A range of 30%-62% of functioning multiciliated airway cells is sufficient to maintain ciliary airway clearance.

BACKGROUND: Primary ciliary dyskinesia (PCD) is a genetic disorder caused by aberrant motile cilia function that results in defective ciliary airway clearance and subsequently to recurrent airway infections and bronchiectasis. QUESTION: How many functional multiciliated airway cells are sufficient to maintain ciliary airway clearance? METHODS: To answer this question we exploited the molecular defects of the X-linked recessive PCD variant caused by pathogenic variants in DNAAF6 (PIH1D3), characterized by immotile cilia in the affected males. We carefully analyzed the clinical phenotype, molecular defect (immunofluorescence and transmission-electron microscopy) and performed in vitro (particle tracking in air-liquid interface cultures) and in vivo (radiolabeled tracer studies) studies to assess ciliary clearance of respiratory cells from females with heterozygous and males with hemizygous pathogenic DNAAF6 variants. RESULTS: PCD males with hemizygous pathogenic DNAAF6 variants displayed exclusively immotile cilia, absence of ciliary clearance and severe PCD symptoms. Due to random or skewed X-chromosome inactivation in six females with heterozygous pathogenic DNAAF6 variants, 54.3%±10 (range 38%-70%) of multiciliated cells were defective. Nevertheless, in vitro and in vivo assessment of the ciliary airway clearance was normal or slightly abnormal. Consistently, heterozygous female individuals showed no or only mild respiratory symptoms. CONCLUSIONS: Our findings indicate that 30%-62% of functioning multiciliated respiratory cells are able to generate either normal or slightly reduced ciliary clearance. Because heterozygous females displayed either no or subtle respiratory symptoms, complete correction of 30% of cells by precision medicine might be able to improve ciliary airway clearance in PCD individuals as well as clinical symptoms.

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

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.

Analyses of 1,236 genotyped primary ciliary dyskinesia individuals identify regional clusters of distinct DNA variants and significant genotype-phenotype correlations.

BACKGROUND: Primary ciliary dyskinesia (PCD) represents a group of rare hereditary disorders characterized by deficient ciliary airway clearance that can be associated with laterality defects. We aimed to describe the underlying gene defects, geographical differences in genotypes and their relationship to diagnostic findings and clinical phenotypes. METHODS: Genetic variants and clinical findings (age, sex, body mass index, laterality defects, FEV1) were collected from 19 countries using the ERN LUNG International PCD Registry. Genetic data were evaluated according to ACMG guidelines. We assessed regional distribution of implicated genes and genetic variants as well as genotype correlations with laterality defects and FEV1. RESULTS: 1236 individuals carried 908 distinct pathogenic DNA variants in 46 PCD genes. We found considerable variation in the distribution of PCD genotypes across countries due to the presence of distinct founder variants. The prevalence of PCD genotypes associated with pathognomonic ultrastructural defects (mean 72%; 47-100%) and laterality defects (mean 42%; 28-69%) varied widely among the countries. The prevalence of laterality defects was significantly lower in PCD individuals without pathognomonic ciliary ultrastructure defects (18%). The PCD cohort had a reduced median FEV1 z-score (-1.66). In the group of individuals with CCNO (-3.26), CCDC39 (-2.49), and CCDC40 (-2.96) variants, FEV1 z-scores were significantly lower, while the group of DNAH11 (-0.83) and ODAD1 (-0.85) variant individuals had significantly milder FEV1 z-score reductions compared to the whole PCD cohort. CONCLUSION: This unprecedented multinational dataset of DNA variants and information on their distribution across countries facilitates interpretation of genetic epidemiology of PCD and provides prediction of diagnostic and phenotypic features such as the course of lung function.

[Primary ciliary dyskinesia]. / Primäre Ciliäre Dyskinesie.

Primary ciliary dyskinesia (PCD) is a rare genetic disorder with a variable clinical phenotype that is accompanied by reduced motility of the cilia in the respiratory tract and numerous other organs. This leads to various characteristic symptoms and disease manifestations, primarily affecting the lungs (chronic persistent productive cough, bronchiectasis), the nose and paranasal sinuses (chronic persistent rhinitis or rhinosinusitis) as well as the middle ear (chronic otitis media, middle ear effusion). Moreover, PCD is associated with impaired fertility or lateralization defects (situs anomalies, congenital heart defects). The diagnostics of PCD are complex and require a combination of several sophisticated instrument-based diagnostic procedures. Through thorough history taking and evaluation, suspected cases can be comparatively well identified based on typical clinical features and referred to further diagnostics. In recent years, molecular genetic analysis through panel diagnostics or whole exome and whole genome sequencing, has gained in importance as this enables affected individuals to participate in disease-specific and genotype-specific clinical trials. Although the current treatment is purely symptomatic, the earliest possible diagnosis is crucial for connecting patients to specialized PCD centers, which can have a significant impact on the clinical course of the affected individuals.

