Axonemal structures reveal mechanoregulatory and disease mechanisms.

Motile cilia and flagella beat rhythmically on the surface of cells to power the flow of fluid and to enable spermatozoa and unicellular eukaryotes to swim. In humans, defective ciliary motility can lead to male infertility and a congenital disorder called primary ciliary dyskinesia (PCD), in which impaired clearance of mucus by the cilia causes chronic respiratory infections1. Ciliary movement is generated by the axoneme, a molecular machine consisting of microtubules, ATP-powered dynein motors and regulatory complexes2. The size and complexity of the axoneme has so far prevented the development of an atomic model, hindering efforts to understand how it functions. Here we capitalize on recent developments in artificial intelligence-enabled structure prediction and cryo-electron microscopy (cryo-EM) to determine the structure of the 96-nm modular repeats of axonemes from the flagella of the alga Chlamydomonas reinhardtii and human respiratory cilia. Our atomic models provide insights into the conservation and specialization of axonemes, the interconnectivity between dyneins and their regulators, and the mechanisms that maintain axonemal periodicity. Correlated conformational changes in mechanoregulatory complexes with their associated axonemal dynein motors provide a mechanism for the long-hypothesized mechanotransduction pathway to regulate ciliary motility. Structures of respiratory-cilia doublet microtubules from four individuals with PCD reveal how the loss of individual docking factors can selectively eradicate periodically repeating structures.

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.

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.

Nasal nitric oxide measurement in children for the diagnosis of primary ciliary dyskinesia: European Respiratory Society technical standard.

Nasal nitric oxide (nNO) is extremely low in most people with primary ciliary dyskinesia (PCD) and its measurement is an important contributor to making the diagnosis. Existing guidelines and technical standards focus on nNO measurements in older, cooperative children using chemiluminescence analysers. However, measurements of nNO in pre-school-age children (age 2-5 years) may facilitate early diagnosis and electrochemical rather than chemiluminescence analysers are widely used. Pre-schoolers often need different methods to be employed when measuring nNO. Hence, a European Respiratory Society Task Force has developed this technical standard as the first step towards standardising sampling, analysis and reporting of nNO measured as part of the diagnostic testing for PCD in all age groups, including pre-school-age children. Furthermore, we considered both chemiluminescence and electrochemical analysers that are in use worldwide. There was a paucity of quality evidence for electrochemical analysers and sampling methods used in young children, and the Task Force proposes future research priorities to allow updates of this technical standard.

Primary ciliary dyskinesia in Volendam: Diagnostic and phenotypic features in patients with a CCDC114 mutation.

Primary ciliary dyskinesia (PCD) is a heterogeneous disease, with impaired mucociliary clearance causing respiratory tract infections. A founding CCDC114 mutation has led to a relatively homogeneous and large Dutch PCD population in Volendam. Our aim was to describe their phenotype. Therefore, all Volendam PCD patients seen at the Amsterdam UMC were included in this study. Data were collected on lung function, microbiology, radiology, and ear-nose-throat (ENT) symptoms. A mixed effects model estimated lung function decline in %point per year (95% confidence interval [CI]). Thirty-three (60%) out of approximately 56 Volendam PCD patients were treated at our center and included in this study. Only 30% of patients had situs inversus. FEV1 declined in children (-1.43%/year, CI: -1.80/-1.05), but not in adults (0.01%/year, CI: -0.36/0.38). Pseudomonas aeruginosa was cultured in 21% of children and 60% of adults, respectively. Patients who have been infected at some point with P. aeruginosa had a steeper decline in FEV1 as compared to patients that have never been infected. Neonatal symptoms (79%) and ENT problems (94%) were common; fertility issues however, were not (11%) common. Compared to other PCD cohorts, the Volendam/CCDC114 patients have a moderately severe phenotype with lung function decline predominantly occurring in childhood.

Lung function from school age to adulthood in primary ciliary dyskinesia.

