Skewed X-chromosome inactivation drives the proportion of DNAAF6-defective airway motile cilia and variable expressivity in primary ciliary dyskinesia.

BACKGROUND: Primary ciliary dyskinesia (PCD) is a rare airway disorder caused by defective motile cilia. Only male patients have been reported with pathogenic mutations in X-linked DNAAF6, which result in the absence of ciliary dynein arms, whereas their heterozygous mothers are supposedly healthy. Our objective was to assess the possible clinical and ciliary consequences of X-chromosome inactivation (XCI) in these mothers. METHODS: XCI patterns of six mothers of male patients with DNAAF6-related PCD were determined by DNA-methylation studies and compared with their clinical phenotype (6/6 mothers), as well as their ciliary phenotype (4/6 mothers), as assessed by immunofluorescence and high-speed videomicroscopy analyses. The mutated X chromosome was tracked to assess the percentage of cells with a normal inactivated DNAAF6 allele. RESULTS: The mothers' phenotypes ranged from absence of symptoms to mild/moderate or severe airway phenotypes, closely reflecting their XCI pattern. Analyses of the symptomatic mothers' airway ciliated cells revealed the coexistence of normal cells and cells with immotile cilia lacking dynein arms, whose ratio closely mirrored their XCI pattern. CONCLUSION: This study highlights the importance of searching for heterozygous pathogenic DNAAF6 mutations in all female relatives of male PCD patients with a DNAAF6 defect, as well as in females consulting for mild chronic respiratory symptoms. Our results also demonstrate that about one-third-ranging from 20% to 50%-normal ciliated airway cells sufficed to avoid severe PCD, a result paving the way for gene therapy.

Results and perinatal outcomes from 189 ICSI cycles of couples with asthenozoospermic men and flagellar defects assessed by transmission electron microscopy.

RESEARCH QUESTION: Do patients presenting with flagella ultrastructural defects as assessed by electron microscopy, and defined within three phenotypes (dysplasia of the fibrous sheath [DFS], primary flagellar dyskinesia [PFD] and non-specific flagellar abnormalities [NSFA]), have decreased chances of success in intracytoplasmic sperm injection (ICSI) or adverse obstetric and neonatal outcomes? DESIGN: Retrospective analysis of 189 ICSI cycles from 80 men with spermatozoa flagellum ultrastructural defects (DFS [n = 16]; PFD [n = 14]; NSFA [n = 50] compared with a control group (n = 97). Cycles were cumulatively analysed. All fresh and frozen embryo transfers resulting from each ICSI attempt were included. The effect of transmission electron microscopy (TEM) phenotype on the main ICSI outcomes was assessed by a multivariate logistic regression combined with a generalized linear mixed model to account for the non-independence of the observations. RESULTS: No predictive value of TEM phenotype was found on the main outcomes of ICSI, namely fertilization rates, pregnancy and delivery rates, and cumulative pregnancy and delivery rates. Cumulative pregnancy rates ranged from 29.0-43.3% in the different TEM phenotype subgroups compared with 36.8% in the control group. Cumulative live birth rates ranged from 24.6-36.7% compared with 31.4% in the control group. No increase was found in miscarriages, preterm births, low birth weights or birth abnormalities. CONCLUSIONS: Data on the cumulative chances of success in ICSI of patients with ultrastructural flagellar defects, a rare cause of male infertility often associated with an underlying genetic cause, are reassuring, as are obstetrical and neonatal outcomes in this population.

Otological Manifestations in Adults with Primary Ciliary Dyskinesia: A Controlled Radio-Clinical Study.