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 biallelic pathogenic variants in WAP four-disulfide core domain 2 (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.

Association between upper and lower respiratory disease among patients with primary ciliary dyskinesia: an international study.

Introduction: Nearly all patients with primary ciliary dyskinesia (PCD) report ear-nose-throat (ENT) symptoms. However, scarce evidence exists about how ENT symptoms relate to pulmonary disease in PCD. We explored possible associations between upper and lower respiratory disease among patients with PCD in a multicentre study. Methods: We included patients from the ENT Prospective International Cohort (EPIC-PCD). We studied associations of several reported ENT symptoms and chronic rhinosinusitis (defined using patient-reported information and examination findings) with reported sputum production and shortness of breath, using ordinal logistic regression. In a subgroup with available lung function results, we used linear regression to study associations of chronic rhinosinusitis and forced expiratory volume in 1 s (FEV1) accounting for relevant factors. Results: We included 457 patients (median age 15 years, interquartile range 10-24 years; 54% males). Shortness of breath associated with reported nasal symptoms and ear pain of any frequency, often or daily hearing problems, headache when bending down (OR 2.1, 95% CI 1.29-3.54) and chronic rhinosinusitis (OR 2.3, 95% CI 1.57-3.38) regardless of polyp presence. Sputum production associated with daily reported nasal (OR 2.2, 95% CI 1.20-4.09) and hearing (OR 2.0, 95% CI 1.10-3.64) problems and chronic rhinosinusitis (OR 2.1, 95% CI 1.48-3.07). We did not find any association between chronic rhinosinusitis and FEV1. Conclusion: Reported upper airway symptoms and signs of chronic rhinosinusitis associated with reported pulmonary symptoms, but not with lung function. Our results emphasise the assessment and management of upper and lower respiratory disease as a common, interdependent entity among patients with PCD.

Molecular defects in primary ciliary dyskinesia are associated with agenesis of the frontal and sphenoid paranasal sinuses and chronic rhinosinusitis.

Background: Primary ciliary dyskinesia (PCD; MIM 242650) is a rare genetic disorder characterized by malfunction of the motile cilia resulting in reduced mucociliary clearance of the airways. Together with recurring infections of the lower respiratory tract, chronic rhinosinusitis (CRS) is a hallmark symptom of PCD. Data on genotype-phenotype correlations in the upper airways are scarce. Materials and methods: We investigated the prevalence, radiologic severity, and impact on health-related quality of life (HrQoL) of CRS in 58 individuals with genetically confirmed PCD. Subgroup analysis was performed according to the predicted ultrastructural phenotype based on genetic findings. Results: Among 58 individuals harboring pathogenic variants in 22 distinct genes associated with PCD, all were diagnosed with CRS, and 47% underwent sinus surgery. A total of 36 individuals answered a German-adapted version of the 20-item Sinonasal Outcome Test (SNOT-20-GAV) with a mean score of 35.8 ± 17, indicating a remarkably reduced HrQoL. Paranasal sinus imaging of 36 individuals showed moderate-to-severe opacification with an elevated Lund-Mackay Score (LMS) of 10.2 ± 4.4. Bilateral agenesis of frontal sinus (19%) and sphenoid sinus (9.5%) was a frequent finding in individuals aged 16 years or older. Subgroup analysis for predicted ultrastructural phenotypes did not identify differences in HrQoL, extent of sinus opacification, or frequency of aplastic paranasal sinuses. Conclusion: PCD is strongly associated with CRS. The high burden of disease is indicated by decreased HrQoL. Therefore, the upper airways of PCD individuals should be evaluated and managed by ear-nose-throat (ENT) specialists. Genetically determined PCD groups with predicted abnormal versus (near) normal ultrastructure did not differ in disease severity. Further studies are needed to gain evidence-based knowledge of the phenotype and management of upper airway manifestations in PCD. In addition, individuals with agenesis of the frontal and sphenoid paranasal sinuses and chronic respiratory symptoms should be considered for a diagnostic evaluation of PCD.

Primary ciliary dyskinesia.