Primary ciliary dyskinesia (PCD) presents with symptoms early in life and the disease course may be progressive, but longitudinal data on lung function are scarce. This multinational cohort study describes lung function trajectories in children, adolescents and young adults with PCD. We analysed data from 486 patients with repeated lung function measurements obtained between the age of 6 and 24 years from the International PCD Cohort and calculated z-scores for forced expiratory volume in 1 s (FEV1), forced vital capacity (FVC) and FEV1/FVC ratio using the Global Lung Function Initiative 2012 references. We described baseline lung function and change of lung function over time and described their associations with possible determinants in mixed-effects linear regression models. Overall, FEV1, FVC and FEV1/FVC z-scores declined over time (average crude annual FEV1 decline was -0.07 z-scores), but not at the same rate for all patients. FEV1 z-scores improved over time in 21% of patients, remained stable in 40% and declined in 39%. Low body mass index was associated with poor baseline lung function and with further decline. Results differed by country and ultrastructural defect, but we found no evidence of differences by sex, calendar year of diagnosis, age at diagnosis, diagnostic certainty or laterality defect. Our study shows that on average lung function in PCD declines throughout the entire period of lung growth, from childhood to young adult age, even among patients treated in specialised centres. It is essential to develop strategies to reverse this tendency and improve prognosis.

Topological data analysis reveals genotype-phenotype relationships in primary ciliary dyskinesia.

BACKGROUND: Primary ciliary dyskinesia (PCD) is a heterogeneous inherited disorder caused by mutations in approximately 50 cilia-related genes. PCD genotype-phenotype relationships have mostly arisen from small case series because existing statistical approaches to investigating relationships have been unsuitable for rare diseases. METHODS: We applied a topological data analysis (TDA) approach to investigate genotype-phenotype relationships in PCD. Data from separate training and validation cohorts included 396 genetically defined individuals carrying pathogenic variants in PCD genes. To develop the TDA models, 12 clinical and diagnostic variables were included. TDA-driven hypotheses were subsequently tested using traditional statistics. RESULTS: Disease severity at diagnosis, measured by forced expiratory volume in 1 s (FEV1) z-score, was significantly worse in individuals with CCDC39 mutations (compared to other gene mutations) and better in those with DNAH11 mutations; the latter also reported less neonatal respiratory distress. Patients without neonatal respiratory distress had better preserved FEV1 at diagnosis. Individuals with DNAH5 mutations were phenotypically diverse. Cilia ultrastructure and beat pattern defects correlated closely to specific causative gene groups, confirming these tests can be used to support a genetic diagnosis. CONCLUSIONS: This large scale, multi-national study presents PCD as a syndrome with overlapping symptoms and variations in phenotype according to genotype. TDA modelling confirmed genotype-phenotype relationships reported by smaller studies (e.g. FEV1 worse with CCDC39 mutation) and identified new relationships, including FEV1 preservation with DNAH11 mutations and diversity of severity with DNAH5 mutations.

Current and Future Treatments in Primary Ciliary Dyskinesia.

Primary ciliary dyskinesia (PCD) is a rare genetic ciliopathy in which mucociliary clearance is disturbed by the abnormal motion of cilia or there is a severe reduction in the generation of multiple motile cilia. Lung damage ensues due to recurrent airway infections, sometimes even resulting in respiratory failure. So far, no causative treatment is available and treatment efforts are primarily aimed at improving mucociliary clearance and early treatment of bacterial airway infections. Treatment guidelines are largely based on cystic fibrosis (CF) guidelines, as few studies have been performed on PCD. In this review, we give a detailed overview of the clinical studies performed investigating PCD to date, including three trials and several case reports. In addition, we explore precision medicine approaches in PCD, including gene therapy, mRNA transcript and read-through therapy.

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.

Prevalence and course of disease after lung resection in primary ciliary dyskinesia: a cohort & nested case-control study.

