Zebrafish Motile Cilia as a Model for Primary Ciliary Dyskinesia

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.

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Mutation of CFAP57 causes primary ciliary dyskinesia by disrupting the asymmetric targeting of a subset of ciliary inner dynein arms

10.1101/773028 ◽

2019 ◽

Author(s):

Ximena M. Bustamante-Marin ◽

Amjad Horani ◽

Mihaela Stoyanova ◽

Wu-Lin Charng ◽

Mathieu Bottier ◽

...

Keyword(s):

Epithelial Cells ◽

Primary Ciliary Dyskinesia ◽

Large Population ◽

Exome Capture ◽

Swimming Velocity ◽

Population Sampling ◽

Dynein Arms ◽

Motile Cilia ◽

Ciliary Dyskinesia ◽

Sampling Probability

AbstractPrimary ciliary dyskinesia (PCD) is characterized by chronic airway disease, male infertility, and randomization of the left/right body axis, and is caused by defects of motile cilia and sperm flagella. We screened a cohort of affected individuals that lack an obvious TEM structural phenotype for pathogenic variants using whole exome capture and next generation sequencing. The population sampling probability (PSAP) algorithm identified one subject with a homozygous nonsense variant [(c.1762C>T) p.(Arg588*) exon 11] in the uncharacterized CFAP57 gene. In normal human nasal epithelial cells, CFAP57 localizes throughout the ciliary axoneme. Analysis of cells from the PCD patient shows a loss of CFAP57, reduced beat frequency, and an alteration in the ciliary waveform. Knockdown of CFAP57 in human tracheobronchial epithelial cells (hTECs) recapitulates these findings. Phylogenetic analysis showed that CFAP57 is conserved in organisms that assemble motile cilia, and CFAP57 is allelic with the BOP2 gene identified previously in Chlamydomonas. Two independent, insertional fap57 Chlamydomonas mutant strains show reduced swimming velocity and altered waveforms. Tandem mass spectroscopy showed that CFAP57 is missing, and the “g” inner dyneins (DHC7 and DHC3) and the “d” inner dynein (DHC2) are reduced. Our data demonstrate that the FAP57 protein is required for the asymmetric assembly of inner dyneins on only a subset of the microtubule doublets, and this asymmetry is essential for the generation of an effective axonemal waveform. Together, our data identifies mutations in CFAP57 as a cause of PCD with a specific defect in the inner dynein arm assembly process.SignificanceMotile cilia are found throughout eukaryotic organisms and performs essential functions. Primary ciliary dyskinesia (PCD) is a rare disease that affects the function of motile cilia. By applying a novel population sampling probability algorithm (PSAP) that uses large population sequencing databases and pathogenicity prediction algorithms, we identified a variant in an uncharacterized gene, CFAP57. This is the first reported example of PCD caused by a mutation that affects only a subset of the inner dynein arms, which are needed to generate the waveform. CFAP57 identifies an address for specific dynein arms. These findings demonstrate the effectiveness of the PSAP algorithm, expand our understanding of the positioning of dynein arms, and identify mutations in CFAP57 as a cause of PCD.

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Truncating mutations in exons 20 and 21 of OFD1 can cause primary ciliary dyskinesia without associated syndromic symptoms

Journal of Medical Genetics ◽

10.1136/jmedgenet-2018-105918 ◽

2019 ◽

Vol 56 (11) ◽

pp. 769-777 ◽

Cited By ~ 6

Author(s):

Zuzanna Bukowy-Bieryllo ◽

Alicja Rabiasz ◽

Maciej Dabrowski ◽

Andrzej Pogorzelski ◽

Alina Wojda ◽

...

Keyword(s):

Magnetic Resonance ◽

Brain Imaging ◽

Primary Ciliary Dyskinesia ◽

Primary Cilia ◽

Mutation Screening ◽

Syndrome Type ◽

Airway Infections ◽

Motile Cilia ◽

Magnetic Resonance Brain Imaging ◽

Ciliary Dyskinesia

BackgroundPrimary ciliary dyskinesia (PCD) is a motile ciliopathy, whose symptoms include airway infections, male infertility and situs inversus. Apart from the typical forms of PCD, rare syndromic PCD forms exist. Mutations of the X-linked OFD1 gene cause several syndromic ciliopathies, including oral-facial-digital syndrome type 1, Joubert syndrome type 10 (JBTS10), and Simpson-Golabi-Behmel syndrome type 2, the latter causing the X-linked syndromic form of PCD. Neurological and skeletal symptoms are characteristic for these syndromes, with their severity depending on the location of the mutation within the gene.ObjectivesTo elucidate the role of motile cilia defects in the respiratory phenotype of PCD patients with C-terminal OFD1 mutations.MethodsWhole-exome sequencing in a group of 120 Polish PCD patients, mutation screening of the OFD1 coding sequence, analysis of motile cilia, and magnetic resonance brain imaging.ResultsFour novel hemizygous OFD1 mutations, in exons 20 and 21, were found in men with a typical PCD presentation but without severe neurological, skeletal or renal symptoms characteristic for other OFD1-related syndromes. Magnetic resonance brain imaging in two patients did not show a molar tooth sign typical for JBTS10. Cilia in the respiratory epithelium were sparse, unusually long and displayed a defective motility pattern.ConclusionConsistent with the literature, truncations of the C-terminal part of OFD1 (exons 16–22) almost invariably cause a respiratory phenotype (due to motile cilia defects) while their impact on the primary cilia function is limited. We suggest that exons 20–21 should be included in the panel for regular mutation screening in PCD.

