We aimed to examine the correlation between clinical characteristics and the pathogenic gene variants in patients with Primary Ciliary Dyskinesia (PCD). We conducted a retrospective single-center study in patients with PCD followed at the University Hospitals Leuven. We included patients with genetically confirmed PCD and described their genotype, data from ultrastructural ciliary evaluation and clinical characteristics. Genotype/phenotype correlations were studied in patients with the most frequently involved genes. We enrolled 74 patients with a median age of 25.58 years. The most frequently involved genes were DNAH11 (n = 23) and DNAH5 (n = 19). The most frequent types of pathogenic variants were missense (n = 42) and frameshift variants (n = 36) and most patients had compound heterozygous variants (n = 44). Ciliary ultrastructure (p < 0.001), situs (p = 0.015) and age at diagnosis (median 9.50 vs 4.71 years, p = 0.037) differed between DNAH11 and DNAH5. When correcting for situs this difference in age at diagnosis was no longer significant (p = 0.973). Patients with situs inversus were diagnosed earlier (p = 0.031). Respiratory tract microbiology (p = 0.161), lung function (cross-sectional, p = 0.829 and longitudinal, p = 0.329) and chest CT abnormalities (p = 0.202) were not significantly different between DNAH11 and DNAH5 variants. This study suggests a genotype–phenotype correlation for some of the evaluated clinical characteristics of the two most frequently involved genes in this study, namely DNAH11 and DNAH5.

We report the use of reconstituted 3D-human airway epithelium cells of bronchial origin (HuAEC) in an air-liquid interface to study respiratory syncytial virus (RSV) infection and to assess the efficacy of RSV inhibitors in (pre-)clinical development. RSV-A replicates efficiently in HuAEC and viral RNA is shed for weeks after infection. RSV infection reduces the ciliary beat frequency of the ciliated cells as of 4 days post infection, with complete ciliary dyskinesia observed by day 10. Treatment with RSV fusion inhibitors resulted in an antiviral effect only when added at the time of infection. In contrast, the use of replication inhibitors (both nucleoside and non-nucleosides) elicited a marked antiviral effect even when start of treatment was delayed until one or even three days after infection. Levels of the inflammation marker RANTES (mRNA) increased ∼200-fold in infected-untreated cultures (at three weeks post infection), but levels were comparable to those of uninfected cultures in the presence of PC-876, a RSV-replication inhibitor, demonstrating that an efficient antiviral treatment inhibits virus induced inflammation in this model. Overall, HuAEC offer a robust and physiologically relevant model to study RSV replication and to assess the efficacy of antiviral compounds.

Primary ciliary dyskinesia (PCD) is a rare autosomal recessive disorder (prevalence 1:10 000 to 1:40 000 births) characterised by impaired mucociliary clearance because of abnormal motile ciliary function [1, 2]. Five main ultrastructural PCD phenotypes have been described. Most result from a lack of dynein arms (DAs): no outer and inner DAs (2DAs), outer DAs alone (ODA) or inner DAs with microtubular disorganisation (IDA/MTD); or defects yielding an abnormal central complex (CC). Some patients with genetically confirmed PCD have apparently normal ciliary structure on electron microscopy (nEM). More than 30 genes encoding proteins involved in the structure or assembly of the axoneme, the ciliary internal cytoskeleton, are implicated in PCD [3]; their analysis enables identification of bi-allelic disease-causing mutations in 50–75% of patients. Approximately half of PCD cases are associated with situs inversus, thereby defining Kartagener's syndrome. Moreover, because motile cilia and sperm flagella share common axonemal structures, most PCD-affected males are thought to be infertile [4]. According to the literature, male infertility is caused by severe or total asthenozoospermia and is currently treated by recourse to in vitro fertilisation or intracytoplasmic sperm injection [5, 6]. However, spontaneous fatherhood of PCD patients has been reported. Infertility, observed in 75% of male and 61% of female PCD patients, is dependent on ultrastructural and gene defects http://ow.ly/P4K030fPnPp

Primary ciliary dyskinesia (PCD) is a rare autosomal-recessive disorder characterized by abnormal motile ciliary function. Because motile cilia and sperm flagella share common axonemal structures, male individuals are supposed to be infertile and female may have hypofertility. We hypothesize that a correlation may exist between the risk of hypofertility and the genotype in PCD. We retrospectively recorded the fertility status of adult patients with a definite PCD diagnosis, based on ultrastructural phenotype and an established genotype based on the identification of two non-ambiguous mutations in a known PCD gene. Fertility was considered as normal in case of a spontaneous pregnancy. Hypofertility was defined when attempts for spontaneous conception were unsuccessful for at least one year and/or pregnancy was obtained after assisted reproductive technologies. Data on fertility were available for 83 (50 %) patients. Among the 34 included females, 14 (35.8%) had been spontaneously pregnant and among the 49 male patients, 10 (20.4%) had spontaneously fathered a child. When looking at the different genes involved, in groups containing at least 3 patients, we observed that patients with mutations in CCDC39 or CCDC40 (associated with inner dynein arms with microtubular disorganization defects) and those with mutations in DNAAF1 (LRRC50), or LRRC6 (associated with absence of outer and inner dynein arms) were more likely to be hypofertile (chi-squared test 17.5, p=0.004) and whereas those with RSPH4A mutations (associated with abnormal central complex) are more likely to be fertile. Hypofertility is not always observed in PCD and is dependent of gene defect.

