Recessively Inherited Deficiency of Secreted WFDC2 (HE4) Causes Nasal Polyposis and Bronchiectasis.

RATIONALE: Bronchiectasis is a pathological dilatation of the bronchi in the respiratory airways associated with environmental or genetic causes (e.g., cystic fibrosis, primary ciliary dyskinesia and primary immunodeficiency disorders), but most cases remain idiopathic. OBJECTIVES: To identify novel genetic defects in unsolved cases of bronchiectasis presenting with severe rhinosinusitis, nasal polyposis, and pulmonary Pseudomonas aeruginosa infection. METHODS: DNA was analyzed by next-generation or targeted Sanger sequencing. RNA was analyzed by quantitative PCR and single-cell RNA sequencing. Patient-derived, cells, cell cultures and secretions (mucus, saliva, seminal fluid) were analyzed by Western blotting and immunofluorescence microscopy, and mucociliary activity was measured. Blood serum was analyzed by electrochemiluminescence immunoassay. Protein structure and proteomic analyses were used to assess the impact of a disease-causing founder variant. MEASUREMENTS AND MAIN RESULTS: We identified bi-allelic pathogenic variants in WFDC2 in 11 individuals from 10 unrelated families originating from the United States, Europe, Asia, and Africa. Expression of WFDC2 was detected predominantly in secretory cells of control airway epithelium and also in submucosal glands. We demonstrate that WFDC2 is below the limit of detection in blood serum and hardly detectable in samples of saliva, seminal fluid, and airway surface liquid from WFDC2-deficient individuals. Computer simulations and deglycosylation assays indicate that the disease-causing founder variant p.Cys49Arg structurally hampers glycosylation and thus secretion of mature WFDC2. CONCLUSIONS: WFDC2 dysfunction defines a novel molecular etiology of bronchiectasis characterized by the deficiency of a secreted component of the airways. A commercially available blood test combined with genetic testing allows its diagnosis. This article is open access and distributed under the terms of the Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/).

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

PURPOSE: Primary 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. METHODS: In addition to studies in mouse and planaria, clinical exome sequencing and functional analyses in human were performed. RESULTS: In 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. CONCLUSION: Our 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.

Mutations in TP73 cause impaired mucociliary clearance and lissencephaly.

TP73 belongs to the TP53 family of transcription factors and has therefore been well studied in cancer research. Studies in mice, however, have revealed non-oncogenic activities related to multiciliogenesis. Utilizing whole-exome sequencing analysis in a cohort of individuals with a mucociliary clearance disorder and cortical malformation, we identified homozygous loss-of-function variants in TP73 in seven individuals from five unrelated families. All affected individuals exhibit a chronic airway disease as well as a brain malformation consistent with lissencephaly. We performed high-speed video microscopy, immunofluorescence analyses, and transmission electron microscopy in respiratory epithelial cells after spheroid or air liquid interface culture to analyze ciliary function, ciliary length, and number of multiciliated cells (MCCs). The respiratory epithelial cells studied display reduced ciliary length and basal bodies mislocalized within the cytoplasm. The number of MCCs is severely reduced, consistent with a reduced number of cells expressing the transcription factors crucial for multiciliogenesis (FOXJ1, RFX2). Our data demonstrate that autosomal-recessive deleterious variants in the TP53 family member TP73 cause a mucociliary clearance disorder due to a defect in MCC differentiation.

Motility of efferent duct cilia aids passage of sperm cells through the male reproductive system.

Motile cilia line the efferent ducts of the mammalian male reproductive tract. Several recent mouse studies have demonstrated that a reduced generation of multiple motile cilia in efferent ducts is associated with obstructive oligozoospermia and fertility issues. However, the sole impact of efferent duct cilia dysmotility on male infertility has not been studied so far either in mice or human. Using video microscopy, histological- and ultrastructural analyses, we examined male reproductive tracts of mice deficient for the axonemal motor protein DNAH5: this defect exclusively disrupts the outer dynein arm (ODA) composition of motile cilia but not the ODA composition and motility of sperm flagella. These mice have immotile efferent duct cilia that lack ODAs, which are essential for ciliary beat generation. Furthermore, they show accumulation of sperm in the efferent duct. Notably, the ultrastructure and motility of sperm from these males are unaffected. Likewise, human individuals with loss-of-function DNAH5 mutations present with reduced sperm count in the ejaculate (oligozoospermia) and dilatations of the epididymal head but normal sperm motility, similar to DNAH5 deficient mice. The findings of this translational study demonstrate, in both mice and men, that efferent duct ciliary motility is important for male reproductive fitness and uncovers a novel pathomechanism distinct from primary defects of sperm motility (asthenozoospermia). If future work can identify environmental factors or defects in genes other than DNAH5 that cause efferent duct cilia dysmotility, this will help unravel other causes of oligozoospermia and may influence future practices in genetic and fertility counseling as well as ART.

