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Newly synthesised glycoproteins enter the rough endoplasmic reticulum through a translocation pore. The translocon associated protein (TRAP) complex is located close to the pore. In a patient with a homozygous start codon variant in TRAPγ (SSR3), absence of TRAPγ causes disruption of the TRAP complex, impairs protein translocation into the endoplasmic reticulum and affects transport, for example, into the brush-border membrane. Furthermore, we observed an unbalanced non-occupancy of N-glycosylation sites. The major clinical features are intrauterine growth retardation, facial dysmorphism, congenital diarrhoea, failure to thrive, pulmonary disease and severe psychomotor disability.

Motile Zilien sind Ausstulpungen der Zelloberflache, die in der Lage sind, aktive Bewegungen auszufuhren und dadurch Zellen (z.B. Spermien) oder Flussigkeiten (z. B. in den Atemwegen) in Bewegung zu setzen. Der Begriff Primare ziliare Dyskinesie (PCD) beschreibt eine Gruppe genetisch und klinisch heterogener hereditarer Erkrankungen, bei denen die Dysfunktion motiler Zilien zu einer gravierenden Storung der Atemwegs-Clearance fuhrt. Dies hat eine chronische Sekretretention in den oberen und unteren Atemwegen zur Folge, auf deren Basis es zu chronisch-rezidivierenden Infektionen sowie Inflammationsprozessen und schlieslich zu einer irreversiblen Gewebsdestruktion kommt.

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.

Primary Ciliary Dyskinesia (PCD, MIM 242650) is a rare, hereditary multiorgan disease characterized by malfunction of motile cilia. Hallmark symptom is a chronic airway infection due to mucostasis leading to irreversible lung damage that may progress to respiratory failure. There is no cure for this genetic disease and evidence-based treatment is limited. Until recently, there were no randomized controlled trials performed in PCD, but this year, data of the first placebo-controlled trial on pharmacotherapy in PCD were published. This cornerstone in the management of PCD was decisive for reviewing currently used treatment strategies. This article is a consensus of patient representatives and clinicians, which are highly experienced in care of PCD-patients and provides an overview of the management of PCD. Treatments are mainly based on expert opinions, personal experiences, or are deduced from other lung diseases, notably cystic fibrosis (CF), COPD or bronchiectasis. Most strategies focus on routine airway clearance and treatment of recurrent respiratory tract infections. Non-respiratory symptoms are treated organ specific. To generate further evidence-based knowledge, other projects are under way, e. g. the International PCD-Registry. Participating in patient registries facilitates access to clinical and research studies and strengthens networks between centers. In addition, knowledge of genotype-specific course of the disease will offer the opportunity to further improve and individualize patient care.Die Primäre Ciliäre Dyskinesie (PCD, MIM 242650) ist eine seltene hereditäre Multisystemerkrankung mit klinisch heterogenem Phänotyp. Leitsymptom ist eine chronische Sekretretention der oberen und unteren Atemwege, welche durch die Dysfunktion motiler respiratorischer Zilien entsteht. In der Folge kommt es zur Ausbildung von Bronchiektasen, häufig zu einer Infektion durch Pseudomonas aeruginosa sowie einer abnehmenden Lungenfunktion bis hin zum Lungenversagen.Bislang gibt es kaum evidenzbasierte Therapieempfehlungen, da randomisierte Langzeitstudien zur Behandlung der PCD fehlten. In diesem Jahr wurden die Daten einer ersten placebokontrollierten Medikamentenstudie bei PCD veröffentlicht. Anlässlich dieses Meilensteins im Management der PCD wurde der vorliegende Übersichtsartikel als Konsens von Patientenvertretern sowie Klinikern, die langjährige Erfahrung in der Behandlung der PCD haben, verfasst. Diese Arbeit bietet eine Zusammenfassung aktuell eingesetzter Behandlungsverfahren, die überwiegend auf persönlichen Erfahrungen und Expertenmeinungen beruhen oder von anderen Atemwegserkrankungen wie der Cystischen Fibrose (CF), COPD oder Bronchiektasen-Erkrankung abgeleitet werden. Da es derzeit keine kurative Therapie für PCD gibt, stehen symptomatische Maßnahmen wie die regelmäßige Reinigung der Atemwege und die Behandlung von rezidivierenden Atemwegsinfektionen im Fokus. Nicht respiratorische Manifestationen werden organspezifisch behandelt. Um neben der ersten Medikamentenstudie mehr evidenzbasiertes Wissen zu generieren, werden weitere Projekte etabliert, u. a. ein internationales PCD-Register. Hierüber wird Patienten der Zugang zu klinischen und wissenschaftlichen Studien erleichtert und die Vernetzung behandelnder Zentren gefördert. Des Weiteren können Erkenntnisse über eine Genotyp-spezifische Erkrankungsschwere erlangt werden, um folglich die therapeutische Versorgung der Patienten zu verbessern und somit zu individualisieren.

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 mechanism by which adenosine monophosphate modulates dynein ATPase-mediated ciliary and flagellar beating remains obscure. Here the authors identify an axonemal module including cilia and flagella associated protein 45 that supports adenine nucleotide homeostasis and underlies a human ciliopathy

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 FEV 1 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 (

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.

