Pathogenic variants in CLXN encoding the outer dynein arm docking–associated calcium-binding protein calaxin cause primary ciliary dyskinesia

PurposePrimary 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.MethodsIn addition to studies in mouse and planaria, clinical exome sequencing and functional analyses in human were performed.ResultsIn 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.ConclusionOur 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.     

Clinical and genetic aspects of primary ciliary dyskinesia/Kartagener syndrome

Primary ciliary dyskinesia is a genetically heterogeneous disorder of motile cilia. Most of the disease-causing mutations identified to date involve the heavy (dynein axonemal heavy chain 5) or intermediate (dynein axonemal intermediate chain 1) chain dynein genes in ciliary outer dynein arms, although a few mutations have been noted in other genes. Clinical molecular genetic testing for primary ciliary dyskinesia is available for the most common mutations. The respiratory manifestations of primary ciliary dyskinesia (chronic bronchitis leading to bronchiectasis, chronic rhino-sinusitis, and chronic otitis media) reflect impaired mucociliary clearance owing to defective axonemal structure. Ciliary ultrastructural analysis in most patients (>80%) reveals defective dynein arms, although defects in other axonemal components have also been observed. Approximately 50% of patients with primary ciliary dyskinesia have laterality defects (including situs inversus totalis and, less commonly, heterotaxy, and congenital heart disease), reflecting dysfunction of embryological nodal cilia. Male infertility is common and reflects defects in sperm tail axonemes. Most patients with primary ciliary dyskinesia have a history of neonatal respiratory distress, suggesting that motile cilia play a role in fluid clearance during the transition from a fetal to neonatal lung. Ciliopathies involving sensory cilia, including autosomal dominant or recessive polycystic kidney disease, Bardet-Biedl syndrome, and Alstrom syndrome, may have chronic respiratory symptoms and even bronchiectasis suggesting clinical overlap with primary ciliary dyskinesia.     

Genetic Analysis of Korean Adult Patients with Nontuberculous Mycobacteria Suspected of Primary Ciliary Dyskinesia Using Whole Exome Sequencing

PurposeNontuberculous mycobacteria (NTM) is ubiquitous in the environment, but NTM lung disease (NTM-LD) is uncommon. Since exposure to NTM is inevitable, patients who develop NTM-LD are likely to have specific susceptibility factors, such as primary ciliary dyskinesia (PCD). PCD is a genetically heterogeneous disorder of motile cilia and is characterized by chronic respiratory tract infection, organ laterality defect, and infertility. In this study, we performed whole exome sequencing (WES) and investigated the genetic characteristics of adult NTM patients with suspected PCD.Materials and MethodsWES was performed in 13 NTM-LD patients who were suspected of having PCD by clinical symptoms and/or ultrastructural ciliary defect observed by transmission electron microscopy. A total of 45 PCD-causing genes, 23 PCD-candidate genes, and 990 ciliome genes were analyzed.ResultsFour patients were found to have biallelic loss-of-function (LoF) variants in the following PCD-causing genes: CCDC114, DNAH5, HYDIN, and NME5. In four other patients, only one LoF variant was identified, while the remaining five patients did not have any LoF variants.ConclusionAt least 30.8% of NTM-LD patients who were suspected of having PCD had biallelic LoF variants, and an additional 30.8% of patients had one LoF variant. Therefore, PCD should be considered in patients with NTM-LD with symptoms or signs suspicious of PCD.     

