCD with normal ultrastructure, 13 (22%) had two (biallelic) mutations in DNAH11; and two patients without ultrastructural analysis had biallelic mutations. All mutations were novel and private. None of the patients with dynein arm or radial spoke/central pair defects, or isolated situs abnormalities, had mutations in DNAH11. Of the 35 identified mutant alleles, 24 (69%) were nonsense, insertion/deletion or loss-of-function splice-site mutations.Mutations in DNAH11 are a common cause of PCD in patients without ciliary ultrastructural defects; thus, genetic analysis can be used to ascertain the diagnosis of PCD in this challenging group of patients.
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Project description:Cilia are essential for fertilization, respiratory clearance, cerebrospinal fluid circulation and establishing laterality. Cilia motility defects cause primary ciliary dyskinesia (PCD, MIM244400), a disorder affecting 1:15,000-30,000 births. Cilia motility requires the assembly of multisubunit dynein arms that drive ciliary bending. Despite progress in understanding the genetic basis of PCD, mutations remain to be identified for several PCD-linked loci. Here we show that the zebrafish cilia paralysis mutant schmalhans (smh(tn222)) encodes the coiled-coil domain containing 103 protein (Ccdc103), a foxj1a-regulated gene product. Screening 146 unrelated PCD families identified individuals in six families with reduced outer dynein arms who carried mutations in CCDC103. Dynein arm assembly in smh mutant zebrafish was rescued by wild-type but not mutant human CCDC103. Chlamydomonas Ccdc103/Pr46b functions as a tightly bound, axoneme-associated protein. These results identify Ccdc103 as a dynein arm attachment factor that causes primary ciliary dyskinesia when mutated.
| S-EPMC3371652 | biostudies-literature