Dysfunction of motile monocilia, altering the leftward flow at the embryonic node essential for determination of left-right body asymmetry, is a major cause of laterality defects. Laterality defects are also often associated with reduced mucociliary clearance caused by defective multiple motile cilia of the airway and are responsible for destructive airway disease. Outer dynein arms (ODAs) are essential for ciliary beat generation, and human respiratory cilia contain different ODA heavy chains (HCs): the panaxonemally distributed gamma-HC DNAH5, proximally located beta-HC DNAH11 (defining ODA type 1), and the distally localized beta-HC DNAH9 (defining ODA type 2). Here we report loss-of-function mutations in DNAH9 in five independent families causing situs abnormalities associated with subtle respiratory ciliary dysfunction. Consistent with the observed subtle respiratory phenotype, high-speed video microscopy demonstrates distally impaired ciliary bending in DNAH9 mutant respiratory cilia. DNAH9-deficient cilia also lack other ODA components such as DNAH5, DNAI1, and DNAI2 from the distal axonemal compartment, demonstrating an essential role of DNAH9 for distal axonemal assembly of ODAs type 2. Yeast two-hybrid and co-immunoprecipitation analyses indicate interaction of DNAH9 with the ODA components DNAH5 and DNAI2 as well as the ODA-docking complex component CCDC114. We further show that during ciliogenesis of respiratory cilia, first proximally located DNAH11 and then distally located DNAH9 is assembled in the axoneme. We propose that the beta-HC paralogs DNAH9 and DNAH11 achieved specific functional roles for the distinct axonemal compartments during evolution with human DNAH9 function matching that of ancient beta-HCs such as that of the unicellular Chlamydomonas reinhardtii.

OBJECTIVES The lung clearance index (LCI) is determined by multiple-breath washout lung function (MBW). It is increasingly used as an endpoint in clinical trials. Chest physiotherapy (CP) is part of routine cystic fibrosis (CF) care. Whether the LCI is useful in detecting short-term treatment effects of CP has not been sufficiently investigated. We assessed the short-term influence of CP with highly standardized high-frequency chest wall oscillation (HFCWO) on the LCI in CF patients. METHODS In this randomized controlled study, the LCI was obtained in 20 CF patients (7-34 years) hospitalized for infective pulmonary exacerbation prior to and immediately after a single treatment of HFCWO. Twenty-one control group CF patients (7-51 years) received no treatment. We calculated the coefficient of repeatability (CR) to estimate the clinical relevance of possible treatment effects. RESULTS HFCWO improved (ie, decreased) the LCI by a median of 0.9 (range −0.45; 3.47; P = 0.002); the LCI decreased in 15 of 20 intervention group patients. In five patients the decrease in LCI exceeded the CR (2.15), indicating a clinically relevant treatment effect; in five patients the LCI increased but did not exceed the CR. The LCI did not change significantly in the control group patients. CONCLUSIONS HFCWO can have a short-term decreasing effect on the LCI, but the treatment response is heterogeneous. In future trials using LCI as an endpoint, the timing of CP in relation to MBW should be considered a possible bias.

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.

Introduction: Use of maintenance therapy with the macrolide antibiotic azithromycin (AZI) is increasing in different chronic respiratory disorders incl. primary ciliary dyskinesia (PCD), although no clear evidence exists in PCD. Aims and Objectives: The primary objective was to determine efficacy of AZI maintenance therapy on respiratory exacerbations in PCD. The secondary objectives were to determine efficacy on lung function, health-related quality of life, sputum microbiology, and hearing impairment, and assess safety. Methods: Patients with confirmed diagnosis of PCD, age 7-50 years, were enrolled in this European multi-centre, parallel group, double-blind, randomized, placebo-controlled trial. Participants were randomly assigned to AZI 250/500 mg according to body weight ( Results: A total of 90 participants were randomized - 49 assigned to AZI and 41 to placebo. The AZI group had significant lower rate of exacerbations (rate ratio 0.46, 95%CI: 0.27; 0.80, p=0.006) and pathogenic bacterial species in sputum (rate ratio 0.39, 95%CI: 0.24; 0.64, p=0.0002), compared to the placebo group. There was no significant difference between the treatment groups in lung function, hearing level, or Respiratory symptoms, Sinus symptoms and Ear & hearing symptoms on the quality of life measure. Gastrointestinal complaints were more common in the AZI group. Conclusions: This first multi-national randomized controlled trial on pharmacotherapy in PCD showed that maintenance AZI for 6 months was safe and reduced rate of respiratory exacerbations and rate of pathogenic bacterial species in sputum to less than half.