BACKGROUND: Lung resection is a controversial and understudied therapeutic modality in Primary Ciliary Dyskinesia (PCD). We assessed the prevalence of lung resection in PCD across countries and compared disease course in lobectomised and non-lobectomised patients. METHODS: In the international iPCD cohort, we identified lobectomised and non-lobectomised age and sex-matched PCD patients and compared their characteristics, lung function and BMI cross-sectionally and longitudinally. RESULTS: Among 2896 patients in the iPCD cohort, 163 from 20 centers (15 countries) underwent lung resection (5.6%). Among adult patients, prevalence of lung resection was 8.9%, demonstrating wide variation among countries. Compared to the rest of the iPCD cohort, lobectomised patients were more often females, older at diagnosis, and more often had situs solitus. In about half of the cases (45.6%) lung resection was performed before presentation to specialized PCD centers for diagnostic work-up. Compared to controls (n = 197), lobectomised patients had lower FVC z-scores (- 2.41 vs - 1.35, p = 0.0001) and FEV1 z-scores (- 2.79 vs - 1.99, p = 0.003) at their first post-lung resection assessment. After surgery, lung function continued to decline at a faster rate in lobectomised patients compared to controls (FVC z-score slope: - 0.037/year Vs - 0.009/year, p = 0.047 and FEV1 z-score slope: - 0.052/year Vs - 0.033/year, p = 0.235), although difference did not reach statistical significance for FEV1. Within cases, females and patients with multiple lobe resections had lower lung function. CONCLUSIONS: Prevalence of lung resection in PCD varies widely between countries, is often performed before PCD diagnosis and overall is more frequent in patients with delayed diagnosis. After lung resection, compared to controls most lobectomised patients have poorer and continuing decline of lung function despite lung resection. Further studies benefiting from prospective data collection are needed to confirm these findings.

Diagnostic yield of a targeted gene panel in primary ciliary dyskinesia patients.

We aimed to determine the diagnostic yield of a targeted-exome panel in a cohort of 74 Dutch primary ciliary dyskinesia (PCD) patients. The panel consisted of 26 PCD-related and 284 candidate genes. To prioritize PCD candidate genes, we investigated the transcriptome of human airway cells of 12 healthy volunteers during in vitro ciliogenesis and hypothesized that PCD-related genes show significant upregulation. We compared gene expression in epithelial precursor cells grown as collagen monolayer and ciliated cells grown in suspension by RNA sequencing. All genes reported as PCD causative, except NME8, showed significant upregulation during in vitro ciliogenesis. We observed 67.6% diagnostic yield when testing the targeted-exome panel in our cohort. There was relatively high percentage of DNAI and HYDIN mutations compared to other countries. The latter may be due to our solution for the problem of the confounding HYDIN2 pseudogene. Candidate genes included two recently published PCD-related genes DNAJB13 and PIH1D3; identification of the latter was a direct result of this study. In conclusion, we demonstrate 67.6% diagnostic yield by targeted exome sequencing in a Dutch PCD population and present a highly sensitive and moderately specific approach for identification of PCD-related genes, based on significant upregulation during in vitro ciliogenesis.

Clinical and inflammatory phenotyping by breathomics in chronic airway diseases irrespective of the diagnostic label.

Asthma and chronic obstructive pulmonary disease (COPD) are complex and overlapping diseases that include inflammatory phenotypes. Novel anti-eosinophilic/anti-neutrophilic strategies demand rapid inflammatory phenotyping, which might be accessible from exhaled breath.Our objective was to capture clinical/inflammatory phenotypes in patients with chronic airway disease using an electronic nose (eNose) in a training and validation set.This was a multicentre cross-sectional study in which exhaled breath from asthma and COPD patients (n=435; training n=321 and validation n=114) was analysed using eNose technology. Data analysis involved signal processing and statistics based on principal component analysis followed by unsupervised cluster analysis and supervised linear regression.Clustering based on eNose resulted in five significant combined asthma and COPD clusters that differed regarding ethnicity (p=0.01), systemic eosinophilia (p=0.02) and neutrophilia (p=0.03), body mass index (p=0.04), exhaled nitric oxide fraction (p<0.01), atopy (p<0.01) and exacerbation rate (p<0.01). Significant regression models were found for the prediction of eosinophilic (R2=0.581) and neutrophilic (R2=0.409) blood counts based on eNose. Similar clusters and regression results were obtained in the validation set.Phenotyping a combined sample of asthma and COPD patients using eNose provides validated clusters that are not determined by diagnosis, but rather by clinical/inflammatory characteristics. eNose identified systemic neutrophilia and/or eosinophilia in a dose-dependent manner.

Lung function in patients with primary ciliary dyskinesia: an iPCD Cohort study.