CFAP45 deficiency causes situs abnormalities and asthenospermia by disrupting an axonemal adenine nucleotide homeostasis module.

Axonemal dynein ATPases direct ciliary and flagellar beating via adenosine triphosphate (ATP) hydrolysis. The modulatory effect of adenosine monophosphate (AMP) and adenosine diphosphate (ADP) on flagellar beating is not fully understood. Here, we describe a deficiency of cilia and flagella associated protein 45 (CFAP45) in humans and mice that presents a motile ciliopathy featuring situs inversus totalis and asthenospermia. CFAP45-deficient cilia and flagella show normal morphology and axonemal ultrastructure. Proteomic profiling links CFAP45 to an axonemal module including dynein ATPases and adenylate kinase as well as CFAP52, whose mutations cause a similar ciliopathy. CFAP45 binds AMP in vitro, consistent with structural modelling that identifies an AMP-binding interface between CFAP45 and AK8. Microtubule sliding of dyskinetic sperm from Cfap45-/- mice is rescued with the addition of either AMP or ADP with ATP, compared to ATP alone. We propose that CFAP45 supports mammalian ciliary and flagellar beating via an adenine nucleotide homeostasis module.

The centrosomal/basal body protein OFD1 is required for microtubule organization and cell cycle progression.

Oral-Facial-Digital type I (OFD1) is a rare inherited form of renal cystic disease associated with ciliary dysfunction. This disorder is due to mutations in the OFD1 gene that encodes a protein localized to centrosomes and basal bodies in different cell types. Immunofluorescence analysis demonstrated that OFD1 displays a dynamic distribution during cell cycle. High-content microscopy analysis of Ofd1-depleted fibroblasts revealed impaired cell cycle progression. Immunofluorescence analysis and cell proliferation assays also indicated the presence of a variety of defects such as centrosome accumulation, nuclear abnormalities and aneuploidy. In addition, Ofd1-depleted cells displayed an abnormal microtubule network that may underlie these defects. All together our results suggest that OFD1 contributes to the function of the microtubule organizing center (MTOC) in the cell, controlling cell cycle progression both in vitro and in vivo.

A human ciliopathy reveals essential functions for NEK10 in airway mucociliary clearance.

Mucociliary clearance, the physiological process by which mammalian conducting airways expel pathogens and unwanted surface materials from the respiratory tract, depends on the coordinated function of multiple specialized cell types, including basal stem cells, mucus-secreting goblet cells, motile ciliated cells, cystic fibrosis transmembrane conductance regulator (CFTR)-rich ionocytes, and immune cells1,2. Bronchiectasis, a syndrome of pathological airway dilation associated with impaired mucociliary clearance, may occur sporadically or as a consequence of Mendelian inheritance, for example in cystic fibrosis, primary ciliary dyskinesia (PCD), and select immunodeficiencies3. Previous studies have identified mutations that affect ciliary structure and nucleation in PCD4, but the regulation of mucociliary transport remains incompletely understood, and therapeutic targets for its modulation are lacking. Here we identify a bronchiectasis syndrome caused by mutations that inactivate NIMA-related kinase 10 (NEK10), a protein kinase with previously unknown in vivo functions in mammals. Genetically modified primary human airway cultures establish NEK10 as a ciliated-cell-specific kinase whose activity regulates the motile ciliary proteome to promote ciliary length and mucociliary transport but which is dispensable for normal ciliary number, radial structure, and beat frequency. Together, these data identify a novel and likely targetable signaling axis that controls motile ciliary function in humans and has potential implications for other respiratory disorders that are characterized by impaired mucociliary clearance.

Author Correction: A human ciliopathy reveals essential functions for NEK10 in airway mucociliary clearance.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

SPEF2- and HYDIN-Mutant Cilia Lack the Central Pair-associated Protein SPEF2, Aiding Primary Ciliary Dyskinesia Diagnostics.