Primary Ciliary Dyskinesia (PCD) is rare and its features in Israel have not been described.to assess prevalence utilizing state-of-the-art diagnostic techniques, and describe clinical features, diagnostic and management practices in Israel.A national multicenter study from 2012 to 2013 recruited patients diagnosed or suspected of having PCD. Diagnosis was verified using: nasal Nitric Oxide (nNO); High-speed Video Microscope Analysis (HVMA); Transmission Electron Microscopy (TEM) of cilia; Immuno-fluorescence staining (IF) for ciliary proteins, and genetic analysis.Of the 203 patients recruited from 14 pediatric centers, 150 had a PCD diagnosis verified. Median age was 15.05y, with range 0.15-60.5y. PCD prevalence was 1:54,000 for the general population and 1:25,000 in children (5-14 y). For the non-Jewish (mainly Druze and Arab Moslem) compared to Jewish populations, prevalence was 1:16,500 and 1:139,000 respectively (p 0.0001) and parental consanguinity was 85.4% and 21.9% respectively (p 0.0001). Clinical features included bronchiectasis (88%), rhinitis (81%), recurrent pneumonia (78%), recurrent otitis (62%), neonatal pneumonia (60%) and situs inversus (42%). Prior diagnostic practices varied widely between centers with TEM assessed in 55% and abnormal in 61% of these. Management included antibiotics and airway clearance. Diagnostic verification revealed for 150 PCD patients: 81% nNO233 ppb, 62% abnormal HVMA, 51% diagnostic TEM, 58% diagnostic IF and, 57% genetic diagnosis.PCD in Israel is rare, with comprehensive diagnostic tests showing prevalence in children similar to Europe. Prevalence was higher in non-Jews, associated with parental consanguinity. Diagnostic and management practices vary. Referral centers providing comprehensive diagnostic and care capabilities should be established.

Primary ciliary dyskinesia (PCD) is a recessively inherited disease that leads to chronic respiratory disorders owing to impaired mucociliary clearance. Conventional transmission electron microscopy (TEM) is a diagnostic standard to identify ultrastructural defects in respiratory cilia but is not useful in approximately 30% of PCD cases, which have normal ciliary ultrastructure. DNAH11 mutations are a common cause of PCD with normal ciliary ultrastructure and hyperkinetic ciliary beating, but its pathophysiology remains poorly understood. We therefore characterized DNAH11 in human respiratory cilia by immunofluorescence microscopy (IFM) in the context of PCD. We used whole-exome and targeted next-generation sequence analysis as well as Sanger sequencing to identify and confirm eight novel loss-of-function DNAH11 mutations. We designed and validated a monoclonal antibody specific to DNAH11 and performed high-resolution IFM of both control and PCD-affected human respiratory cells, as well as samples from green fluorescent protein (GFP)-left-right dynein mice, to determine the ciliary localization of DNAH11. IFM analysis demonstrated native DNAH11 localization in only the proximal region of wild-type human respiratory cilia and loss of DNAH11 in individuals with PCD with certain loss-of-function DNAH11 mutations. GFP-left-right dynein mice confirmed proximal DNAH11 localization in tracheal cilia. DNAH11 retained proximal localization in respiratory cilia of individuals with PCD with distinct ultrastructural defects, such as the absence of outer dynein arms (ODAs). TEM tomography detected a partial reduction of ODAs in DNAH11-deficient cilia. DNAH11 mutations result in a subtle ODA defect in only the proximal region of respiratory cilia, which is detectable by IFM and TEM tomography.

Multiprotein complexes referred to as outer dynein arms (ODAs) develop the main mechanical force to generate the ciliary and flagellar beat. ODA defects are the most common cause of primary ciliary dyskinesia (PCD), a congenital disorder of ciliary beating, characterized by recurrent infections of the upper and lower airways, as well as by progressive lung failure and randomization of left-right body asymmetry. Using a whole-exome sequencing approach, we identified recessive loss-of-function mutations within TTC25 in three individuals from two unrelated families affected by PCD. Mice generated by CRISPR/Cas9 technology and carrying a deletion of exons 2 and 3 in Ttc25 presented with laterality defects. Consistently, we observed immotile nodal cilia and missing leftward flow via particle image velocimetry. Furthermore, transmission electron microscopy (TEM) analysis in TTC25-deficient mice revealed an absence of ODAs. Consistent with our findings in mice, we were able to show loss of the ciliary ODAs in humans via TEM and immunofluorescence (IF) analyses. Additionally, IF analyses revealed an absence of the ODA docking complex (ODA-DC), along with its known components CCDC114, CCDC151, and ARMC4. Co-immunoprecipitation revealed interaction between the ODA-DC component CCDC114 and TTC25. Thus, here we report TTC25 as a new member of the ODA-DC machinery in humans and mice.

Reduced generation of multiple motile cilia (RGMC) is a novel chronic destructive airway disease within the group of mucociliary clearance disorders with only few cases reported. Mutations in two genes, CCNO and MCIDAS, have been identified as a cause of this disease, both leading to a greatly reduced number of cilia and causing impaired mucociliary clearance. This study was designed to identify the prevalence of CCNO mutations in Israel and further delineate the clinical characteristics of RGMC. We analyzed 170 families with mucociliary clearance disorders originating from Israel for mutations in CCNO and identified two novel mutations (c.165delC, p.Gly56Alafs*38; c.638T>C, p.Leu213Pro) and two known mutations in 15 individuals from 10 families (6% prevalence). Pathogenicity of the missense mutation (c.638T>C, p.Leu213Pro) was demonstrated by functional analyses in Xenopus. Combining these 15 patients with the previously reported CCNO case reports revealed rapid deterioration in lung function, an increased prevalence of hydrocephalus (10%) as well as increased female infertility (22%). Consistent with these findings, we demonstrate that CCNO expression is present in murine ependyma and fallopian tubes. CCNO is mutated more frequently than expected from the rare previous clinical case reports, leads to severe clinical manifestations, and should therefore be considered an important differential diagnosis of mucociliary clearance disorders.