Primary ciliary dyskinesia (PCD) is a rare genetical disease characterized by an abnormal structure or function of the cilia, causing sinusitis, otitis, and bronchiectasis. Hearing loss affects 60% of PCD patients, but data are lacking concerning hearing and temporal bone imaging in adults. Our aim was to describe clinical and radiological ear disease in adults with genetically confirmed PCD. Data were recorded from January 2018 to December 2019. PCD patients were compared with controls with bronchiectasis without PCD. Clinical examination included otomicroscopy and auditory tests. A temporal bone CT scan (TBCT) was systematically performed. Seventeen patients (34 ears) were included in each group. The eardrums were abnormal in 25 (74%) PCD ears versus 8 (24%) ears in the controls (p < 0.05). Conductive hearing loss was more frequent in the PCD group (24% vs. 12% in controls). TBCT were abnormal in 94% PCD patients vs. 32% in the controls (p < 0.05). The Main CT-scan images in PCD were middle ear inflammation (65%), mastoid condensation (62%), or ossicular anomalies (35%). With its excellent sensitivity, TBCT gives typical arguments for PCD diagnosis, adding otological signs to the usual sinus CT signs (hypoplasia, aplasia). Systematic TBCT could be useful in the initial evaluation of patients with suspicion of PCD.

Pathogenesis of chronic rhinosinusitis with nasal polyps: role of IL-6 in airway epithelial cell dysfunction.

BACKGROUND: Chronic rhinosinusitis with nasal polyps (CRSwNP) is characterized by an alteration in airway epithelial cell functions including barrier function, wound repair mechanisms, mucociliary clearance. The mechanisms leading to epithelial cell dysfunction in nasal polyps (NPs) remain poorly understood. Our hypothesis was that among the inflammatory cytokines involved in NPs, IL-6 could alter epithelial repair mechanisms and mucociliary clearance. The aim of this study was to evaluate the in vitro effects of IL-6 on epithelial repair mechanisms in a wound repair model and on ciliary beating in primary cultures of Human Nasal Epithelial Cells (HNEC). METHODS: Primary cultures of HNEC taken from 38 patients during surgical procedures for CRSwNP were used in an in vitro model of wound healing. Effects of increasing concentrations of IL-6 (1 ng/mL, 10 ng/mL, and 100 ng/mL) and other ILs (IL-5, IL-9, IL-10) on wound closure kinetics were compared to cultures without IL-modulation. After wound closure, the differentiation process was characterized under basal conditions and after IL supplementation using cytokeratin-14, MUC5AC, and ßIV tubulin as immunomarkers of basal, mucus, and ciliated cells, respectively. The ciliated edges of primary cultures were analyzed on IL-6 modulation by digital high-speed video-microscopy to measure: ciliary beating frequency (CBF), ciliary length, relative ciliary density, metachronal wavelength and the ciliary beating efficiency index. RESULTS: Our results showed that: (i) IL-6 accelerated airway wound repair in vitro, with a dose-response effect whereas no effect was observed after other ILs-stimulation. After 24 h, 79% of wounded wells with IL6-100 were fully repaired, vs 46% in the IL6-10 group, 28% in the IL6-1 group and 15% in the control group; (ii) specific migration analyses of closed wound at late repair stage (Day 12) showed IL-6 had the highest migration compared with other ILs (iii) The study of the IL-6 effect on ciliary function showed that CBF and metachronal wave increased but without significant modifications of ciliary density, length of cilia and efficiency index. CONCLUSION: The up-regulated epithelial cell proliferation observed in polyps could be induced by IL-6 in the case of prior epithelial damage. IL-6 could be a major cytokine in NP physiopathology.

TTC12 Loss-of-Function Mutations Cause Primary Ciliary Dyskinesia and Unveil Distinct Dynein Assembly Mechanisms in Motile Cilia Versus Flagella.