Biallelic variants in CEP164 cause a motile ciliopathy-like syndrome

Ciliopathies may be classed as primary or motile depending on the underlying ciliary defect and are usually considered distinct clinical entities. Primary ciliopathies are associated with multisystem syndromes typically affecting the brain, kidney, and eye, as well as other organ systems such as the liver, skeleton, auditory system, and metabolism. Motile ciliopathies are a heterogenous group of disorders with defects in specialised motile ciliated tissues found within the lung, brain, and reproductive system, and are associated with primary ciliary dyskinesia, bronchiectasis, infertility and rarely hydrocephalus. Primary and motile cilia share defined core ultra-structures with an overlapping proteome, and human disease phenotypes can reflect both primary and motile ciliopathies. CEP164 encodes a centrosomal distal appendage protein vital for primary ciliogenesis. Human CEP164 mutations are typically described in patients with nephronophthisis-related primary ciliopathies but have also been implicated in motile ciliary dysfunction. Here we describe a patient with an atypical motile ciliopathy phenotype and biallelic CEP164 variants. This work provides further evidence that CEP164 mutations can contribute to both primary and motile ciliopathy syndromes, supporting their functional and clinical overlap, and informs the investigation and management of CEP164 ciliopathy patients.     

Exome sequencing in individuals with cardiovascular laterality defects identifies potential candidate genes

The birth prevalence of laterality defects is about 1.1/10,000 comprising different phenotypes ranging from situs inversus totalis to heterotaxy, mostly associated with complex congenital heart defects (CHD) and situs abnormalities such as intestinal malrotation, biliary atresia, asplenia, or polysplenia. A proportion of laterality defects arise in the context of primary ciliary dyskinesia (PCD) accompanied by respiratory symptoms or infertility. In this study, exome sequencing (ES) was performed in 14 case-parent trios/quattros with clinical exclusion of PCD prior to analysis. Moreover, all cases and parents underwent detailed clinical phenotyping including physical examination, echocardiography by a skilled paediatric cardiologist and abdominal ultrasound examinations not to miss mildly affected individuals. Subsequent survey of the exome data comprised filtering for monoallelic de novo, rare biallelic, and X-linked recessive variants. In two families, rare variants of uncertain significance (VUS) in PKD1L1 and ZIC3 were identified. Both genes have been associated with laterality defects. In two of the remaining families, biallelic variants in LMBRD1 and DNAH17, respectively, were prioritized. In another family, an ultra-rare de novo variant in WDR47 was found. Extensive exome survey of 2,109 single exomes of individuals with situs inversus totalis, heterotaxy, or isolated CHD identified two individuals with novel monoallelic variants in WDR47, but no further individuals with biallelic variants in DNAH17 or LMBRD1. Overall, ES of 14 case-parent trios/quattros with cardiovascular laterality defects identified rare VUS in two families in known disease-associated genes PKD1L1 and ZIC3 and suggests DNAH17, LMBRD1, and WDR47 as potential genes involved in laterality defects.Subject terms: Disease genetics, Genetic counselling, Biological sciences     

Transport of the outer dynein arm complex to cilia requires a cytoplasmic protein Lrrc6

Lrrc6 encodes a cytoplasmic protein that is expressed specifically in cells with motile cilia including the node, trachea and testes of the mice. A mutation of Lrrc6 has been identified in human patients with primary ciliary dyskinesia (PCD). Mutant mice lacking Lrrc6 show typical PCD defects such as hydrocephalus and laterality defects. We found that in the absence of Lrrc6, the morphology of motile cilia remained normal, but their motility was completely lost. The 9 + 2 arrangement of microtubules remained normal in Lrrc6^‐/‐ mice, but the outer dynein arms (ODAs), the structures essential for the ciliary beating, were absent from the cilia. In the absence of Lrrc6, ODA proteins such as DNAH5, DNAH9 and IC2, which are assembled in the cytoplasm and transported to the ciliary axoneme, remained in the cytoplasm and were not transported to the ciliary axoneme. The IC2–IC1 interaction, which is the first step of ODA assembly, was normal in Lrrc6^?/? mice testes. Our results suggest that ODA proteins may be transported from the cytoplasm to the cilia by an Lrrc6‐dependent mechanism.     