In asthmatic airways secondary ciliary dyskinesia contributes to impaired mucociliary clearance. To investigate underlying mechanisms, we studied the effects of cytokines associated with asthma phenotype on the ciliary beat frequency (CBF) in a cell culture model of ciliated human respiratory epithelial cells. Nasal respiratory epithelial cells of 21 patients were used to prepare multicellular cells (spheroids) in the presence of the T helper (TH) 2 cytokines interleukin (IL)-4, IL-5, IL-9 and IL-13, and the TH1 cytokine interferon gamma (IFN-γ). CBF was determined by high-speed video microscopy. Addition of IL-4 and IL-13 and IL-4 + IL-13 decreased the mean CBF by 17, 21, and 22%, respectively, compared with untreated controls. Addition of IL-5 and IL-9 lead to an increase in mean CBF (20 and 10%, respectively). Lower concentrations of IFN-γ (0.1 and 1 ng/ml) decreased mean CBF and higher concentrations (10 ng/ml) increased CBF by 6%. Addition of IFN-γ to IL-13 reversed the effect of IL-13 on the CBF of spheroids. Cytokines directly influence the ciliary function of respiratory epithelium and contribute to the impaired mucociliary clearance in asthmatic disease. Our study encourages further research to investigate IFN-γ as a treatment option in diseases with impaired mucociliary clearance like asthma.

Pulmonary artery sling (PAS) is a rare disease frequently associated with severe malacia and stenosis of the trachea. We present a two-day-old newborn that underwent urgent surgery for PAS and needed prolonged respiratory support afterward. Temporary airway stenting above the level of the tracheal bifurcation was performed five days after surgery to overcome severe airway obstruction caused by tracheomalacia and laceration of the tracheal mucosa after diagnostic bronchoscopy. Two days after the procedure, the child could be extubated and after two weeks the stent was removed without complications. Temporary tracheal stenting after PAS repair can be an effective therapeutic strategy in newborns with tracheal obstruction and allows timely weaning from ventilator support.

ObjectiveTo determine if survival rates of preterm infants receiving active perinatal care improve over time.DesignThe German Neonatal Network is a cohort study of preterm infants with birth weight Setting43 German level III neonatal intensive care units (NICUs).Patients8222 preterm infants with a gestational age between 22/0 and 28/6 weeks who received active perinatal care.InterventionsParticipating NICUs were grouped according to their specific survival rate from 2011 to 2013 to high (percentile >P75), intermediate (P25–P75) and low (Main outcome measuresDeath by any cause before discharge.ResultsTotal survival increased from 85.8% in 2011–2013 to 87.4% in 2014–2016. This increase was due to reduced mortality of NICUs with low survival rates in 2011–2013. Survival increased in these centres from 53% to 64% in the 22–24 weeks strata and from 73% to 84% in the 25–26 weeks strata.ConclusionsOur data support previous reports that active perinatal care of very immature infants improves outcomes at the border of viability and survival rates at higher gestational ages. The high total number of surviving infants below 24 weeks of gestation challenges national recommendations exclusively referring to gestational age as the single criterion for providing active care. However, more data are needed before recommendations for parental counselling should be reconsidered.Trial registrationApproval by the local institutional review board for research in human subjects of the University of Lübeck (file number 08–022) and by the local ethic committees of all participating centres has been given.

Rational Lung resection in Primary Ciliary Dyskinesia (PCD) is a controversial therapeutic modality. We aimed to assess the prevalence of lung resection in PCD across countries and compare disease course in lobectomised and non-lobectomised patients. Methods: In a study nested in the international iPCD cohort (n=2855), we identified lobectomised (cases) and non-lobectomised age, center and sex-matched (controls) PCD patients and compared their clinical characteristics, lung function and BMI cross-sectionally and longitudinally. Results: Data from 163 lobectomised PCD patients from 20 centers (15 countries) were available. The overall prevalence of lung resection in iPCD cohort was 5.7% (8.9% among adults), with wide variation among centers (range: 0% to 20%). Compared to the rest of iPCD cohort, lobectomised patients were more often females, older at diagnosis, and had more often situs solitus. Compared to controls (n=265), lobectomised patients had lower FVC (-2.15 vs -1.34, p Conclusion: Prevalence of lung resection in PCD varies widely between countries and is more frequent in patients with a delayed diagnosis. Most lobectomised PCD patients have poorer and steeper decline of lung function after intervention compared to controls.