Primary ciliary dyskinesia (PCD) has been considered a relatively mild disease, especially compared to cystic fibrosis (CF), but studies on lung function in PCD patients have been few and small.This study compared lung function from spirometry of PCD patients to normal reference values and to published data from CF patients. We calculated z-scores and % predicted values for forced expiratory volume in 1 s (FEV1) and forced vital capacity (FVC) using the Global Lung Function Initiative 2012 values for 991 patients from the international PCD Cohort. We then assessed associations with age, sex, country, diagnostic certainty, organ laterality, body mass index and age at diagnosis in linear regression models. Lung function in PCD patients was reduced compared to reference values in both sexes and all age groups. Children aged 6-9 years had the smallest impairment (FEV1 z-score -0.84 (-1.03 to -0.65), FVC z-score -0.31 (-0.51 to -0.11)). Compared to CF patients, FEV1 was similarly reduced in children (age 6-9 years PCD 91% (88-93%); CF 90% (88-91%)), but less impaired in young adults (age 18-21 years PCD 79% (76-82%); CF 66% (65-68%)). The results suggest that PCD affects lung function from early in life, which emphasises the importance of early standardised care for all patients.

A randomised controlled trial on the effect of inhaled hypertonic saline on quality of life in primary ciliary dyskinesia.

Hypertonic saline inhalation lowers airway mucous viscosity. Increased cough transportability may improve quality of life (QoL) in primary ciliary dyskinesia (PCD).In this randomised controlled trial (RCT), PCD patients received twice-daily inhalations of hypertonic (7%) saline or isotonic (0.9%) saline for 12 weeks, with 4 weeks washout during crossover. Primary outcome was change in QoL measured by the St George's Respiratory Questionnaire (SGRQ) total score. Secondary outcomes were SGRQ subscores, Quality of Life Questionnaire-Bronchiectasis (QoL-B) scores, lower respiratory tract infection symptoms, exacerbations, spirometry, systemic and sputum inflammatory markers, adherence, and adverse events.There was no significant change in median (interquartile range) SGRQ total score between hypertonic saline (-2.6 (-9.0-1.5)) and isotonic saline (-0.3 (-8.1-6.1)) in 22 patients (age range 22-73 years) (p=0.38). QoL-B Health Perception scale improved with hypertonic saline (p=0.03). Adverse events occurred more frequently with hypertonic saline, but were mild.12 weeks of inhaled hypertonic saline did not improve SGRQ total score in adult PCD patients in this RCT, but the sample size was small. On the secondary and more disease-specific end-point of the QoL-B, a significant improvement was observed in the Health Perception scale. This study found little evidence to support the hypothesis that hypertonic saline improves QoL in PCD patients. We advise the use of disease-specific outcome measures in future trials.

Growth and nutritional status, and their association with lung function: a study from the international Primary Ciliary Dyskinesia Cohort.

Chronic respiratory disease can affect growth and nutrition, which can influence lung function. We investigated height, body mass index (BMI), and lung function in patients with primary ciliary dyskinesia (PCD).In this study, based on the international PCD (iPCD) Cohort, we calculated z-scores for height and BMI using World Health Organization (WHO) and national growth references, and assessed associations with age, sex, country, diagnostic certainty, age at diagnosis, organ laterality and lung function in multilevel regression models that accounted for repeated measurements.We analysed 6402 measurements from 1609 iPCD Cohort patients. Height was reduced compared to WHO (z-score -0.12, 95% CI -0.17 to -0.06) and national references (z-score -0.27, 95% CI -0.33 to -0.21) in male and female patients in all age groups, with variation between countries. Height and BMI were higher in patients diagnosed earlier in life (p=0.026 and p<0.001, respectively) and closely associated with forced expiratory volume in 1 s and forced vital capacity z-scores (p<0.001).Our study indicates that both growth and nutrition are affected adversely in PCD patients from early life and are both strongly associated with lung function. If supported by longitudinal studies, these findings suggest that early diagnosis with multidisciplinary management and nutritional advice could improve growth and delay disease progression and lung function impairment in PCD.

The international primary ciliary dyskinesia cohort (iPCD Cohort): methods and first results.