A human laterality disorder associated with a homozygous WDR16 deletion

The laterality in the embryo is determined by left-right asymmetric gene expression driven by the flow of extraembryonic fluid, which is maintained by the rotary movement of monocilia on the nodal cells. Defects manifest by abnormal formation and arrangement of visceral organs. The genetic etiology of defects not associated with primary ciliary dyskinesia is largely unknown. In this study, we investigated the cause of situs anomalies, including heterotaxy syndrome and situs inversus totalis, in a consanguineous family. Whole-exome analysis revealed a homozygous deleterious deletion in the WDR16 gene, which segregated with the phenotype. WDR16 protein was previously proposed to play a role in cilia-related signal transduction processes; the rat Wdr16 protein was shown to be confined to cilia-possessing tissues and severe hydrocephalus was observed in the wdr16 gene knockdown zebrafish. The phenotype associated with the homozygous deletion in our patients suggests a role for WDR16 in human laterality patterning. Exome analysis is a valuable tool for molecular investigation even in cases of large deletions.     

Secondary defects detected by transmission electron microscopy in primary ciliary dyskinesia diagnostics

ABSTRACT

Primary ciliary dyskinesia (PCD) is predominantly an autosomal recessively inherited condition that affects ~1 in 15,000 people. Diagnosis of PCD can be complex and is ordinarily based on the results of multiple investigations. These investigations include nasal nitric oxide, high-speed video microscopy, genotyping, and electron microscopy analysis of ciliary ultrastructure. A diagnosis is ultimately confirmed by the presence of a hallmark defect identified by transmission electron microscopy or biallelic variants in a known PCD gene.

Secondary ciliary defects are commonly seen in samples submitted for diagnosis of PCD. Acquired secondary ciliary ultrastructural abnormalities, which are not caused by a variant in a ciliary gene, are usually transient and reversible however failure to separate primary versus secondary defects can lead to misdiagnosis. In this review, we describe causes of secondary ciliary defects, identify the ultrastructural appearances associated with secondary ciliary dyskinesia and finally suggest methods to avoid misdiagnosis of PCD due to these acquired ciliary defects.     

Motile cilia defects in diseases other than primary ciliary dyskinesia: The contemporary diagnostic and research role for transmission electron microscopy

ABSTRACT

Ultrastructural studies have underpinned the cell biological and clinical investigations of the varied roles of motile cilia in health and disease, with a long history since the 1950s. Recent developments from transmission electron microscopy (TEM; cryo-electron microscopy, electron tomography) have yielded higher resolution and fresh insights into the structure and function of these complex organelles. Microscopy in ciliated organisms, disease models, and in patients with ciliopathy diseases has dramatically expanded our understanding of the ubiquity, multisystem involvement, and importance of cilia in normal human development. Here, we review the importance of motile cilia ultrastructural studies in understanding the basis of diseases other than primary ciliary dyskinesia.     

Unveiling the genetic etiology of primary ciliary dyskinesia: When standard genetic approach is not enough

PurposePrimary 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/methodsA 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.ResultsHere 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.ConclusionsThis 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.     

Mutations in CCDC11, which Encodes a Coiled‐Coil Containing Ciliary Protein,Causes Situs Inversus Due to Dysmotility of Monocilia in the Left–Right Organizer

In vertebrates, establishment of left–right (LR) asymmetry is dependent on cilia‐driven fluid flow within the LR organizer. Mutations in CCDC11 disrupt LR asymmetry in humans, but how the gene functions in LR patterning is presently unknown. We describe a patient with situs inversus totalis carrying homozygous loss‐of‐function mutations in CCDC11. We show that CCDC11 is an axonemal protein in respiratory cilia, but is largely dispensable for their structure and motility. To investigate the role of CCDC11 in LR development, we studied the zebrafish homolog of the gene. Like in human respiratory cilia, loss of Ccdc11 causes minor defects in the motility of zebrafish kidney cilia, although the protein localizes to their axonemes and base. By contrast, Ccdc11 localizes exclusively to the basal bodies of cilia within Kupffer's vesicle, the organ of laterality of teleost fishes, and within the spinal canal. Moreover, the rotational motion of the cilia in these tissues of ccdc11‐deficient embryos was strongly impaired. Our findings demonstrate that CCDC11 has a conserved essential function in cilia of the vertebrate LR organizer. To the best of our knowledge, this is the first ciliary component, which has a differential localization and function in different kinds of motile cilia.     