Patients with interstitial lung disease due to surfactant protein C (SFTPC) mutations are rare and not well characterised.We report on all subjects collected over a 15-year period in the kids-lung register with interstitial lung disease and a provenSFTPCmutation. We analysed clinical courses, interventions and outcomes, as well as histopathological and radiological interrelations.17 patients (seven male) were followed over a median of 3 years (range 0.3–19). All patients were heterozygous carriers of autosomal dominantSFTPCmutations. Three mutations (p.L101P, p.E191 K and p.E191*) have not been described before in the context of surfactant protein C deficiency. Patients with alterations in the BRICHOS domain of the protein (amino acids 94–197) presented earlier. At follow-up, one patient was healthy (2 years), six patients were “sick-better” (2.8 years, range 0.8–19), seven patients were “sick-same” (6.5 years, 1.3–15.8) and three patients were “sick-worse” (0.3 years, 0.3–16.9). Radiological findings changed from ground-glass to increasing signs of fibrosis and cyst formation with increasing age. Empiric treatments had variable effects, also in patients with the same genotype.Prospective studies with randomised interventions are urgently needed and can best be performed in the framework of international registers.

The diagnostic work-up for primary ciliary dyskinesia (PCD) traditionally includes ciliary ultrastructural assessment using transmission electron microscopy (TEM). However, the identification of genetic variants of PCD that are missed by TEM, along with the development of novel diagnostic modalities for PCD that allow structural evaluation of cilia, such as immunofluorescence analysis and the increased availability of genetic testing, calls into questioning the contemporary role of TEM in the diagnostic work-up for PCD. In this manuscript, we describe the evidence for and against the use of TEM in PCD diagnosis, in light of recent developments of PCD.

Background: Lower airway infections (LAI) occur frequently in children with a tracheostostoma. Whether airway pathogens are risk factors for LAI is not sufficiently studied. Methods: We retrospectively analysed patient records of tracheostomised children and young adults, who were treated between 2004 to 2014 in the children´s hospital of the University of Muenster, Germany. The correlation between lower airway pathogens and the number of LAI per year (Y) was assessed by Spearman correlation coefficient (r). Results: 78 patients were enrolled into the study and had a tracheostoma (median of 58.5 months (interquartile range (IQR): 26; 73.5). The median age was 7.5 years (IQR: 3.75; 7.5), 43 (55%) patients were male. We cultured 329 lower airway secretions and found Staphylococcus aureus (n=135), Pseudomonas aeruginosa (n=90), Klebsiella pneumoniae (n=46), Hamophilus influenzae (n=37), Escherichia coli (n=37), Stenotrophomonas maltophilia (n=29), Acinetobacter baumannii (n=21), Moraxella catarrhalis (n=20) und Candida albicans (n=37). The presence of Acinetobacter baumanii (r= 0.355; p=0.001) and Stenotrophomonas maltophilia (r=0.274; p=0.015) was positively correlated to LAI/Y; Pseudomonas aeruginosa (r=0.109; p=0.342), Staphylococcus aureus (r=0.086; p=0.448) or Klebsiella pneumonia (r=0.050; p=0.664) was not correlated to LAI/Y. The duration of long term ventilation was negatively correlated to the number of LAI/Y (r=-0.549; p Discussion: Acinetobacter baumanii or Stenotrophomonas maltophilia in airway secretions may be risk factors and long term ventilation may be protective for LAI in children and young adults with a tracheostoma.

Introduction: Nasal NO is recommended to be used in the diagnostic work-up of PCD. Objective: To determine whether nNO measurements using portable electrochemical device NIOX VERO ® could distinguish subjects with PCD from healthy subjects. The primary endpoint was mean nNO measurements in subjects with at least 2 measurements. Method: A multi-center, single visit, non-randomised study involved subjects ≥5 years with known PCD vs. age matched healthy controls (HC). Subjects attempted nNO using both tidal breathing method (TB-nNO) and expiration against resisitance method (ER-nNO). Descriptive analyses were performed, including a Receiver Operating Characteristic curve analysis to assess optimal cut-off values. Results: In this study, 98.1% (49 PCD [mean age 17.5 (SD 11.66)/105 HC 19.4 (SD 14.22)] completed at least one measurement in both methods; 96.8% completed at least two measurements from one nostril using either method. Mean TB-nNO values were 50.7 (SD 50.28) ppb and 575.3 (SD 236.88) ppb in PCD vs HC and mean ER-nNO values were 80.5 (SD 101.73) ppb and 915.9 (SD 391.68) ppb in PCD vs HC. In subjects with two measurements from both nostrils using either method, opitimal cut-off values were 171 ppb (51.3 nL/min) for TB-nNO (AUC 99.8%; specificity 100%; sensitivity 98.0%) and 356 ppb (106.8 nL/min) for ER-nNO (AUC 98.7%; specificity 96.3%; sensitivity 97.8%). Conclusions: The new NIOX VERO ® nasal application can be used to differentiate patients with PCD from healthy individuals. The obtained cut-off TB-nNO and ER-nNO values show that both methods in NIOX VERO ® can be used in children and adult patients as part of the diagnostic work-up of PCD.