Data on primary ciliary dyskinesia (PCD) epidemiology is scarce and published studies are characterised by low numbers. In the framework of the European Union project BESTCILIA we aimed to combine all available datasets in a retrospective international PCD cohort (iPCD Cohort).We identified eligible datasets by performing a systematic review of published studies containing clinical information on PCD, and by contacting members of past and current European Respiratory Society Task Forces on PCD. We compared the contents of the datasets, clarified definitions and pooled them in a standardised format.As of April 2016 the iPCD Cohort includes data on 3013 patients from 18 countries. It includes data on diagnostic evaluations, symptoms, lung function, growth and treatments. Longitudinal data are currently available for 542 patients. The extent of clinical details per patient varies between centres. More than 50% of patients have a definite PCD diagnosis based on recent guidelines. Children aged 10-19 years are the largest age group, followed by younger children (≤9 years) and young adults (20-29 years).This is the largest observational PCD dataset available to date. It will allow us to answer pertinent questions on clinical phenotype, disease severity, prognosis and effect of treatments, and to investigate genotype-phenotype correlations.

Splice-site mutations in the axonemal outer dynein arm docking complex gene CCDC114 cause primary ciliary dyskinesia.

Defects in motile cilia and sperm flagella cause primary ciliary dyskinesia (PCD), characterized by chronic airway disease, infertility, and left-right laterality disturbances, usually as a result of loss of the outer dynein arms (ODAs) that power cilia/flagella beating. Here, we identify loss-of-function mutations in CCDC114 causing PCD with laterality malformations involving complex heart defects. CCDC114 is homologous to DCC2, an ODA microtubule-docking complex component of the biflagellate alga Chlamydomonas. We show that CCDC114 localizes along the entire length of human cilia and that its deficiency causes a complete absence of ciliary ODAs, resulting in immotile cilia. Thus, CCDC114 is an essential ciliary protein required for microtubular attachment of ODAs in the axoneme. Fertility is apparently not greatly affected by CCDC114 deficiency, and qPCR shows that this may explained by low transcript expression in testis compared to ciliated respiratory epithelium. One CCDC114 mutation, c.742G>A, dating back to at least the 1400s, presents an important diagnostic and therapeutic target in the isolated Dutch Volendam population.

An international registry for primary ciliary dyskinesia.

Primary ciliary dyskinesia (PCD) is a rare autosomal recessive disorder leading to chronic upper and lower airway disease. Fundamental data on epidemiology, clinical presentation, course and treatment strategies are lacking in PCD. We have established an international PCD registry to realise an unmet need for an international platform to systematically collect data on incidence, clinical presentation, treatment and disease course.The registry was launched in January 2014. We used internet technology to ensure easy online access using a web browser under www.pcdregistry.eu. Data from 201 patients have been collected so far. The database is comprised of a basic data form including demographic and diagnostic information, and visit forms designed to monitor the disease course.To establish a definite PCD diagnosis, we used strict diagnostic criteria, which required two to three diagnostic methods in addition to classical clinical symptoms. Preliminary analysis of lung function data demonstrated a mean annual decline of percentage predicted forced expiratory volume in 1 s of 0.59% (95% CI 0.98-0.22).Here, we present the development of an international PCD registry as a new promising tool to advance the understanding of this rare disorder, to recruit candidates for research studies and ultimately to improve PCD care.

Glucocorticoid-Induced Proliferation in Untreated Pediatric Acute Myeloid Leukemic Blasts.

We evaluated the in vitro glucocorticoid (GC) responsiveness of 117 pediatric acute myeloid leukemia cells by considering GC resistance, GC-induced proliferation, and GC-induced differentiation. None of the samples was highly GC sensitive, and only 15% were intermediately sensitive. GC-induced differentiation was not observed, while GC-induced proliferation was observed in 27% of the samples. Samples with French-American-British classification (FAB) type M5 or activating Fms-like tyrosine kinase 3 (FLT3) mutations were significantly more prone to this phenomenon. Although we could not confirm this in our study, if induced proliferation in vitro is paralleled in vivo, GCs during consolidation may have adverse effects on minimal residual leukemic cells, which might increase relapse risk.