Cilia and flagella are evolutionarily conserved organelles whose motility relies on the outer and inner dynein arm complexes (ODAs and IDAs). Defects in ODAs and IDAs result in primary ciliary dyskinesia (PCD), a disease characterized by recurrent airway infections and male infertility. PCD mutations in assembly factors have been shown to cause a combined ODA-IDA defect, affecting both cilia and flagella. We identified four loss-of-function mutations in TTC12, which encodes a cytoplasmic protein, in four independent families in which affected individuals displayed a peculiar PCD phenotype characterized by the absence of ODAs and IDAs in sperm flagella, contrasting with the absence of only IDAs in respiratory cilia. Analyses of both primary cells from individuals carrying TTC12 mutations and human differentiated airway cells invalidated for TTC12 by a CRISPR-Cas9 approach revealed an IDA defect restricted to a subset of single-headed IDAs that are different in flagella and cilia, whereas TTC12 depletion in the ciliate Paramecium tetraurelia recapitulated the sperm phenotype. Overall, our study, which identifies TTC12 as a gene involved in PCD, unveils distinct dynein assembly mechanisms in human motile cilia versus flagella.

Primary ciliary dyskinesia gene contribution in Tunisia: Identification of a major Mediterranean allele.

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous disease of motile cilia. Even though PCD is widely studied, North-African patients have been rarely explored. In this study, we aim at confirming the clinical diagnosis and explore the genetic spectrum of PCD in a cohort of Tunisian patients. Forty clinically diagnosed patients with PCD belonging to 34 families were recruited from Tunisian pediatric departments. In each proband, targeted capture PCD panel sequencing of the 40 PCD genes was performed. PCD panel sequencing identified bi-allelic mutations in 82% of the families in eight PCD genes. Remarkably, 23.5% of patients carried the same c.2190del CCDC39 mutation. Single nucleotide polymorphism profiling in six unrelated patients carrying this mutation has revealed a founder effect in North-African patients. This mutation is estimated to date back at least 1,400-1,750 years ago. The identification of this major allele allowed us to suggest a cost-effective genetic diagnostic strategy in North-African patients with PCD.

Critical Evaluation of Sinonasal Disease in 64 Adults with Primary Ciliary Dyskinesia.

To date, no study precisely described ear, nose and throat (ENT) disease in adults with primary ciliary dyskinesia (PCD) and its relationship with ciliary function/ultrastructure. A retrospective study of standardized ENT data (exam, audiogram, sinus Computed tomography (CT), and bacteriology) was conducted in 64 adults with confirmed PCD who were followed in two ENT reference centers. Rhinorrhoea and hearing loss were the main symptoms. Symptom scores were higher in older patients. Nasal endoscopy was abnormal in all patients except one, showing nasal polyps in one-third of the patients and stagnant nasal mucus secretions in 87.5% of the patients. Sinus CT opacities were mainly incomplete and showed one-third of the patients with sinus hypoplasia and/or agenesis. Middle meatus mainly grew Haemophilus influenzae, Streptoccocus pneumoniae and Pseudomonas aeruginosa. Otitis media with effusion (OME), which is constant in childhood, was diagnosed in less than one-quarter of the patients. In two-thirds of the patients, audiogram showed hearing loss that was sensorineural in half of the patients. ENT disease severity was not correlated with ciliary function and ultrastructure, but the presence of OME was significantly associated with a forced expiratory volume (FEV1) < 70%. Rhinosinusitis is the most common clinical feature of PCD in adults, while OME is less frequent. The presence of active OME in adults with PCD could be a severity marker of lung function and lead to closer monitoring.

FcRn-Dependent Transcytosis of Monoclonal Antibody in Human Nasal Epithelial Cells In Vitro: A Prerequisite for a New Delivery Route for Therapy?