Cilia are organelles specialized for movement and signaling. To infer when during animal evolution signaling pathways became associated with cilia, we characterized the proteomes of cilia from three organisms: sea urchins, sea anemones and choanoflagellates. From these ciliomes, we identified 437 high confidence ciliary candidate proteins conserved in mammals, including known regulators of Hh, GPCR and TRP channel signaling. The phylogenetic profiles of their ciliary association indicate that the Hh and GPCR pathways were linked to cilia before the origin of bilateria and TRP channels before the origin of animals. We demonstrated that some of the candidates not previously implicated in ciliary biology localized to cilia and further investigated ENKUR, a TRP channel-interacting protein that we identified in the cilia of all three organisms. In animals, ENKUR is expressed by cells with motile cilia, ENKUR localizes to cilia in diverse organisms and, in bothXenopus laevisand mice, ENKUR is required for patterning the left/right axis. Moreover, mutation ofENKURcauses situs inversus in humans. Thus, proteomic profiling of cilia from diverse eukaryotes defines a conserved ciliary proteome, reveals ancient connections to Hh, GPCR and TRP channel signaling, and uncovers a novel ciliary protein that controls vertebrate development and human disease.

International audience; The diagnosis of primary ciliary dyskinesia is often confirmed with standard, albeit complex and expensive, tests. In many cases, however, the diagnosis remains difficult despite the array of sophisticated diagnostic tests. There is no “gold standard” reference test. Hence, a Task Force supported by the European Respiratory Society has developed this guideline to provide evidence-based recommendations on diagnostic testing, especially in light of new developments in such tests, and the need for robust diagnoses of patients who might enter randomised controlled trials of treatments. The guideline is based on pre-defined questions relevant for clinical care, a systematic review of the literature, and assessment of the evidence using the GRADE (Grading of Recommendations, Assessment, Development and Evaluation) approach. It focuses on clinical presentation, nasal nitric oxide, analysis of ciliary beat frequency and pattern by high-speed video-microscopy analysis, transmission electron microscopy, genotyping and immunofluorescence. It then used a modified Delphi survey to develop an algorithm for the use of diagnostic tests to definitively confirm and exclude the diagnosis of primary ciliary dyskinesia; and to provide advice when the diagnosis was not conclusive. Finally, this guideline proposes a set of quality criteria for future research on the validity of diagnostic methods for primary ciliary dyskinesia.

Primary ciliary dyskinesia (PCD) is a rare genetic disorder leading to recurrent respiratory tract infections. High-speed video-microscopy analysis (HVMA) of ciliary beating, currently the first-line diagnostic tool for PCD in most centres, is challenging because recent studies have expanded the spectrum of HVMA findings in PCD from grossly abnormal to very subtle. The objective of this study was to describe the diversity of HVMA findings in genetically confirmed PCD individuals.HVMA was performed as part of the routine work-up of individuals with suspected PCD. Subsequent molecular analysis identified biallelic mutations in the PCD-related genes of 66 individuals. 1072 videos of these subjects were assessed for correlation with the genotype.Biallelic mutations (19 novel) were found in 17 genes: DNAI1, DNAI2, DNAH5, DNAH11, CCDC103, ARMC4, KTU/DNAAF2, LRRC50/DNAAF1, LRRC6, DYX1C1, ZMYND10, CCDC39, CCDC40, CCDC164, HYDIN, RSPH4A and RSPH1. Ciliary beat pattern variations correlated well with the genetic findings, allowing the classification of typical HVMA findings for different genetic groups. In contrast, analysis of ciliary beat frequency did not result in additional diagnostic impact.In conclusion, this study provides detailed knowledge about the diversity of HVMA findings in PCD and may therefore be seen as a guide to the improvement of PCD diagnostics.

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous, autosomal-recessive disorder, characterized by oto-sino-pulmonary disease and situs abnormalities. PCD-causing mutations have been identified in 20 genes, but collectively they account for only ∼65% of all PCDs. To identify mutations in additional genes that cause PCD, we performed exome sequencing on three unrelated probands with ciliary outer and inner dynein arm (ODA+IDA) defects. Mutations in SPAG1 were identified in one family with three affected siblings. Further screening of SPAG1 in 98 unrelated affected individuals (62 with ODA+IDA defects, 35 with ODA defects, 1 without available ciliary ultrastructure) revealed biallelic loss-of-function mutations in 11 additional individuals (including one sib-pair). All 14 affected individuals with SPAG1 mutations had a characteristic PCD phenotype, including 8 with situs abnormalities. Additionally, all individuals with mutations who had defined ciliary ultrastructure had ODA+IDA defects. SPAG1 was present in human airway epithelial cell lysates but was not present in isolated axonemes, and immunofluorescence staining showed an absence of ODA and IDA proteins in cilia from an affected individual, thus indicating that SPAG1 probably plays a role in the cytoplasmic assembly and/or trafficking of the axonemal dynein arms. Zebrafish morpholino studies of spag1 produced cilia-related phenotypes previously reported for PCD-causing mutations in genes encoding cytoplasmic proteins. Together, these results demonstrate that mutations in SPAG1 cause PCD with ciliary ODA+IDA defects and that exome sequencing is useful to identify genetic causes of heterogeneous recessive disorders.

Charakteristika Die primare ziliare Dyskinesie (PCD) ist eine seltene angeborene Erkrankung der Zilien, die sich zumeist im respiratorischen System manifestiert.