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous chronic destructive airway disease. PCD is traditionally diagnosed by nasal nitric oxide measurement, analysis of ciliary beating, transmission electron microscopy (TEM), and/or genetic testing. In most genetic PCD variants, laterality defects can occur. However, it is difficult to establish a diagnosis in individuals with PCD and central pair (CP) defects, and alternative strategies are required because of very subtle ciliary beating abnormalities, a normal ciliary ultrastructure, and normal situs composition. Mutations in HYDIN are known to cause CP defects, but the genetic analysis of HYDIN variants is confounded by the pseudogene HYDIN2, which is almost identical in terms of intron/exon structure. We have previously shown that several types of PCD can be diagnosed via immunofluorescence (IF) microscopy analyses. Here, using IF microscopy, we demonstrated that in individuals with PCD and CP defects, the CP-associated protein SPEF2 is absent in HYDIN-mutant cells, revealing its dependence on functional HYDIN. Next, we performed IF analyses of SPEF2 in respiratory cells from 189 individuals with suspected PCD and situs solitus. Forty-one of the 189 individuals had undetectable SPEF2 and were subjected to a genetic analysis, which revealed one novel loss-of-function mutation in SPEF2 and three reported and 13 novel HYDIN mutations in 15 individuals. The remaining 25 individuals are good candidates for new, as-yet uncharacterized PCD variants that affect the CP apparatus. SPEF2 mutations have been associated with male infertility but have not previously been identified to cause PCD. We identified a mutation of SPEF2 that is causative for PCD with a CP defect. We conclude that SPEF2 IF analyses can facilitate the detection of CP defects and evaluation of the pathogenicity of HYDIN variants, thus aiding the molecular diagnosis of CP defects.

Acinetobacter baumannii Is a Risk Factor for Lower Respiratory Tract Infections in Children and Adolescents With a Tracheostomy.

BACKGROUND: Lower respiratory tract infections (LRIs) are a major cause of hospitalization for children and adolescents with a tracheostomy. The aim of this study was to identify risk factors for LRI. METHODS: In this retrospective study, we assessed the number of LRI and hospitalizations for LRI from 2004 to 2014 at the University Hospital Muenster Pediatric Department. We analyzed associations between LRI and clinical findings, and we cultured pathogens in tracheal aspirates (TAs) during noninfection periods. Univariable and multivariable negative, binomial regression analyses were applied to identify associations between possible risk factors and LRI. RESULTS: Seventy-eight patients had 148 LRI, of which 99 were treated in hospital. The median number of LRI per year was 0.4. Six-hundred thirteen pathogens were detected in 315 specimens; Staphylococcus aureus (22.5%), Pseudomonas aeruginosa (14.8%) and Haemophilus influenzae (6.2%) were most frequently detected. Acinetobacter baumannii is an independent risk factor for LRI (rate ratio, 1.792; P = 0.030) and hospital admissions for LRI (rate ratio, 1.917; P = 0.011). CONCLUSIONS: Children with a tracheostomy have frequent LRI. A. baumannii but not P. aeruginosa or S. aureus in TA is a risk factor for LRI in children with a long-term tracheostomy. This supports repetitive culture of TA for microbiologic workup to identify children and adolescents with an increased risk for LRI.

De Novo Mutations in FOXJ1 Result in a Motile Ciliopathy with Hydrocephalus and Randomization of Left/Right Body Asymmetry.

Hydrocephalus is one of the most prevalent form of developmental central nervous system (CNS) malformations. Cerebrospinal fluid (CSF) flow depends on both heartbeat and body movement. Furthermore, it has been shown that CSF flow within and across brain ventricles depends on cilia motility of the ependymal cells lining the brain ventricles, which play a crucial role to maintain patency of the narrow sites of CSF passage during brain formation in mice. Using whole-exome and whole-genome sequencing, we identified an autosomal-dominant cause of a distinct motile ciliopathy related to defective ciliogenesis of the ependymal cilia in six individuals. Heterozygous de novo mutations in FOXJ1, which encodes a well-known member of the forkhead transcription factors important for ciliogenesis of motile cilia, cause a motile ciliopathy that is characterized by hydrocephalus internus, chronic destructive airway disease, and randomization of left/right body asymmetry. Mutant respiratory epithelial cells are unable to generate a fluid flow and exhibit a reduced number of cilia per cell, as documented by high-speed video microscopy (HVMA), transmission electron microscopy (TEM), and immunofluorescence analysis (IF). TEM and IF demonstrate mislocalized basal bodies. In line with this finding, the focal adhesion protein PTK2 displays aberrant localization in the cytoplasm of the mutant respiratory epithelial cells.

Randomization of Left-right Asymmetry and Congenital Heart Defects: The Role of DNAH5 in Humans and Mice.