BACKGROUND AND OBJECTIVES: Primary ciliary dyskinesia (PCD, ORPHA:244) is a group of rare genetic disorders characterized by dysfunction of motile cilia. It is phenotypically and genetically heterogeneous, with more than 50 genes involved. Thanks to genetic, clinical, and functional characterization, immense progress has been made in the understanding and diagnosis of PCD. Nevertheless, it is underdiagnosed due to the heterogeneous phenotype and complexity of diagnosis. This review aims to help clinicians navigate this heterogeneous group of diseases. Here, we describe the broad spectrum of phenotypes associated with PCD and address pitfalls and difficult-to-interpret findings to avoid misinterpretation. METHOD: Review of literature CONCLUSION: PCD diagnosis is complex and requires integration of history, clinical picture, imaging, functional and structural analysis of motile cilia and, if available, genetic analysis to make a definitive diagnosis. It is critical that we continue to expand our knowledge of this group of rare disorders to improve the identification of PCD patients and to develop evidence-based therapeutic approaches.

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.

The disease-specific clinical trial network for primary ciliary dyskinesia: PCD-CTN.

Primary ciliary dyskinesia (PCD) is a rare genetic disorder characterised by impaired mucociliary clearance leading to irreversible lung damage. In contrast to other rare lung diseases like cystic fibrosis (CF), there are only few clinical trials and limited evidence-based treatments. Management is mainly based on expert opinions and treatment is challenging due to a wide range of clinical manifestations and disease severity. To improve clinical and translational research and facilitate development of new treatments, the clinical trial network for PCD (PCD-CTN) was founded in 2020 under the framework of the European Reference Network (ERN)-LUNG PCD Core. Applications from European PCD sites interested in participating in the PCD-CTN were requested. Inclusion criteria consisted of patient numbers, membership of ERN-LUNG PCD Core, use of associated standards of care, experience in PCD and/or CF clinical research, resources to run clinical trials, good clinical practice (GCP) certifications and institutional support. So far, applications from 22 trial sites in 18 European countries have been approved, including >1400 adult and >1600 paediatric individuals with PCD. The PCD-CTN is headed by a coordinating centre and consists of a steering and executive committee, a data safety monitoring board and committees for protocol review, training and standardisation. A strong association with patient organisations and industrial companies are further cornerstones. All participating trial sites agreed on a code of conduct. As CTNs from other diseases have demonstrated successfully, this newly formed PCD-CTN operates to establish evidence-based treatments for this orphan disease and to bring new personalised treatment approaches to patients.

Limitations of Nasal Nitric Oxide Measurement for Diagnosis of Primary Ciliary Dyskinesia with Normal Ultrastructure.

Rationale: Primary ciliary dyskinesia (PCD) is a heterogeneous, multisystem disorder characterized by defective ciliary beating. Diagnostic guidelines of the American Thoracic Society and European Respiratory Society recommend measurement of nasal nitric oxide (nNO) for PCD diagnosis. Several studies demonstrated low nNO production rates in PCD individuals, but underlying causes remain elusive. Objectives: To determine nNO production rates in a well-characterized PCD cohort, including subgroup analyses with regard to ultrastructural and ciliary beating phenotypes. Methods: This study included 301 individuals assessed according to European Respiratory Society guidelines. Diagnostic cutoffs for nNO production rates for this study cohort and subgroups with normal and abnormal ultrastructure were determined. Diagnostic accuracy was also tested for the widely used 77 nl/min cutoff in this study cohort. The relationship between nNO production rates and ciliary beat frequencies (CBFs) was evaluated. Results: The study cohort comprised 180 individuals with definite PCD diagnosis, including 160 individuals with genetic diagnosis, 16 individuals with probable PCD diagnosis, and 105 disease controls. The 77 nl/min nNO cutoff showed a test sensitivity of 0.92 and specificity of 0.86. Test sensitivity was lower (0.85) in the subgroup of 47 PCD individuals with normal ultrastructure compared with 133 PCD individuals with abnormal ultrastructure (0.95). The optimal diagnostic cutoff for the nNO production rate for the whole study cohort was 69.8 nl/min (sensitivity, 0.92; specificity, 0.89); however, it was 107.8 nl/min (sensitivity, 0.89; specificity, 0.78) for the subgroup of PCD with normal ultrastructure. PCD individuals with normal ultrastructure compared with abnormal ultrastructure showed higher ciliary motility. Consistently, PCD individuals with higher CBFs showed higher nNO production rates. In addition, laterality defects occurred less frequently in PCD with normal ultrastructure. Conclusions: Measurements of nNO below the widely used 77 nl/min cutoff are less sensitive in detecting PCD individuals with normal ultrastructure. Our findings indicate that higher nNO production in this subgroup with a higher cutoff for the nNO production rate (107.8 nl/min) and higher residual ciliary motility is dependent on the underlying molecular PCD defect. Higher nNO production rates, higher residual CBFs, and the lower prevalence of laterality defects hamper diagnosis of PCD with normal ultrastructure. Adjusting the cutoff of nNO production rate to 107.8 nl/min might promote diagnosing PCD with normal ultrastructure.

