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13 pages, 2882 KiB

Open AccessArticle

Primary Ciliary Dyskinesia Associated Disease-Causing Variants in CCDC39 and CCDC40 Cause Axonemal Absence of Inner Dynein Arm Heavy Chains DNAH1, DNAH6, and DNAH7

by Alina Wilken, Inga Marlena Höben, Alexander Wolter, Niki Tomas Loges, Heike Olbrich, Isabella Aprea, Bernd Dworniczak, Johanna Raidt and Heymut Omran

Cells 2024, 13(14), 1200; https://doi.org/10.3390/cells13141200 - 15 Jul 2024

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Abstract

Disease-causing bi-allelic DNA variants in CCDC39 and CCDC40 are frequent causes of the hereditary disorder of primary ciliary dyskinesia (PCD). The encoded proteins form a molecular ruler complex, crucial for maintaining the 96 nm repeat units along the ciliary axonemes. Defects of those [...] Read more.

Disease-causing bi-allelic DNA variants in CCDC39 and CCDC40 are frequent causes of the hereditary disorder of primary ciliary dyskinesia (PCD). The encoded proteins form a molecular ruler complex, crucial for maintaining the 96 nm repeat units along the ciliary axonemes. Defects of those proteins cause a stiff, rapid, and flickery ciliary beating pattern, recurrent respiratory infections, axonemal disorganization, and abnormal assembly of GAS8, CCDC39, and DNALI1. We performed molecular characterization of the defects in the 96 nm axonemal ruler due to disease-causing variants in CCDC39 and CCDC40 and analyzed the effect on additional axonemal components. We identified a cohort of 51 individuals with disease-causing variants in CCDC39 and CCDC40 via next-generation sequencing techniques and demonstrated that the IDA heavy chains DNAH1, DNAH6, and DNAH7 are conspicuously absent within the respiratory ciliary axonemes by immunofluorescence analyses. Hence, we show for the first time that the centrin2 (CETN2) containing IDAs are also affected. These findings underscore the crucial role of CCDC39 and CCDC40 in the assembly and function of IDAs in human respiratory cilia. Thus, our data improve the diagnostics of axonemal ruler defects by further characterizing the associated molecular IDA defects. Full article

(This article belongs to the Special Issue The Role of Cilia in Health and Diseases)

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20 pages, 7681 KiB

Open AccessArticle

A Short Sequence Targets Transmembrane Proteins to Primary Cilia

by Viviana Macarelli, Edward C. Harding, David C. Gershlick and Florian T. Merkle

Cells 2024, 13(13), 1156; https://doi.org/10.3390/cells13131156 - 6 Jul 2024

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Abstract

Primary cilia are finger-like sensory organelles that extend from the bodies of most cell types and have a distinct lipid and protein composition from the plasma membrane. This partitioning is maintained by a diffusion barrier that restricts the entry of non-ciliary proteins, and [...] Read more.

Primary cilia are finger-like sensory organelles that extend from the bodies of most cell types and have a distinct lipid and protein composition from the plasma membrane. This partitioning is maintained by a diffusion barrier that restricts the entry of non-ciliary proteins, and allows the selective entry of proteins harboring a ciliary targeting sequence (CTS). However, CTSs are not stereotyped and previously reported sequences are insufficient to drive efficient ciliary localisation across diverse cell types. Here, we describe a short peptide sequence that efficiently targets transmembrane proteins to primary cilia in all tested cell types, including human neurons. We generate human-induced pluripotent stem cell (hiPSC) lines stably expressing a transmembrane construct bearing an extracellular HaloTag and intracellular fluorescent protein, which enables the bright, specific labeling of primary cilia in neurons and other cell types to facilitate studies of cilia in health and disease. We demonstrate the utility of this resource by developing an image analysis pipeline for the automated measurement of primary cilia to detect changes in their length associated with altered signaling or disease state. Full article

(This article belongs to the Special Issue The Role of Cilia in Health and Diseases)

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19 pages, 3846 KiB

Open AccessArticle

The Odad3 Gene Is Necessary for Spermatozoa Development and Male Fertility in Mice

by Miriam Pasquini, Francesco Chiani, Alessia Gambadoro, Chiara Di Pietro, Renata Paoletti, Tiziana Orsini, Sabrina Putti, Ferdinando Scavizzi, Gina La Sala and Olga Ermakova

Cells 2024, 13(12), 1053; https://doi.org/10.3390/cells13121053 - 18 Jun 2024

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Abstract

Odad3 gene loss-of-function mutation leads to Primary Ciliary Dyskinesia (PCD), a disease caused by motile cilia dysfunction. Previously, we demonstrated that knockout of the Odad3 gene in mice replicates several features of PCD, such as hydrocephalus, defects in left–right body symmetry, and male [...] Read more.