Respiratory viruses are an important yet underestimated cause of infectious morbidity and mortality in immunocompromised children and adolescents. Here, we report the occurrence of fatal lower respiratory tract disease associated with human metapneumovirus (HMPV) infection in a 10-year-old girl with chronic graft-versus-host disease following allogeneic hematopoietic stem cell transplantation (HSCT) for secondary chronic myeloid leukemia. Symptoms occurred 8 months after HSCT while on immunosuppression with 0.2 mg/kg/day of prednisone, and presented as dry cough, bilateral pneumonitis, and progressive respiratory distress. Non-invasive and invasive microbiological investigations revealed HMPV type B as the sole pathogen. Histopathological findings showed interstitial and intra-alveolar pneumonitis with profound alveolar cell damage. The patient was treated with intravenous and oral ribavirin and polyvalent immunoglobulins, but ultimately died from respiratory failure. The case reflects the potentially fatal impact of infections by respiratory viruses in immunocompromised patients and the need for effective approaches to their prevention and treatment.

Summary 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.

Introduction Primary Ciliary Dyskiensia (PCD) describes a clinically and genetically heterogeneous group of hereditary disorders characterized by chronic mucopurulent infections of the airways caused by dysfunction of multiple motile cilia. Mutations in > 30 genes have been described to cause PCD. We present a new genetic PCD-variant caused by defects of the nexin-dynein regulatory complex (N-DRC). Objectives PCD is widely underdiagnosed because of the great heterogeneity of phenotypes. We wanted to reveal rare PCD-variants with only subtle changes in ciliary beating or ultrastructure by further methods such as immunofluorescence microscopy (IF) and genetic analysis. Methods Patient material was supplied for routine diagnostic work-up including high-speed videomicroscopy (HVMA), transmission electron microscopy (TEM), IF and genetics. Within IF we used an antibody directed against the protein GAS8 (human DRC4). In case of abnormal results we performed whole-exome sequencing and confirmed findings with Sanger sequencing. Results IF showed the absence of GAS8 from the ciliary axoneme in three patients. We excluded mutations in the genes CCDC65 and CCDC164 , which lead to isolated nexin link-defects. Subsequent genetic analysis showed recessive loss-of-function mutations in GAS8 encoding for human protein DRC4 in the three families . HVMA and TEM did not show obvious abnormalities. Conclusions Defects of the N-DRC were difficult to diagnose because routinely performed HVMA and TEM showed almost normal findings. We could show that IF and genetic analysis in those patients can be very useful to detect rare PCD-variants e.g. with defects of the N-DRCs.

Objectives: In Cystic fibrosis (CF) and Primary ciliary dyskinesia (PCD) medical history of cough is important to identify disease progress, infective exacerbations and response to therapy. Thus treatment decisions are based on highly subjective patient´s reports what emphasises the need for objective cough monitoring. Methods: We investigated 28 healthy subjects (age 22.5 years (12; 43); median (min; max)), 21 patients with CF (26 (10; 44)) and 17 PCD patients (21 (9; 43)) at the University children´s hospital Muenster, Germany. In all patients cough-sounds were recorded and analysed with LEOSound-Monitor® (Heinen und Lowenstein, Bad Ems, Germany) during sleep. Kruskal-Wallis, Mann Whitney U test and Spearman´s correlation were applied. Results: The median (min; max) recorded time for healthy subjects, CF and PCD was 6.58 hours (5.42; 8.28), 7:58 (6:02; 9:46) and 7:51 (5:50; 9:34), respectively; cough counts per hour (cough/h median (min; max)) was 0 (0.0; 1.14), 2.15 (0.0; 62.6) and 0.37 (0.00; 7.75), respectively. Cough/h was statistically different between healthy subjects and CF (p Conclusions: Cough detection with LEOSound-Monitor® is feasible in CF, PCD and healthy subjects. CF and PCD patients cough significantly more than healthy subjects. Cough in CF and PCD correlates with parameters of disease severity, which indicates the usefulness of cough monitoring in CF and PCD.

Introduction: Quality of life is an important parameter in the evaluation of health care and treatments. We have developed the first primary ciliary dyskinesia (PCD) health-related quality of life (HRQoL) measure for children, parents, teenagers and adults (QOL-PCD) in English. Clinical trials on rare diseases like PCD must be conducted in international settings to enrol enough patients, and outcome measures only assess the same thing if translation accounts for the socio-cultural context in which it is understood. This study aims to conduct socio-cultural translations of QOL-PCD into German, Swiss German, Danish, Dutch, French, and Greek. Methodology: We conducted (a) forward translations by 2 independent translators (b) back translation to English of the consensus versions by 1 translator, and (c) cognitive interviews of each age/language version by >5 patients to ensure the “meaning” attributed to each item is similar in different languages and cross-culturally understood. This involved 20 translators, 24 chaired meetings and interviews with 120 patients. Results: Each stage of translation involved comprehensive discussions (>2 hours), resulting in adjustments. 172 items (39-49 items across 4 age groups) were translated averaging 65 working hours per language. Cognitive interviews demonstrated face validity and no missing aspect of HRQoL were identified. International testing additionally provided valuable input on how to proceed for future improvement of QOL-PCD. Conclusion: To meet standards set by international guidelines, this extensive process has produced practical, cross-cultural and equivalent measures for future use as outcome measures in clinical trials.