Disrupted intraflagellar transport due to IFT74 variants causes Joubert syndrome

PurposeCiliopathies are a group of disorders caused by defects of the cilia. Joubert syndrome (JBTS) is a recessive and pleiotropic ciliopathy that causes cerebellar vermis hypoplasia and psychomotor delay. Although the intraflagellar transport (IFT) complex serves as a key module to maintain the ciliary structure and regulate ciliary signaling, the function of IFT in JBTS remains largely unknown. We aimed to explore the impact of IFT dysfunction in JBTS.MethodsExome sequencing was performed to screen for pathogenic variants in IFT genes in a JBTS cohort. Animal model and patient-derived fibroblasts were used to evaluate the pathogenic effects of the variants.ResultsWe identified IFT74 as a JBTS-associated gene in three unrelated families. All the affected individuals carried truncated variants and shared one missense variant (p.Q179E) found only in East Asians. The expression of the human p.Q179E-IFT74 variant displayed compromised rescue effects in zebrafish ift74 morphants. Attenuated ciliogenesis; altered distribution of IFT proteins and ciliary membrane proteins, including ARL13B, INPP5E, and GPR161; and disrupted hedgehog signaling were observed in patient fibroblasts with IFT74 variants.ConclusionIFT74 is identified as a JBTS-related gene. Cellular and biochemical mechanisms are also provided.

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Mutations in GAS8, a Gene Encoding a Nexin‐Dynein Regulatory Complex Subunit,Cause Primary Ciliary Dyskinesia with Axonemal Disorganization

Primary ciliary dyskinesia (PCD) is an autosomal recessive disease characterized by chronic respiratory infections of the upper and lower airways, hypofertility, and, in approximately half of the cases, situs inversus. This complex phenotype results from defects in motile cilia and sperm flagella. Among the numerous genes involved in PCD, very few—including CCDC39 and CCDC40—carry mutations that lead to a disorganization of ciliary axonemes with microtubule misalignment. Focusing on this particular phenotype, we identified bi‐allelic loss‐of‐function mutations in GAS8, a gene that encodes a subunit of the nexin‐dynein regulatory complex (N‐DRC) orthologous to DRC4 of the flagellated alga Chlamydomonas reinhardtii. Unlike the majority of PCD patients, individuals with GAS8 mutations have motile cilia, which, as documented by high‐speed videomicroscopy, display a subtle beating pattern defect characterized by slightly reduced bending amplitude. Immunofluorescence studies performed on patients’ respiratory cilia revealed that GAS8 is not required for the proper expression of CCDC39 and CCDC40. Rather, mutations in GAS8 affect the subcellular localization of another N‐DRC subunit called DRC3. Overall, this study, which identifies GAS8 as a PCD gene, unveils the key importance of the corresponding protein in N‐DRC integrity and in the proper alignment of axonemal microtubules in humans.     

Different frequency of cilia with transposition in human nasal and bronchial mucosa. A case of acquired ciliary dyskinesia

Nasal and bronchial cilia and spermatozoa of a patient with a high clinical suspicion of a ciliary dyskinesia syndrome were ultrastructurally studied and quantified. Defective cilia showed two types of axonemal patterns: 9d+0s and 8d+1d. Of these, 9d+0s cilia prevailed in the proximal region, whereas 8d+1d prevailed in the distal region. Translocation of a peripheral doublet to the central position occurred at the middle region of cilia lacking the central pair, probably to compensate for its absence. Quantitative analysis showed that the percentages of anomalous cilia were 5.32±0.93 in nasal samples and 43.17±2.34 in bronchial samples. Spermatozoa without the central pair or with a translocated microtubular doublet were rarely observed, but a variety of nonspecific defects were seen. Even though transposition is generally considered to be an inherited ciliary defect and one of the causes of primary ciliary dyskinesia, in this case quantitative ultrastructural analysis and clinical data indicate that this is an acquired ciliary defect. Received: 10 November 1999 / Accepted: 10 March 2000     