INTRODUCTION Nebulized drugs are used in the treatment of cystic fibrosis (CF) lung disease, asthma, and COPD, and increasingly also in other chronic lung diseases. Their use in CF is reasonably evidence based, but this is not so for use in other orphan diseases. Potential side effects often have not been studied. Therefore, we evaluated the influence of nebulized drugs on ciliary activity in an in vitro model. METHODS We constructed an in vitro nebulization model to examine the effect of drugs on ciliary activity. The model was validated by testing solutions with known neutral, positive, or negative effect on ciliary beat frequency (CBF). Next, the influence on CBF of other inhaled drugs was tested. RESULTS Nebulization of NaCl 0.9% had no influence on CBF, and was used as paired neutral control in further experiments. Salbutamol (Ventolin(®)) had a ciliostimulatory effect (CBF +18%, CBF at t0-t10-t60 7.1-8.5-8.6 Hz, p = 0.002), while hypertonic saline (CBF - 11%, CBF at t0-t10-t60 6.5-5.1-5.9 Hz, p = 0.018) and dry air (CBF -10%, CBF at t0-t10-t60 6.8-5.8-6.1 Hz, p = 0.008) had a cilioinhibitory effect. Nebulization of tobramycin inhaled solution (TOBI(®)) (p = 0.662), colistimethate (Colistineb(®)) (p = 0.369), rhDNAse (Pulmozyme(®)) (p = 0.069), ceftazidim (Glazidim(®)) (p = 0.875), and aztreonam (Cayston(®)) (p = 0.435) did not affect CBF. Obracin(®), a tobramycin containing solution manufactured for intravenous use, had a negative effect on CBF (CBF - 21%, CBF at t0-t10-t60 6.9-5.2-4.5 Hz, p = 0.004). CONCLUSION Inhaled drugs that are used off-label might have an influence on ciliary activity. This must be taken into account when prescribing these drugs for non-CF indications.

Introduction The efficacy of several inhaled drugs is well studied in the context of cystic fibrosis (CF). Their use is however extrapolated to other chronic diseases, without clear evidence of benefit. In addition, the effect of inhaled drugs on ciliary activity has rarely been studied. Previously, we validated an in vitro model for testing the effect of nebulized drugs on ciliary beat frequency (CBF) and showed negative influence of Obracin® but not of Tobi®. Objectives To examine the influence of 2 antibiotics on CBF: the approved inhalation drug Cayston® (C) (provided by Gilead) and Glazidim® (G), used off-label for inhalation in patients with specific infections, e.g. Achromobacter. Methods Non-CF nasal epithelial cells were cultured as monolayer. On day 4 to 6, confluent cell monolayers were exposed to nebulization in an aerosol chamber for 10 minutes. C was nebulized with an eFlow nebulizer plus Altera head, G with a Pari boy nebulizer plus Pari LC star head. Isotonic saline (S) was used as paired, neutral control. Images were acquired with a high-speed camera, and CBF was expressed as % of the starting value. CBF until 1 hour after nebulization was compared between the test condition and S using repeated measure mixed effect model statistics. Results Compared to S, C (n = 4) did not influence CBF (p 0.355): CBF was 96% 1 hour after nebulization of C, versus 98% for S. G (n = 3) neither influenced CBF (p 0.346): CBF was 99% 1 hour after nebulization of G, versus 109% for S. Results on days 2 and 3 were similar. Conclusion Nebulization of Cayston® and Glazidim® did not influence ciliary activitity in vitro. This may be relevant to their in vivo efficacy and safety.