Background Chronic rhinosinusitis is a hallmark of Cystic fibrosis (CF) impairing the patients' quality of life and overall health. However, therapeutic options have not been sufficiently evaluated. Bronchial inhalation of mucolytic substances is a gold standard in CF therapy. Previously, we found that sinonasal inhalation of dornase alfa as vibrating aerosol reduces symptoms of chronic rhinosinusitis more effectively than NaCl 0.9% (net treatment benefit: −5.87±2.3 points, p=0.017; SNOT-20 total score). This multicenter study compares the effect of NaCl 6.0% vs. NaCl 0.9% following the protocol from our preceding study with dornase alfa. Methods Sixty nine CF patients with chronic rhinosinusitis in eleven German CF centers were randomized to receive sinonasal vibrating inhalation of either NaCl 6.0% or NaCl 0.9% for 28days. After 28days of wash-out, patients crossed over to the alternative treatment. The primary outcome parameter was symptom score in the disease-specific quality of life Sino-Nasal Outcome Test-20 (SNOT-20). Additionally, pulmonary function was assessed, as well as rhinomanometry and inflammatory markers in nasal lavage (neutrophil elastase, interleukin (IL)-1β, IL-6, and IL-8) in a subgroup. Results Both therapeutic arms were well tolerated and showed slight improvements in SNOT-20 total scores (NaCl 6.0%: −3.1±6.5 points, NaCl 0.9%: −5.1±8.3 points, ns). In both treatment groups, changes of inflammatory parameters in nasal lavage from day 1 to day 29 were not significant. We suppose that the irritating properties of NaCl 6.0% reduced the suitability of the SNOT-20 scores as an outcome parameter. Alternative primary outcome parameters such as MR-imaging or the quantity of sinonasal secretions mobilized with both saline concentrations were, however, not feasible. Conclusion Sinonasal inhalation with NaCl 6.0% did not lead to superior results vs. NaCl 0.9%, whereas dornase alfa had been significantly more effective than NaCl 0.9%.

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous recessive disorder characterized by defective cilia and flagella motility. Chronic destructive-airway disease is caused by abnormal respiratory-tract mucociliary clearance. Abnormal propulsion of sperm flagella contributes to male infertility. Genetic defects in most individuals affected by PCD cause randomization of left-right body asymmetry; approximately half show situs inversus or situs ambiguous. Almost 70 years after the hy3 mouse possessing Hydin mutations was described as a recessive hydrocephalus model, we report HYDIN mutations in PCD-affected persons without hydrocephalus. By homozygosity mapping, we identified a PCD-associated locus, chromosomal region 16q21-q23, which contains HYDIN. However, a nearly identical 360 kb paralogous segment (HYDIN2) in chromosomal region 1q21.1 complicated mutational analysis. In three affected German siblings linked to HYDIN, we identified homozygous c.3985G>T mutations that affect an evolutionary conserved splice acceptor site and that subsequently cause aberrantly spliced transcripts predicting premature protein termination in respiratory cells. Parallel whole-exome sequencing identified a homozygous nonsense HYDIN mutation, c.922A>T (p.Lys307∗), in six individuals from three Faroe Island PCD-affected families that all carried an 8.8 Mb shared haplotype across HYDIN, indicating an ancestral founder mutation in this isolated population. We demonstrate by electron microscopy tomography that, consistent with the effects of loss-of-function mutations, HYDIN mutant respiratory cilia lack the C2b projection of the central pair (CP) apparatus; similar findings were reported in Hydin-deficient Chlamydomonas and mice. High-speed videomicroscopy demonstrated markedly reduced beating amplitudes of respiratory cilia and stiff sperm flagella. Like the hy3 mouse model, all nine PCD-affected persons had normal body composition because nodal cilia function is apparently not dependent on the function of the CP apparatus.

Die primare ziliare Dyskinesie (PCD) ist eine klinisch und genetisch heterogene hereditare Erkrankung. Der Phanotyp der Erkrankung erklart sich durch angeborene Defekte respiratorischer Flimmerharchen (Zilien). Aufgrund einer verminderten mukoziliaren Reinigung der Atemwege kommt es zu rezidivierenden Infektionen der oberen und unteren Atemwege. Die Halfte der PCD-Patienten weist aufgrund einer zufalligen Anordnung der Links/Rechts-Korperasymmetrie einen Situs inversus (Kartagener Syndrom) auf. Bei klinischem Verdacht kann die Diagnose durch Elektronenmikroskopie, hochauflosende Immunfluoreszenzmikroskopie und/oder direktmikroskopische Evaluation des Zilienschlages bestatigt werden. Kurzlich konnten Mutationen bei rezessiv vererbter PCD mit auseren Dyneinarmdefekten in den Genen DNAI1, DNAH5, DNAH11, TXNDC3 nachgewiesen werden. Hierbei finden sich DNAH5-Mutationen in mehr als 50% der Falle. Selten finden sich Mutationen bei Jungen mit X-chromosomal rezessiver PCD, assoziiert mit Retinitis pigmentosa (RPGR) oder einem komplexen mentalen Retardierungssyndrom mit Makrozephalie (OFD1).