Monoclonal antibodies (mAbs) are promising therapies to treat airway chronic inflammatory disease (asthma or nasal polyps). To date, no study has specifically assessed, in vitro, the potential function of neonatal Fc receptor (FcRn) in IgG transcytosis through the human nasal airway epithelium. The objective of this study was to report the in vitro expression and function of FcRn in nasal human epithelium. FcRn expression was studied in an air⁻liquid interface (ALI) primary culture model of human nasal epithelial cells (HNEC) from polyps. FcRn expression was characterized by quantitative RT-PCR, western blot, and immunolabeling. The ability of HNECs to support mAb transcytosis via FcRn was assessed by transcytosis assay. This study demonstrates the expression of FcRn mRNA and protein in HNEC. We report a high expression of FcRn in the cytosol of ciliated, mucus, and basal cells by immunohistochemistry with a higher level of FcRn proteins in differentiated HNEC. We also proved in vitro transepithelial delivery of an IgG1 therapeutic mAb with a dose⁻response curve. This is the first time that FcRn expression and mAb transcytosis has been shown in a model of human nasal respiratory epithelium in vitro. This study is a prerequisite for FcRn-dependent nasal administration of mAbs.

Lack of GAS2L2 Causes PCD by Impairing Cilia Orientation and Mucociliary Clearance.

Primary ciliary dyskinesia (PCD) is a genetic disorder in which impaired ciliary function leads to chronic airway disease. Exome sequencing of a PCD subject identified an apparent homozygous frameshift variant, c.887_890delTAAG (p.Val296Glyfs∗13), in exon 5; this frameshift introduces a stop codon in amino acid 308 of the growth arrest-specific protein 2-like 2 (GAS2L2). Further genetic screening of unrelated PCD subjects identified a second proband with a compound heterozygous variant carrying the identical frameshift variant and a large deletion (c.867_∗343+1207del; p.?) starting in exon 5. Both individuals had clinical features of PCD but normal ciliary axoneme structure. In this research, using human nasal cells, mouse models, and X.laevis embryos, we show that GAS2L2 is abundant at the apical surface of ciliated cells, where it localizes with basal bodies, basal feet, rootlets, and actin filaments. Cultured GAS2L2-deficient nasal epithelial cells from one of the affected individuals showed defects in ciliary orientation and had an asynchronous and hyperkinetic (GAS2L2-deficient = 19.8 Hz versus control = 15.8 Hz) ciliary-beat pattern. These results were recapitulated in Gas2l2-/- mouse tracheal epithelial cell (mTEC) cultures and in X. laevis embryos treated with Gas2l2 morpholinos. In mice, the absence of Gas2l2 caused neonatal death, and the conditional deletion of Gas2l2 impaired mucociliary clearance (MCC) and led to mucus accumulation. These results show that a pathogenic variant in GAS2L2 causes a genetic defect in ciliary orientation and impairs MCC and results in PCD.

Mutations in Outer Dynein Arm Heavy Chain DNAH9 Cause Motile Cilia Defects and Situs Inversus.

Motile cilia move body fluids and gametes and the beating of cilia lining the airway epithelial surfaces ensures that they are kept clear and protected from inhaled pathogens and consequent respiratory infections. Dynein motor proteins provide mechanical force for cilia beating. Dynein mutations are a common cause of primary ciliary dyskinesia (PCD), an inherited condition characterized by deficient mucociliary clearance and chronic respiratory disease coupled with laterality disturbances and subfertility. Using next-generation sequencing, we detected mutations in the ciliary outer dynein arm (ODA) heavy chain gene DNAH9 in individuals from PCD clinics with situs inversus and in one case male infertility. DNAH9 and its partner heavy chain DNAH5 localize to type 2 ODAs of the distal cilium and in DNAH9-mutated nasal respiratory epithelial cilia we found a loss of DNAH9/DNAH5-containing type 2 ODAs that was restricted to the distal cilia region. This confers a reduced beating frequency with a subtle beating pattern defect affecting the motility of the distal cilia portion. 3D electron tomography ultrastructural studies confirmed regional loss of ODAs from the distal cilium, manifesting as either loss of whole ODA or partial loss of ODA volume. Paramecium DNAH9 knockdown confirms an evolutionarily conserved function for DNAH9 in cilia motility and ODA stability. We find that DNAH9 is widely expressed in the airways, despite DNAH9 mutations appearing to confer symptoms restricted to the upper respiratory tract. In summary, DNAH9 mutations reduce cilia function but some respiratory mucociliary clearance potential may be retained, widening the PCD disease spectrum.