Odad3 gene loss-of-function mutation leads to Primary Ciliary Dyskinesia (PCD), a disease caused by motile cilia dysfunction. Previously, we demonstrated that knockout of the Odad3 gene in mice replicates several features of PCD, such as hydrocephalus, defects in left–right body symmetry, and male infertility, with a complete absence of sperm in the reproductive tract. The majority of Odad3 knockout animals die before sexual maturation due to severe hydrocephalus and failure to thrive, which precludes fertility studies. Here, we performed the expression analysis of the Odad3 gene during gonad development and in adult testes. We showed that Odad3 starts its expression during the first wave of spermatogenesis, specifically at the meiotic stage, and that its expression is restricted to the germ cells in the adult testes, suggesting that Odad3 plays a role in spermatozoa formation. Subsequently, we conditionally deleted the Odad3 gene in adult males and demonstrated that even partial ablation of the Odad3 gene leads to asthenoteratozoospermia with multiple morphological abnormalities of sperm flagella (MMAF) in mice. The analysis of the seminiferous tubules in Odad3-deficient mice revealed defects in spermatogenesis with accumulation of seminiferous tubules at the spermiogenesis and spermiation phases. Furthermore, analysis of fertility in heterozygous Odad3^+/− knockout mice revealed a reduction in sperm count and motility as well as abnormal sperm morphology. Additionally, Odad3^+/− males exhibited a shorter fertile lifespan. Overall, these results suggest the important role of Odad3 and Odad3 gene dosage in male fertility. These findings may have an impact on the genetic and fertility counseling practice of PCD patients carrying Odad3 loss-of-function mutations. Full article

(This article belongs to the Special Issue The Role of Cilia in Health and Diseases)

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11 pages, 1490 KiB

Open AccessArticle

Biallelic Variants in MNS1 Are Associated with Laterality Defects and Respiratory Involvement

by Rim Hjeij, Joseph Leslie, Hoda Rizk, Bernd Dworniczak, Heike Olbrich, Johanna Raidt, Sebastian Felix Nepomuk Bode, Alice Gardham, Karen Stals, Mohammad Al-Haggar, Engy Osman, Andrew Crosby, Tarek Eldesoky, Emma Baple and Heymut Omran

Cells 2024, 13(12), 1017; https://doi.org/10.3390/cells13121017 - 11 Jun 2024

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Abstract

Defects in motile cilia, termed motile ciliopathies, result in clinical manifestations affecting the respiratory and reproductive system, as well as laterality defects and hydrocephalus. We previously defined biallelic MNS1 variants causing situs inversus and male infertility, mirroring the findings in Mns1^−/− mice. [...] Read more.

Defects in motile cilia, termed motile ciliopathies, result in clinical manifestations affecting the respiratory and reproductive system, as well as laterality defects and hydrocephalus. We previously defined biallelic MNS1 variants causing situs inversus and male infertility, mirroring the findings in Mns1^−/− mice. Here, we present clinical and genomic findings in five newly identified individuals from four unrelated families affected by MNS1-related disorder. Ciliopathy panel testing and whole exome sequencing identified one previously reported and two novel MNS1 variants extending the genotypic spectrum of disease. A broad spectrum of laterality defects including situs inversus totalis and heterotaxia was confirmed. Interestingly, a single affected six-year-old girl homozygous for an MNS1 nonsense variant presented with a history of neonatal respiratory distress syndrome, recurrent respiratory tract infections, chronic rhinitis, and wet cough. Accordingly, immunofluorescence analysis showed the absence of MNS1 from the respiratory epithelial cells of this individual. Two other individuals with hypomorphic variants showed laterality defects and mild respiratory phenotype. This study represents the first observation of heterotaxia and respiratory disease in individuals with biallelic MNS1 variants, an important extension of the phenotype associated with MNS1-related motile ciliopathy disorder. Full article

(This article belongs to the Special Issue The Role of Cilia in Health and Diseases)