Discordant organ laterality in monozygotic twins with primary ciliary dyskinesia

Primary ciliary dyskinesia (PCD) is a genetic disease characterized by abnormal ciliary structure and function, impaired mucociliary clearance, and chronic middle ear, sinus, and lung disease. PCD is associated with situs inversus in ∼50% of the patients. One proposed explanation for this relationship is that normal ciliary function plays a role in normal organ orientation, whereas organ orientation in PCD is a random event because of dysfunctional cilia in early embryonic development. Another hypothesis for the association between PCD and situs inversus is that mutated genes in PCD not only cause defective cilia, but are also linked to the control of organ laterality, such that abnormalities in this molecular pathway result in random left-right asymmetry. We report on a set of monozygotic twin women with PCD. In both patients, deficiency of the inner dynein arms was noted on ciliary ultrastructural analysis, associated with a clinical syndrome of bronchiectasis, chronic sinusitis, and middle ear disease. One of the twins has situs solitus, the other has situs inversus totalis. DNA analysis confirmed that the twins are monozygotic. This is consistent with the hypothesis that situs inversus occurring in patients with primary ciliary dyskinesia is a random but “complete” event in the fetal development of patients with PCD. Am. J. Med. Genet. 82:155–160, 1999. © 1999 Wiley-Liss, Inc.     

Mutation of serine/threonine protein kinase 36 (STK36) causes primary ciliary dyskinesia with a central pair defect

Primary ciliary dyskinesia (PCD) is a genetic condition of impaired ciliary beating, characterized by chronic infections of the upper and lower airways and progressive lung failure. Defects of the outer dynein arms are the most common cause of PCD. In about half of the affected individuals, PCD occurs with situs inversus (Kartagener syndrome). A minor PCD subgroup including defects of the radial spokes (RS) and central pair (CP) is hallmarked by the absence of laterality defects, subtle beating abnormalities, and unequivocally apparent ultrastructural defects of the ciliary axoneme, making their diagnosis challenging. We identified homozygous loss‐of‐function mutations in STK36 in one PCD‐affected individual with situs solitus. Transmission electron microscopy analysis demonstrates that STK36 is required for cilia orientation in human respiratory epithelial cells, with a probable localization of STK36 between the RS and CP. STK36 screening can now be included for this rare and difficult to diagnose PCD subgroup.     

Defective Motile Cilia in Prickle2-Deficient Mice

Motile cilia play diverse roles across phyla and cell types, and abnormalities in motile cilia lead to numerous disease states, including hydrocephalus. Although motile ciliary abnormalities in Prickle2 mutants have not yet been described, the planar cell polarity genes, including Prickle2, are implicated in the development and function of motile cilia. This report evaluates Prickle2-deficient mice for dysfunction in processes known to depend on functioning motile cilia. Prickle2-deficient mice do not develop hydrocephalus, but do display abnormal morphology and motility in the motile cilia of the ependyma. The morphology of tracheal motile cilia is also abnormal. Taken together, these results demonstrate that Prickle2 is required for normal ependymal motile cilia development and function.     

Dysfunction of axonemal dynein heavy chain Mdnah5 inhibits ependymal flow and reveals a novel mechanism for hydrocephalus formation

Motility of unicellular organisms occurred early in evolution with the emergence of cilia and flagella. In vertebrates, motile cilia are required for numerous functions such as clearance of the airways and determination of left-right body asymmetry. Ependymal cells lining the brain ventricles also carry motile cilia, but their biological function has remained obscure. Here, we show that ependymal cilia generate a laminar flow of cerebrospinal fluid through the cerebral aqueduct, which we term as 'ependymal flow'. The axonemal dynein heavy chain gene Mdnah5 is specifically expressed in ependymal cells, and is essential for ultrastructural and functional integrity of ependymal cilia. In Mdnah5-mutant mice, lack of ependymal flow causes closure of the aqueduct and subsequent formation of triventricular hydrocephalus during early postnatal brain development. The higher incidence of aqueduct stenosis and hydrocephalus formation in patients with ciliary defects proves the relevance of this novel mechanism in humans.