Gas exchanges in children with cystic fibrosis or primary ciliary dyskinesia: A retrospective study.

Primary ciliary dyskinesia (PCD) and cystic fibrosis (CF) both entail bronchiectasis and pulmonary impairment as measured using spirometry, during childhood. We aimed at looking whether blood gas exchanges progressed differently between CF and PCD children in a retrospective study of repeated measurements. Comparisons between groups (Wilcoxon-Mann-Whitney and Chi-squared tests) and a mixed linear model, adjusted for age, evaluated associations between diseases and PaO2, PaCO2, or PaO2-PaCO2 ratio. Among 42 PCD and 73 CF children, 62% and 59% had respectively bronchiectasis (P = 0.75). Spirometry and blood gases were similar at inclusion (PaO2 median [IQR] PCD -1.80 [-3.40; -0.40]; CF -1.80 [-4.20; 0.60] z-scores; P = 0.72). PaO2 and PaO2-PaCO2 ratio similarly and significantly decreased with age in both groups (P < 0.01) whereas PaCO2 increased more in CF (P = 0.02) remaining within the range of normal (except for one child). To conclude, gas exchange characteristics, similarly initially impaired in PCD and CF children, tended to less deteriorate with time in PCD children who could benefit from an early diagnosis.

Functional characterization of tektin-1 in motile cilia and evidence for TEKT1 as a new candidate gene for motile ciliopathies.

A child presenting with Mainzer-Saldino syndrome (MZSDS), characterized by renal, retinal and skeletal involvements, was also diagnosed with lung infections and airway ciliary dyskinesia. These manifestations suggested dysfunction of both primary and motile cilia, respectively. Targeted exome sequencing identified biallelic mutations in WDR19, encoding an IFT-A subunit previously associated with MZSDS-related chondrodysplasia, Jeune asphyxiating thoracic dysplasia and cranioectodermal dysplasia, linked to primary cilia dysfunction, and in TEKT1 which encodes tektin-1 an uncharacterized member of the tektin family, mutations of which may cause ciliary dyskinesia. Tektin-1 localizes at the centrosome in cycling cells, at basal bodies of both primary and motile cilia and to the axoneme of motile cilia in airway cells. The identified mutations impaired these localizations. In addition, airway cells from the affected individual showed severe motility defects without major ultrastructural changes. Knockdown of tekt1 in zebrafish resulted in phenotypes consistent with a function for tektin-1 in ciliary motility, which was confirmed by live imaging. Finally, experiments in the zebrafish also revealed a synergistic effect of tekt1 and wdr19. Altogether, our data show genetic interactions between WDR19 and TEKT1 likely contributing to the overall clinical phenotype observed in the affected individual and provide strong evidence for TEKT1 as a new candidate gene for primary ciliary dyskinesia.

Clinical characteristics, functional respiratory decline and follow-up in adult patients with primary ciliary dyskinesia.