Background - Nearly one in 100 live births presents with congenital heart defects (CHD). CHD are frequently associated with laterality defects, such as situs inversus totalis (SIT), a mirrored positioning of internal organs. Body laterality is established by a complex process: monocilia at the embryonic left-right organizer (LRO) facilitate both the generation and sensing of a leftward fluid flow. This induces the conserved left-sided Nodal signaling cascade to initiate asymmetric organogenesis. Primary ciliary dyskinesia (PCD) originates from dysfunction of motile cilia, causing symptoms such as chronic sinusitis, bronchiectasis and frequently SIT. The most frequently mutated gene in PCD, DNAH5 is associated with randomization of body asymmetry resulting in SIT in half of the patients; however, its relation to CHD occurrence in humans has not been investigated in detail so far. Methods - We performed genotype / phenotype correlations in 132 PCD patients carrying disease-causing DNAH5 mutations, focusing on situs defects and CHD. Using high speed video microscopy-, immunofluorescence-, and in situ hybridization analyses, we investigated the initial steps of left-right axis establishment in embryos of a Dnah5 mutant mouse model. Results - 65.9% (87 / 132) of the PCD patients carrying disease-causing DNAH5 mutations had laterality defects: 88.5% (77 / 87) presented with SIT, 11.5% (10 / 87) presented with situs ambiguus; and 6.1% (8 / 132) presented with CHD. In Dnah5mut/mut mice, embryonic LRO monocilia lack outer dynein arms resulting in immotile cilia, impaired flow at the LRO, and randomization of Nodal signaling with normal, reversed or bilateral expression of key molecules. Conclusions - For the first time, we directly demonstrate the disease-mechanism of laterality defects linked to DNAH5 deficiency at the molecular level during embryogenesis. We highlight that mutations in DNAH5 are not only associated with classical randomization of left-right body asymmetry but also with severe laterality defects including CHD.

CiliaCarta: An integrated and validated compendium of ciliary genes.

The cilium is an essential organelle at the surface of mammalian cells whose dysfunction causes a wide range of genetic diseases collectively called ciliopathies. The current rate at which new ciliopathy genes are identified suggests that many ciliary components remain undiscovered. We generated and rigorously analyzed genomic, proteomic, transcriptomic and evolutionary data and systematically integrated these using Bayesian statistics into a predictive score for ciliary function. This resulted in 285 candidate ciliary genes. We generated independent experimental evidence of ciliary associations for 24 out of 36 analyzed candidate proteins using multiple cell and animal model systems (mouse, zebrafish and nematode) and techniques. For example, we show that OSCP1, which has previously been implicated in two distinct non-ciliary processes, causes ciliogenic and ciliopathy-associated tissue phenotypes when depleted in zebrafish. The candidate list forms the basis of CiliaCarta, a comprehensive ciliary compendium covering 956 genes. The resource can be used to objectively prioritize candidate genes in whole exome or genome sequencing of ciliopathy patients and can be accessed at http://bioinformatics.bio.uu.nl/john/syscilia/ciliacarta/.

Comparison of Nocturnal Cough Analysis in Healthy Subjects and in Patients with Cystic Fibrosis and Primary Ciliary Dyskinesia: A Prospective Observational Study.

BACKGROUND: Cough is a key symptom in patients with cystic fibrosis (CF) and primary ciliary dyskinesia (PCD). OBJECTIVE: The study objectives were to test whether cough is related to parameters reflecting their disease severity and whether CF and PCD differ in cough frequency. METHODS: In this prospective observational study, we used a microphone-based monitoring system (LEOSound® Monitor) to count the coughs in healthy subjects (HS) and in stable patients with CF and PCD (25 subjects per group) on 2 consecutive nights. RESULTS: The median number of coughs/h in the HS, CF, and PCD groups was 0.0, 1.3, and 0.5 on the first night and 0.0, 2.3, and 0.2 on the second night, respectively. Patients with CF and PCD coughed more than HS (p < 0.001 and p = 0.009, respectively) and CF patients coughed more than PCD patients (p = 0.023). A multivariable mixed model analysis revealed forced expiratory volume in 1 s as an independent risk factor for increased cough frequency in patients. The reliability for repeated measurements was higher for cough epochs/h than for coughs/h (intraclass correlation coefficient: 0.75 and 0.49, respectively). CONCLUSIONS: Patients with CF cough more than patients with PCD. The cough frequency in CF and PCD is associated with parameters reflecting disease severity. Cough frequency is a possible endpoint in clinical trials and cough epochs/h may be more useful than coughs/h.

Homozygous loss-of-function mutations in MNS1 cause laterality defects and likely male infertility.

