Using a whole-exome sequencing strategy, we identified recessive CCNO (encoding cyclin O) mutations in 16 individuals suffering from chronic destructive lung disease due to insufficient airway clearance. Respiratory epithelial cells showed a marked reduction in the number of multiple motile cilia (MMC) covering the cell surface. The few residual cilia that correctly expressed axonemal motor proteins were motile and did not exhibit obvious beating defects. Careful subcellular analyses as well as in vitro ciliogenesis experiments in CCNO-mutant cells showed defective mother centriole generation and placement. Morpholino-based knockdown of the Xenopus ortholog of CCNO also resulted in reduced MMC and centriole numbers in embryonic epidermal cells. CCNO is expressed in the apical cytoplasm of multiciliated cells and acts downstream of multicilin, which governs the generation of multiciliated cells. To our knowledge, CCNO is the first reported gene linking an inherited human disease to reduced MMC generation due to a defect in centriole amplification and migration.

Reduced generation of multiple motile cilia (RGMC) is a rare mucociliary clearance disorder. Affected persons suffer from recurrent infections of upper and lower airways because of highly reduced numbers of multiple motile respiratory cilia. Here we report recessive loss-of-function and missense mutations in MCIDAS-encoding Multicilin, which was shown to promote the early steps of multiciliated cell differentiation in Xenopus. MCIDAS mutant respiratory epithelial cells carry only one or two cilia per cell, which lack ciliary motility-related proteins (DNAH5; CCDC39) as seen in primary ciliary dyskinesia. Consistent with this finding, FOXJ1-regulating axonemal motor protein expression is absent in respiratory cells of MCIDAS mutant individuals. CCNO, when mutated known to cause RGMC, is also absent in MCIDAS mutant respiratory cells, consistent with its downstream activity. Thus, our findings identify Multicilin as a key regulator of CCNO/FOXJ1 for human multiciliated cell differentiation, and highlight the 5q11 region containing CCNO and MCIDAS as a locus underlying RGMC.

We demonstrated that measuring LCI in patients with PCD is of clinical relevance; it was more frequently abnormal than FEV1, correlated well with CFCT and was more sensitive than FEV1 to detect patients with structural abnormalities.

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous recessive disorder caused by several distinct defects in genes responsible for ciliary beating, leading to defective mucociliary clearance often associated with randomization of left/right body asymmetry. Individuals with PCD caused by defective radial spoke (RS) heads are difficult to diagnose owing to lack of gross ultrastructural defects and absence of situs inversus. Thus far, most mutations identified in human radial spoke genes (RSPH) are loss-of-function mutations, and missense variants have been rarely described. We studied the consequences of different RSPH9, RSPH4A, and RSPH1 mutations on the assembly of the RS complex to improve diagnostics in PCD. We report 21 individuals with PCD (16 families) with biallelic mutations in RSPH9, RSPH4A, and RSPH1, including seven novel mutations comprising missense variants, and performed high-resolution immunofluorescence analysis of human respiratory cilia. Missense variants are frequent genetic defects in PCD with RS defects. Absence of RSPH4A due to mutations in RSPH4A results in deficient axonemal assembly of the RS head components RSPH1 and RSPH9. RSPH1 mutant cilia, lacking RSPH1, fail to assemble RSPH9, whereas RSPH9 mutations result in axonemal absence of RSPH9, but do not affect the assembly of the other head proteins, RSPH1 and RSPH4A. Interestingly, our results were identical in individuals carrying loss-of-function mutations, missense variants, or one amino acid deletion. Immunofluorescence analysis can improve diagnosis of PCD in patients with loss-of-function mutations as well as missense variants. RSPH4A is the core protein of the RS head.

Respiratory infections, situs inversus and male infertility are typical manifestations of PCD, a rare autosomal recessive disorder.

BackgroundPrimary ciliary dyskinesia (PCD) is a rare disorder with variable disease progression. To date, mutations in more than 20 different genes have been found. At present, PCD subtypes are described according to the ultrastructural defect on transmission electron microscopy (TEM) of the motile cilia. PCD with normal ultrastructure (NU) is rarely reported because it requires additional testing. Biallelic mutations in DNAH11 have been described as one cause of PCD with NU.The aim of our study was to describe the clinical characteristics of a large population of patients with PCD, in relation to the ultrastructural defect. Additionally, we aimed to demonstrate the need for biopsy and cell culture to reliably diagnose PCD, especially the NU subtype.MethodsWe retrospectively analyzed data from 206 patients with PCD. We compared the clinical characteristics, lung function, microbiology and imaging results of 68 patients with PCD and NU to those of 90 patients with dynein deficiencies and 41 patients with central pair abnormalities. In addition, we aimed to demonstrate the robustness of the diagnosis of the NU subtype in cell culture by data from genetic analysis.ResultsPCD with NU comprised 33% (68/206) of all patients with PCD. Compared to other subtypes, patients with PCD and NU had a similar frequency of upper and lower respiratory tract problems, as well as similar lung function and imaging. With the currently widely applied approach, without cell culture, the diagnosis would have been missed in 16% (11/68) of patients with NU. Genetic analysis was performed in 29/68 patients with PCD and NU, and biallelic mutations were found in 79% (23/29) of tested patients.ConclusionsWe reported on the clinical characteristics of a large population of patients with PCD and NU. We have shown that systematic performance of biopsy and cell culture increases sensitivity to detect PCD, especially the subtype with NU.PCD with NU has similar clinical characteristics as other PCD types and requires biopsy plus ciliogenesis in culture for optimal diagnostic yield.

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.

RationaleThe lung clearance index (LCI) is a promising endpoint for use in cystic fibrosis (CF) clinical trials, but correlations with validated clinical endpoints have not yet been established. Objective This study aimed to demonstrate that, in young patients with CF, baseline LCI predicts subsequent pulmonary exacerbation (PE) and correlates with the respiratory domain of the CF Questionnaire-Revised (CFQ-R resp ). Methods Baseline LCI, forced expiratory volume in 1 s (FEV 1 ), CFQ-R resp and PEs over the subsequent year were prospectively recorded in 63 patients aged 5-19 years. The ability of baseline LCI to predict PE was assessed using negative binomial regression models and KaplanMeier plots. Results Twenty-six patients (41%) experienced 48 PEs. Baseline LCI and FEV 1 were predictors of PE. Compared with the quartile with the lowest LCI, the annual PE rate in increasing LCI quartiles was 2.9 (95% CI 0.5 to 16.5, p=0.238), 5.4 (95% CI 1.0 to 29.0, p=0.045) and 13.6 (95% CI 2.8 to 67.1, p=0.001). Similarly, time to first PE decreased with worsening LCI quartiles (log-rank test for trend, p<0.001). Furthermore, LCI correlated with CFQ-R resp (r=−0.43, p<0.001). In the subgroup of 53 patients with normal FEV 1 , LCI was a predictor of PE. In this subgroup, LCI also correlated with CFQ-R resp (r=−0.282, p=0.043). Conclusions Baseline LCI predicts PE in young patients with CF and correlates with CFQ-R resp , a validated patient-reported outcome, even in the subgroup with normal FEV 1 . These data further support the use of LCI as a surrogate outcome measure in CF clinical trials.