INTRODUCTION: Primary ciliary dyskinesia (PCD) is a genetic disease characterised by abnormalities in ciliary function, responsible for chronic pulmonary and sinonasal diseases. Adult clinical features and outcome are poorly described. OBJECTIVES: To assess the clinical characteristics and disease progression in adults with PCD. METHODS: Bicentric retrospective study, focusing on adults (≥18 years) with an asserted diagnosis of PCD based on the presence of bronchiectasis with typical ultrastructural defect of cilia and/or situs inversus (SI). Clinical symptoms, respiratory function, extent of bronchiectasis, microbiology and molecular analysis were assessed. Results are expressed as median (25th; 75th centile). RESULTS: 78 patients were included with a median follow-up of 8.1 years. 91% of patients had respiratory symptoms and 95% had chronic rhinosinusitis. Half of ultrastructural defects concerned dynein arms. Respiratory function was significantly lower in women (FEV1=60% predicted (50; 76), vs 77% (62; 95), p=0.009) and in patients with chronic airway Pseudomonas aeruginosa (PA, n=21) infection (FEV1=60% (48; 71) vs 75% (55; 89), p=0.04). FEV1 was associated with gender (regression coefficient for men =13.8, p=0.009), chest CT score (r=-0.42, p<0.001) but not with age at diagnosis, SI or body mass index. FEV1 decline was -13.4 mL/year (-42.8; +11.9) and was greater in women (-29.3 mL/year, (-59.7; -11.9), vs -2.0 mL/year (-26.9; +25.4), p=0.002). Three patients had severe respiratory failure. CONCLUSIONS: Alteration of respiratory function in adults with PCD is heterogeneous and usually moderate but appears more severe in women and in patients with chronic PA infection. Only 4% of patients develop chronic respiratory failure.

Mutations in GAS8, a Gene Encoding a Nexin-Dynein Regulatory Complex Subunit, Cause Primary Ciliary Dyskinesia with Axonemal Disorganization.

Primary ciliary dyskinesia (PCD) is an autosomal recessive disease characterized by chronic respiratory infections of the upper and lower airways, hypofertility, and, in approximately half of the cases, situs inversus. This complex phenotype results from defects in motile cilia and sperm flagella. Among the numerous genes involved in PCD, very few-including CCDC39 and CCDC40-carry mutations that lead to a disorganization of ciliary axonemes with microtubule misalignment. Focusing on this particular phenotype, we identified bi-allelic loss-of-function mutations in GAS8, a gene that encodes a subunit of the nexin-dynein regulatory complex (N-DRC) orthologous to DRC4 of the flagellated alga Chlamydomonas reinhardtii. Unlike the majority of PCD patients, individuals with GAS8 mutations have motile cilia, which, as documented by high-speed videomicroscopy, display a subtle beating pattern defect characterized by slightly reduced bending amplitude. Immunofluorescence studies performed on patients' respiratory cilia revealed that GAS8 is not required for the proper expression of CCDC39 and CCDC40. Rather, mutations in GAS8 affect the subcellular localization of another N-DRC subunit called DRC3. Overall, this study, which identifies GAS8 as a PCD gene, unveils the key importance of the corresponding protein in N-DRC integrity and in the proper alignment of axonemal microtubules in humans.

A new workflow for proteomic analysis of urinary exosomes and assessment in cystinuria patients.

Cystinuria is a purely renal, rare genetic disease caused by mutations in cystine transporter genes and characterized by defective cystine reabsorption leading to kidney stones. In 14% of cases, patients undergo nephrectomy, but given the difficulty to predict the evolution of the disease, the identification of markers of kidney damage would improve the follow-up of patients with a higher risk. The aim of the present study is to develop a robust, reproducible, and noninvasive methodology for proteomic analysis of urinary exosomes using high resolution mass spectrometry. A clinical pilot study conducted on eight cystinuria patients versus 10 controls highlighted 165 proteins, of which 38 were up-regulated, that separate cystinuria patients from controls and further discriminate between severe and moderate forms of the disease. These proteins include markers of kidney injury, circulating proteins, and a neutrophil signature. Analysis of selected proteins by immunobloting, performed on six additional cystinuria patients, validated the mass spectrometry data. To our knowledge, this is the first successful proteomic study in cystinuria unmasking the potential role of inflammation in this disease. The workflow we have developed is applicable to investigate urinary exosomes in different renal diseases and to search for diagnostic/prognostic markers. Data are available via ProteomeXchange with identifier PXD001430.

Proteomic analysis of nasal epithelial cells from cystic fibrosis patients.