The clinical spectrum of ciliopathies affecting motile cilia spans impaired mucociliary clearance in the respiratory system, laterality defects including heart malformations, infertility and hydrocephalus. Using linkage analysis and whole exome sequencing, we identified two recessive loss-of-function MNS1 mutations in five individuals from four consanguineous families: 1) a homozygous nonsense mutation p.Arg242* in four males with laterality defects and infertility and 2) a homozygous nonsense mutation p.Gln203* in one female with laterality defects and recurrent respiratory infections additionally carrying homozygous mutations in DNAH5. Consistent with the laterality defects observed in these individuals, we found Mns1 to be expressed in mouse embryonic ventral node. Immunofluorescence analysis further revealed that MNS1 localizes to the axonemes of respiratory cilia as well as sperm flagella in human. In-depth ultrastructural analyses confirmed a subtle outer dynein arm (ODA) defect in the axonemes of respiratory epithelial cells resembling findings reported in Mns1-deficient mice. Ultrastructural analyses in the female carrying combined mutations in MNS1 and DNAH5 indicated a role for MNS1 in the process of ODA docking (ODA-DC) in the distal respiratory axonemes. Furthermore, co-immunoprecipitation and yeast two hybrid analyses demonstrated that MNS1 dimerizes and interacts with the ODA docking complex component CCDC114. Overall, we demonstrate that MNS1 deficiency in humans causes laterality defects (situs inversus) and likely male infertility and that MNS1 plays a role in the ODA-DC assembly.

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

Primary ciliary dyskinesia (PCD) is characterized by chronic airway disease, male infertility, and randomization of the left/right body axis as a result of defects of motile cilia and sperm flagella. We identified loss-of-function mutations in the open-reading frame C11orf70 in PCD individuals from five distinct families. Transmission electron microscopy analyses and high-resolution immunofluorescence microscopy demonstrate that loss-of-function mutations in C11orf70 cause immotility of respiratory cilia and sperm flagella, respectively, as a result of the loss of axonemal outer (ODAs) and inner dynein arms (IDAs), indicating that C11orf70 is involved in cytoplasmic assembly of dynein arms. Expression analyses of C11orf70 showed that C11orf70 is expressed in ciliated respiratory cells and that the expression of C11orf70 is upregulated during ciliogenesis, similar to other previously described cytoplasmic dynein-arm assembly factors. Furthermore, C11orf70 shows an interaction with cytoplasmic ODA/IDA assembly factor DNAAF2, supporting our hypothesis that C11orf70 is a preassembly factor involved in the pathogenesis of PCD. The identification of additional genetic defects that cause PCD and male infertility is of great importance for the clinic as well as for genetic counselling.

Evolutionary Proteomics Uncovers Ancient Associations of Cilia with Signaling Pathways.

Cilia are organelles specialized for movement and signaling. To infer when during evolution signaling pathways became associated with cilia, we characterized the proteomes of cilia from sea urchins, sea anemones, and choanoflagellates. We identified 437 high-confidence ciliary candidate proteins conserved in mammals and discovered that Hedgehog and G-protein-coupled receptor pathways were linked to cilia before the origin of bilateria and transient receptor potential (TRP) channels before the origin of animals. We demonstrated that candidates not previously implicated in ciliary biology localized to cilia and further investigated ENKUR, a TRP channel-interacting protein identified in the cilia of all three organisms. ENKUR localizes to motile cilia and is required for patterning the left-right axis in vertebrates. Moreover, mutation of ENKUR causes situs inversus in humans. Thus, proteomic profiling of cilia from diverse eukaryotes defines a conserved ciliary proteome, reveals ancient connections to signaling, and uncovers a ciliary protein that underlies development and human disease.

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.

Mutations in PIH1D3 Cause X-Linked Primary Ciliary Dyskinesia with Outer and Inner Dynein Arm Defects.

Defects in motile cilia and sperm flagella cause primary ciliary dyskinesia (PCD), characterized by chronic airway disease, infertility, and left-right body axis disturbance. Here we report maternally inherited and de novo mutations in PIH1D3 in four men affected with PCD. PIH1D3 is located on the X chromosome and is involved in the preassembly of both outer (ODA) and inner (IDA) dynein arms of cilia and sperm flagella. Loss-of-function mutations in PIH1D3 lead to absent ODAs and reduced to absent IDAs, causing ciliary and flagellar immotility. Further, PIH1D3 interacts and co-precipitates with cytoplasmic ODA/IDA assembly factors DNAAF2 and DNAAF4. This result has clinical and genetic counseling implications for genetically unsolved male case subjects with a classic PCD phenotype that lack additional phenotypes such as intellectual disability or retinitis pigmentosa.