Meeting abstractSince the 1980s, a few case reports described patients with oto-rhino-pulmonary symptoms and respiratory epithelia lacking cilia who were subsequently diagnosed to suffer from "ciliary aplasia" or "acilia syndrome". Via a whole exome sequencing approach, we analyzed "ciliary aplasia" candidates (including patients from previous reports) and identified recessive mutations in CCNO (encoding Cyclin O) and MCIDAS (encoding Multicilin) in 9 and 16 individuals, respectively [1, 2]. All individuals suffered from severe respiratory symptoms of the upper and lower airways and development of bronchiectasis at an early age. Thorough analysis of respiratory epithelial cells obtained by nasal brush biopsy by both transmission electron microscopy (TEM) and immunofluorescence analysis (IF) revealed that respiratory cilia were not completely absent; some cells still retained one or two cilia. These cells not only showed a reduction of cilia by TEM and IF, but also a reduction and mislocalization of basal bodies and rootlets throughout the cytoplasm. Detailed analyses by IF in both man and Xenopus revealed that this reduction of cilia number was due to a centriole amplification defect in the acentriolar pathway, which is specific for multiciliated cells [1, 2].IF showed that MCIDAS functions upstream of CCNO and FOXJ1, which is important for transcriptional control of axonemal motor proteins such as DNAH5 and CCDC39. Whereas cilia in CCNO-mutant cells still contain motility-related proteins such as DNAH5 and CCDC39 and can display normal beating patterns, MCIDAS-mutant cells are immotile and lack those axonemal motor proteins [1, 2] . MCIDAS and CCNO lie adjacently on chromosome 5q11 in a region related to multiciliogenesis, and act in the same pathway underlying multiciliogenesis. Based on these findings, we propose that this disease now should be referred to as "mucociliary clearance disorder with reduced generation of multiple motile cilia" (RGMC).

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.

Introduction: Nasal discharge is very common and has significant impact on quality of life. Several therapies are available, but none has the aim to increase nasal mucociliary clearance by enhancing ciliary activity. Aim: We aimed to study the influence of a new compound for nasal use, Ciliactive®, on ciliary activity in vitro. Methods: Human nasal epithelial cells expressing motile cilia were obtained from surgery specimens and cultured as a monolayer. We studied the influence of Ciliactive® (C), compared to saline (NaCl 0,9%, S). The compounds were diluted in cell culture medium and ciliary beat frequency (CBF) was followed during exposure (0, 1, 5, 24, 48 hours). CBF was measured with a high speed camera, mounted on an inverted microscope: each result was the mean of 5 measurements. All results were expressed as % of the starting value. C and S were used in concentrations of 50, 75 and 100 %, diluted in cell culture medium. Conditions were compared using paired t-tests. Results: Samples from 8 persons were included. C significantly increased CBF in all concentrations, compared to the paired controls with S (p<0.005 for 5h, p<0.002 for 24h, p<0.03 for 48h). C increased CBF with 15, 24 and 15% after 24h, compared to S for a concentration of 50%, 75% and 100 % respectively. Conclusion: Ciliactive® is a new compound, that enhances ciliary activity in vitro. It might be useful to treat nasal mucociliary clearance disorders.

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.

BACKGROUND:Primary 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.METHODS:We 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.RESULTS:PCD 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.CONCLUSIONS:We 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.

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous disorder caused by cilia and sperm dysmotility. About 12% of cases show perturbed 9+2 microtubule cilia structure and inner dynein arm (IDA) loss, historically termed “radial spoke defect.” We sequenced CCDC39 and CCDC40 in 54 “radial spoke defect” families, as these are the two genes identified so far to cause this defect. We discovered biallelic mutations in a remarkable 69% (37/54) of families, including identification of 25 (19 novel) mutant alleles (12 in CCDC39 and 13 in CCDC40). All the mutations were nonsense, splice, and frameshift predicting early protein truncation, which suggests this defect is caused by “null” alleles conferring complete protein loss. Most families (73%; 27/37) had homozygous mutations, including families from outbred populations. A major putative hotspot mutation was identified, CCDC40 c.248delC, as well as several other possible hotspot mutations. Together, these findings highlight the key role of CCDC39 and CCDC40 in PCD with axonemal disorganization and IDA loss, and these genes represent major candidates for genetic testing in families affected by this ciliary phenotype. We show that radial spoke structures are largely intact in these patients and propose this ciliary ultrastructural abnormality be referred to as “IDA and microtubular disorganisation defect,” rather than “radial spoke defect.”

DYX1C1 has been associated with dyslexia and neuronal migration in the developing neocortex. Unexpectedly, we found that deleting exons 2-4 of Dyx1c1 in mice caused a phenotype resembling primary ciliary dyskinesia (PCD), a disorder characterized by chronic airway disease, laterality defects and male infertility. This phenotype was confirmed independently in mice with a Dyx1c1 c.T2A start-codon mutation recovered from an N-ethyl-N-nitrosourea (ENU) mutagenesis screen. Morpholinos targeting dyx1c1 in zebrafish also caused laterality and ciliary motility defects. In humans, we identified recessive loss-of-function DYX1C1 mutations in 12 individuals with PCD. Ultrastructural and immunofluorescence analyses of DYX1C1-mutant motile cilia in mice and humans showed disruptions of outer and inner dynein arms (ODAs and IDAs, respectively). DYX1C1 localizes to the cytoplasm of respiratory epithelial cells, its interactome is enriched for molecular chaperones, and it interacts with the cytoplasmic ODA and IDA assembly factor DNAAF2 (KTU). Thus, we propose that DYX1C1 is a newly identified dynein axonemal assembly factor (DNAAF4).

From the authors : We wish to thank M. Boon and co-workers for their careful reading of our manuscript and to apologise to them and the readers for our mistake in the preparation of table 2 of our original article [ …