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Homozygous truncating NEK10 mutation, associated with primary ciliary dyskinesia: a case report

BMC Pulmonary Medicine ◽

10.1186/s12890-020-1175-1 ◽

2020 ◽

Vol 20 (1) ◽

Cited By ~ 1

Author(s):

Fuad Al Mutairi ◽

Randa Alkhalaf ◽

Abdullah Alkhorayyef ◽

Fayhan Alroqi ◽

Alyafee Yusra ◽

...

Keyword(s):

Primary Ciliary Dyskinesia ◽

Autosomal Recessive ◽

Autosomal Recessive Disorder ◽

Molecular Testing ◽

Case Presentation ◽

Immotile Cilia ◽

Whole Exome ◽

Novel Variant ◽

Pathophysiological Aspect ◽

Ciliary Dyskinesia

Abstract Background Primary Ciliary Dyskinesia (PCD) is also known as immotile-cilia syndrome, an autosomal recessive disorder of ciliary function, leading to mucus retention in the respiratory system in childhood. Our knowledge in the pathophysiological aspect of this devastating disorder is increasing with the advancement of genetic and molecular testing. Case presentation Here in, we report two siblings with a classical clinical and radiological presentation of PCD. Using whole exome sequencing we identified a homozygous truncating variant (c.3402 T > A); p.(Tyr1134*) in the NEK10 gene. Western bolt analysis revealed a decrease in the expression of NEK10 protein in the patient cells. Conclusions NEK10 plays a central role in the post-mitotic process of cilia assembly, regulating ciliary length and functions during physiological and pathological status. This study highlights the challenges of identifying disease-causing variants for a highly heterogeneous disorder and reports on the identification of a novel variant in NEK10 which recently associated with PCD.

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Faculty Opinions recommendation of Mutations in ZMYND10, a gene essential for proper axonemal assembly of inner and outer dynein arms in humans and flies, cause primary ciliary dyskinesia.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.718050584.793504030 ◽

2015 ◽

Author(s):

Nicholas Katsanis

Keyword(s):

Primary Ciliary Dyskinesia ◽

Dynein Arms ◽

Ciliary Dyskinesia

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Unique among ciliopathies: primary ciliary dyskinesia, a motile cilia disorder

F1000Prime Reports ◽

10.12703/p7-36 ◽

2015 ◽

Vol 7 ◽

Cited By ~ 40

Author(s):

Kavita Praveen ◽

Erica E. Davis ◽

Nicholas Katsanis

Keyword(s):

Primary Ciliary Dyskinesia ◽

Motile Cilia ◽

Ciliary Dyskinesia

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Mutations in C11orf70 Cause Primary Ciliary Dyskinesia with Randomization of Left/Right Body Asymmetry Due to Defects of Outer and Inner Dynein Arms

The American Journal of Human Genetics ◽

10.1016/j.ajhg.2018.03.025 ◽

2018 ◽

Vol 102 (5) ◽

pp. 973-984 ◽

Cited By ~ 23

Author(s):

Inga M. Höben ◽

Rim Hjeij ◽

Heike Olbrich ◽

Gerard W. Dougherty ◽

Tabea Nöthe-Menchen ◽

...

Keyword(s):

Primary Ciliary Dyskinesia ◽

Body Asymmetry ◽

Dynein Arms ◽

Ciliary Dyskinesia

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Primary Ciliary Dyskinesia (Immotile Cilia Syndrome)

Nelson Textbook of Pediatrics ◽

10.1016/b978-1-4377-0755-7.00396-1 ◽

2011 ◽

pp. 1497-1497.e6 ◽

Cited By ~ 2

Author(s):

Thomas Ferkol

Keyword(s):

Primary Ciliary Dyskinesia ◽

Immotile Cilia Syndrome ◽

Immotile Cilia ◽

Ciliary Dyskinesia

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Clinical and Genetic Analysis of Children with Kartagener Syndrome

Cells ◽

10.3390/cells8080900 ◽

2019 ◽

Vol 8 (8) ◽

pp. 900 ◽

Cited By ~ 6

Author(s):

Rute Pereira ◽

Telma Barbosa ◽

Luís Gales ◽

Elsa Oliveira ◽

Rosário Santos ◽

...

Keyword(s):

Autosomal Recessive Disorder ◽

Kartagener Syndrome ◽

Mucus Clearance ◽

Pathogenic Variants ◽

Transmission Electron ◽

Whole Exome ◽

Dynein Arms ◽

Organ Laterality ◽

Ciliary Dyskinesia ◽

Laterality Defects

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.

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