The pathophysiology of cystic fibrosis (CF) lung disease remains incompletely understood. New explanations for the pathogenesis of CF lung disease may be discovered by studying the patterns of protein expression in cultured human nasal epithelial cells (HNEC). To that aim, we compared the level of protein expressions in primary cultures of HNEC from nasal polyps secondary to CF (CFNP, n = 4), primary nasal polyps (NP, n = 8) and control mucosa (CTRL, n = 4) using isobaric tag for relative and absolute quantification (iTRAQ) labeling coupled with liquid chromatography (LC)-MS-MS. The analysis of the data revealed 42 deregulated protein expressions in CFNP compared to NP and CTRL, suggesting that these alterations are related to CF. Overall, AmiGo analysis highlighted six major pathways important for cell functions that seem to be impaired: metabolism, G protein process, inflammation and oxidative stress response, protein folding, proteolysis and structural proteins. Among them, glucose and fatty acid metabolic pathways could be impaired in CF with nine deregulated proteins. Our proteomic study provides a reproducible set of differentially expressed proteins in airway epithelial cells from CF patients and reveals many novel deregulated proteins that could lead to further studies aiming to clarify the involvement of such proteins in CF pathophysiology.

Loss-of-function mutations in RSPH1 cause primary ciliary dyskinesia with central-complex and radial-spoke defects.

Primary ciliary dyskinesia (PCD) is a rare autosomal-recessive respiratory disorder resulting from defects of motile cilia. Various axonemal ultrastructural phenotypes have been observed, including one with so-called central-complex (CC) defects, whose molecular basis remains unexplained in most cases. To identify genes involved in this phenotype, whose diagnosis can be particularly difficult to establish, we combined homozygosity mapping and whole-exome sequencing in a consanguineous individual with CC defects. This identified a nonsense mutation in RSPH1, a gene whose ortholog in Chlamydomonas reinhardtii encodes a radial-spoke (RS)-head protein and is mainly expressed in respiratory and testis cells. Subsequent analyses of RSPH1 identified biallelic mutations in 10 of 48 independent families affected by CC defects. These mutations include splicing defects, as demonstrated by the study of RSPH1 transcripts obtained from airway cells of affected individuals. Wild-type RSPH1 localizes within cilia of airway cells, but we were unable to detect it in an individual with RSPH1 loss-of-function mutations. High-speed-videomicroscopy analyses revealed the coexistence of different ciliary beating patterns-cilia with a normal beat frequency but abnormal motion alongside immotile cilia or cilia with a slowed beat frequency-in each individual. This study shows that this gene is mutated in 20.8% of individuals with CC defects, whose diagnosis could now be improved by molecular screening. RSPH1 mutations thus appear as a major etiology for this PCD phenotype, which in fact includes RS defects, thereby unveiling the importance of RSPH1 in the proper building of CCs and RSs in humans.

Primary ciliary dyskinesia presentation in 60 children according to ciliary ultrastructure.

Primary ciliary dyskinesia (PCD) is an inherited disease related to ciliary dysfunction, with heterogeneity in clinical presentation and in ciliary ultrastructural defect. Our study intended to determine if there are phenotypic differences in patients with PCD based on ciliary ultrastructural abnormality. In this retrospective study carried out among 60 children with a definitive diagnosis of PCD, we analyzed clinical, radiological, and functional features at diagnosis and at last recorded visit, according to cilia defect (absence of dynein arms: DAD group, n = 36; abnormalities of the central complex: CCA group, n = 24). Onset of respiratory symptoms occurred later in the CCA than in the DAD group (9.5 versus 0.5 months, p = 0.03). Situs inversus was only observed in the DAD group, while respiratory disease in siblings were more frequent in the CCA group (p = 0.003). At diagnosis, clinical presentation was more severe in the CCA group: frequency of respiratory tract infections (p = 0.008), rhinosinusitis (p = 0.02), otitis complications (p = 0.0001), bilateral bronchiectasis (p = 0.04), and number of hypoxemic patients (p = 0.03). Pulmonary function remained stable in both groups, but outcome was better in the CCA than in the DAD group: less antibiotic therapy and hypoxemic patients (p = 0.004). In conclusion, our results underlined the relationship between the severity of clinical presentation and the ultrastructural ciliary defect.