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.

Defects in the cytoplasmic assembly of axonemal dynein arms cause morphological abnormalities and dysmotility in sperm cells leading to male infertility.

Axonemal protein complexes, such as outer (ODA) and inner (IDA) dynein arms, are responsible for the generation and regulation of flagellar and ciliary beating. Studies in various ciliated model organisms have shown that axonemal dynein arms are first assembled in the cell cytoplasm and then delivered into axonemes during ciliogenesis. In humans, mutations in genes encoding for factors involved in this process cause structural and functional defects of motile cilia in various organs such as the airways and result in the hereditary disorder primary ciliary dyskinesia (PCD). Despite extensive knowledge about the cytoplasmic assembly of axonemal dynein arms in respiratory cilia, this process is still poorly understood in sperm flagella. To better define its clinical relevance on sperm structure and function, and thus male fertility, further investigations are required. Here we report the fertility status in different axonemal dynein preassembly mutant males (DNAAF2/ KTU, DNAAF4/ DYX1C1, DNAAF6/ PIH1D3, DNAAF7/ZMYND10, CFAP300/C11orf70 and LRRC6). Besides andrological examinations, we functionally and structurally analyzed sperm flagella of affected individuals by high-speed video- and transmission electron microscopy as well as systematically compared the composition of dynein arms in sperm flagella and respiratory cilia by immunofluorescence microscopy. Furthermore, we analyzed the flagellar length in dynein preassembly mutant sperm. We found that the process of axonemal dynein preassembly is also critical in sperm, by identifying defects of ODAs and IDAs in dysmotile sperm of these individuals. Interestingly, these mutant sperm consistently show a complete loss of ODAs, while some respiratory cilia from the same individual can retain ODAs in the proximal ciliary compartment. This agrees with reports of solely one distinct ODA type in sperm, compared to two different ODA types in proximal and distal respiratory ciliary axonemes. Consistent with observations in model organisms, we also determined a significant reduction of sperm flagellar length in these individuals. These findings are relevant to subsequent studies on the function and composition of sperm flagella in PCD patients and non-syndromic infertile males. Our study contributes to a better understanding of the fertility status in PCD-affected males and should help guide genetic and andrological counselling for affected males and their families.

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.

Registries and collaborative studies for primary ciliary dyskinesia in Europe.

Primary ciliary dyskinesia (PCD) is a rare inherited disease characterised by malfunctioning cilia leading to a heterogeneous clinical phenotype with many organ systems affected. There is a lack of data on clinical presentation, prognosis and effectiveness of treatments, making it mandatory to improve the scientific evidence base. This article reviews the data resources that are available in Europe for clinical and epidemiological research in PCD, namely established national PCD registries and national cohort studies, plus two large collaborative efforts (the international PCD (iPCD) Cohort and the International PCD Registry), and discusses their strengths, limitations and perspectives. Denmark, Cyprus, Norway and Switzerland have national population-based registries, while England and France conduct multicentre cohort studies. Based on the data contained in these registries, the prevalence of diagnosed PCD is 3-7 per 100 000 in children and 0.2-6 per 100 000 in adults. All registries, together with other studies from Europe and beyond, contribute to the iPCD Cohort, a collaborative study including data from over 4000 PCD patients, and to the International PCD Registry, which is part of the ERN (European Reference Network)-LUNG network. This rich resource of readily available, standardised and contemporaneous data will allow obtaining fast answers to emerging clinical and research questions in PCD.

Efficacy and safety of azithromycin maintenance therapy in primary ciliary dyskinesia (BESTCILIA): a multicentre, double-blind, randomised, placebo-controlled phase 3 trial.

BACKGROUND: Use of maintenance antibiotic therapy with the macrolide azithromycin is increasing in a number of chronic respiratory disorders including primary ciliary dyskinesia (PCD). However, evidence for its efficacy in PCD is lacking. We aimed to determine the efficacy and safety of azithromycin maintenance therapy for 6 months in patients with PCD. METHODS: The Better Experimental Screening and Treatment for Primary Ciliary Dyskinesia (BESTCILIA) trial was a multicentre, double-blind, parallel group, randomised, placebo-controlled phase 3 trial done at 6 European PCD clinics (tertiary paediatric care centres and university hospitals in Denmark, Germany, Netherlands, Switzerland, and UK). Patients with a confirmed diagnosis of PCD, aged 7-50 years old, and predicted FEV1 greater than 40% were recruited. Participants were randomly assigned (1:1), stratified by age and study site, via a web-based randomisation system to azithromycin 250 mg or 500 mg as tablets according to bodyweight (

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.

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.

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.