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11 pages, 2082 KiB

Open AccessCommunication

Advancing Primary Ciliary Dyskinesia Diagnosis through High-Speed Video Microscopy Analysis

by Wilfredo De Jesús-Rojas, Zachary J. Demetriou, José Muñiz-Hernández, Gabriel Rosario-Ortiz, Frances M. Quiñones, Marcos J. Ramos-Benitez and Ricardo A. Mosquera

Cells 2024, 13(7), 567; https://doi.org/10.3390/cells13070567 - 24 Mar 2024

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Abstract

Primary ciliary dyskinesia (PCD) is an inherited disorder that impairs motile cilia, essential for respiratory health, with a reported prevalence of 1 in 16,309 within Hispanic populations. Despite 70% of Puerto Rican patients having the RSPH4A [c.921+3_921+6del (intronic)] founder mutation, the characterization of [...] Read more.

Primary ciliary dyskinesia (PCD) is an inherited disorder that impairs motile cilia, essential for respiratory health, with a reported prevalence of 1 in 16,309 within Hispanic populations. Despite 70% of Puerto Rican patients having the RSPH4A [c.921+3_921+6del (intronic)] founder mutation, the characterization of the ciliary dysfunction remains unidentified due to the unavailability of advanced diagnostic modalities like High-Speed Video Microscopy Analysis (HSVA). Our study implemented HSVA for the first time on the island as a tool to better diagnose and characterize the RSPH4A [c.921+3_921+6del (intronic)] founder mutation in Puerto Rican patients. By applying HSVA, we analyzed the ciliary beat frequency (CBF) and pattern (CBP) in native Puerto Rican patients with PCD. Our results showed decreased CBF and a rotational CBP linked to the RSPH4A founder mutation in Puerto Ricans, presenting a novel diagnostic marker that could be implemented as an axillary test into the PCD diagnosis algorithm in Puerto Rico. The integration of HSVA technology in Puerto Rico substantially enhances the PCD evaluation and diagnosis framework, facilitating prompt detection and early intervention for improved disease management. This initiative, demonstrating the potential of HSVA as an adjunctive test within the PCD diagnostic algorithm, could serve as a blueprint for analogous developments throughout Latin America. Full article

(This article belongs to the Special Issue The Role of Cilia in Health and Diseases)

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15 pages, 10894 KiB

Open AccessArticle

Primary Ciliary Dyskinesia and Retinitis Pigmentosa: Novel RPGR Variant and Possible Modifier Gene

by Noelia Baz-Redón, Laura Sánchez-Bellver, Mónica Fernández-Cancio, Sandra Rovira-Amigo, Thomas Burgoyne, Rai Ranjit, Virginia Aquino, Noemí Toro-Barrios, Rosario Carmona, Eva Polverino, Maria Cols, Antonio Moreno-Galdó, Núria Camats-Tarruella and Gemma Marfany

Cells 2024, 13(6), 524; https://doi.org/10.3390/cells13060524 - 16 Mar 2024

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Abstract

We report a novel RPGR missense variant co-segregated with a familial X-linked retinitis pigmentosa (XLRP) case. The brothers were hemizygous for this variant, but only the proband presented with primary ciliary dyskinesia (PCD). Thus, we aimed to elucidate the role of the RPGR [...] Read more.

We report a novel RPGR missense variant co-segregated with a familial X-linked retinitis pigmentosa (XLRP) case. The brothers were hemizygous for this variant, but only the proband presented with primary ciliary dyskinesia (PCD). Thus, we aimed to elucidate the role of the RPGR variant and other modifier genes in the phenotypic variability observed in the family and its impact on motile cilia. The pathogenicity of the variant on the RPGR protein was evaluated by in vitro studies transiently transfecting the mutated RPGR gene, and immunofluorescence analysis on nasal brushing samples. Whole-exome sequencing was conducted to identify potential modifier variants. In vitro studies showed that the mutated RPGR protein could not localise to the cilium and impaired cilium formation. Accordingly, RPGR was abnormally distributed in the siblings’ nasal brushing samples. In addition, a missense variant in CEP290 was identified. The concurrent RPGR variant influenced ciliary mislocalisation of the protein. We provide a comprehensive characterisation of motile cilia in this XLRP family, with only the proband presenting PCD symptoms. The variant’s pathogenicity was confirmed, although it alone does not explain the respiratory symptoms. Finally, the CEP290 gene may be a potential modifier for respiratory symptoms in patients with RPGR mutations. Full article