CFTR dysfunction induces vascular endothelial growth factor synthesis in airway epithelium.

Peribronchial angiogenesis may occur in cystic fibrosis and vascular endothelial growth factor (VEGF)-A regulates angiogenesis in airways. Peribronchial vascularity and VEGF-A expression were examined using immunocytochemistry and morphometric analysis in lung sections obtained in 10 cystic fibrosis patients at transplantation versus 10 control nonsmokers, and in two strains of Cftr-deficient mice versus wild-type littermates. Airway epithelial NCI-H292 cells and primary cultures of noncystic fibrosis human airway epithelial cells were treated with cystic fibrosis transmembrane conductance regulator (CFTR) inhibitors (CFTR-inh(172) or PPQ-102) or transfected with a CFTR small interfering (si)RNA with or without a selective epidermal growth factor receptor tyrosine kinase inhibitor. Concentrations of VEGF-A and phosphorylated epidermal growth factor receptor were measured by ELISA. Peribronchial vascularity was increased in cystic fibrosis patients, but not in Cftr-deficient mice. VEGF-A immunostaining was localised to airway epithelium and was increased in cystic fibrosis patients and in Cftr-deficient mice. In cultured airway epithelial cells, treatment with CFTR inhibitors or transfection with CFTR siRNA induced a twofold increase in VEGF-A production. CFTR inhibitors triggered epidermal growth factor receptor phosphorylation that was required for VEGF-A synthesis. Cystic fibrosis airways at transplantation showed increased peribronchial vascularity and epithelial VEGF-A expression. CFTR dysfunction triggered epithelial synthesis of VEGF-A, which may contribute to vascular remodelling.

Delineation of CCDC39/CCDC40 mutation spectrum and associated phenotypes in primary ciliary dyskinesia.

BACKGROUND: CCDC39 and CCDC40 genes have recently been implicated in primary ciliary dyskinesia (PCD) with inner dynein arm (IDA) defects and axonemal disorganisation; their contribution to the disease is, however, unknown. Aiming to delineate the CCDC39/CCDC40 mutation spectrum and associated phenotypes, this study screened a large cohort of patients with IDA defects, in whom clinical and ciliary phenotypes were accurately described. METHODS: All CCDC39 and CCDC40 exons and intronic boundaries were sequenced in 43 patients from 40 unrelated families. The study recorded and compared clinical features (sex, origin, consanguinity, laterality defects, ages at first symptoms and at phenotype evaluation, neonatal respiratory distress, airway infections, nasal polyposis, otitis media, bronchiectasis, infertility), ciliary beat frequency, and quantitative ultrastructural analyses of cilia and sperm flagella. RESULTS: Biallelic CCDC39 or CCDC40 mutations were identified in 30/34 (88.2%) unrelated families with IDA defects associated with axonemal disorganisation (22 and eight families, respectively). Fourteen of the 28 identified mutations are novel. No mutation was found in the six families with isolated IDA defects. Patients with identified mutations shared a similar phenotype, in terms of both clinical features and ciliary structure and function. The sperm flagellar ultrastructure, analysed in 4/7 infertile males, showed evidence of abnormalities similar to the ciliary ones. CONCLUSIONS: CCDC39 and CCDC40 mutations represent the major cause of PCD with IDA defects and axonemal disorganisation. Patients carrying CCDC39 or CCDC40 mutations are phenotypically indistinguishable. CCDC39 and CCDC40 analyses in selected patients ensure mutations are found with high probability, even if clinical or ciliary phenotypes cannot prioritise one analysis over the other.