(This article belongs to the Special Issue The Role of Cilia in Health and Diseases)

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14 pages, 9113 KiB

Open AccessArticle

Ciliary Ultrastructure Assessed by Transmission Electron Microscopy in Adults with Bronchiectasis and Suspected Primary Ciliary Dyskinesia but Inconclusive Genotype

by Ben O. Staar, Jan Hegermann, Bernd Auber, Raphael Ewen, Sandra von Hardenberg, Ruth Olmer, Isabell Pink, Jessica Rademacher, Martin Wetzke and Felix C. Ringshausen

Cells 2023, 12(22), 2651; https://doi.org/10.3390/cells12222651 - 18 Nov 2023

Cited by 1 | Viewed by 1508

Abstract

Whole-exome sequencing has expedited the diagnostic work-up of primary ciliary dyskinesia (PCD), when used in addition to clinical phenotype and nasal nitric oxide. However, it reveals variants of uncertain significance (VUS) in established PCD genes or (likely) pathogenic variants in genes of uncertain [...] Read more.

Whole-exome sequencing has expedited the diagnostic work-up of primary ciliary dyskinesia (PCD), when used in addition to clinical phenotype and nasal nitric oxide. However, it reveals variants of uncertain significance (VUS) in established PCD genes or (likely) pathogenic variants in genes of uncertain significance in approximately 30% of tested individuals. We aimed to assess genotype–phenotype correlations in adults with bronchiectasis, clinical suspicion of PCD, and inconclusive whole-exome sequencing results using transmission electron microscopy (TEM) and ciliary image averaging by the PCD Detect software. We recruited 16 patients with VUS in CCDC39, CCDC40, CCDC103, DNAH5, DNAH5/CCDC40, DNAH8/HYDIN, DNAH11, and DNAI1 as well as variants in the PCD candidate genes DNAH1, DNAH7, NEK10, and NME5. We found normal ciliary ultrastructure in eight patients with VUS in CCDC39, DNAH1, DNAH7, DNAH8/HYDIN, DNAH11, and DNAI1. In six patients with VUS in CCDC40, CCDC103, DNAH5, and DNAI1, we identified a corresponding ultrastructural hallmark defect. In one patient with homozygous variant in NME5, we detected a central complex defect supporting clinical relevance. Using TEM as a targeted approach, we established important genotype–phenotype correlations and definite PCD in a considerable proportion of patients. Overall, the PCD Detect software proved feasible in support of TEM. Full article

(This article belongs to the Special Issue The Role of Cilia in Health and Diseases)

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Review

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27 pages, 1273 KiB

Open AccessFeature PaperReview

Primary Ciliary Dyskinesia: A Clinical Review

by Katherine A. Despotes, Maimoona A. Zariwala, Stephanie D. Davis and Thomas W. Ferkol

Cells 2024, 13(11), 974; https://doi.org/10.3390/cells13110974 - 4 Jun 2024

Viewed by 935

Abstract

Primary ciliary dyskinesia (PCD) is a rare, genetically heterogeneous, motile ciliopathy, characterized by neonatal respiratory distress, recurrent upper and lower respiratory tract infections, subfertility, and laterality defects. Diagnosis relies on a combination of tests for confirmation, including nasal nitric oxide (nNO) measurements, high-speed [...] Read more.

Primary ciliary dyskinesia (PCD) is a rare, genetically heterogeneous, motile ciliopathy, characterized by neonatal respiratory distress, recurrent upper and lower respiratory tract infections, subfertility, and laterality defects. Diagnosis relies on a combination of tests for confirmation, including nasal nitric oxide (nNO) measurements, high-speed videomicroscopy analysis (HSVMA), immunofluorescent staining, axonemal ultrastructure analysis via transmission electron microscopy (TEM), and genetic testing. Notably, there is no single gold standard confirmatory or exclusionary test. Currently, 54 causative genes involved in cilia assembly, structure, and function have been linked to PCD; this rare disease has a spectrum of clinical manifestations and emerging genotype–phenotype relationships. In this review, we provide an overview of the structure and function of motile cilia, the emerging genetics and pathophysiology of this rare disease, as well as clinical features associated with motile ciliopathies, novel diagnostic tools, and updates on genotype–phenotype relationships in PCD. Full article

(This article belongs to the Special Issue The Role of Cilia in Health and Diseases)

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