Analyses of 1236 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 characterised 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, forced expiratory volume in 1 s (FEV1)) were collected from 19 countries using the European Reference Network's ERN-LUNG international PCD Registry. Genetic data were evaluated according to American College of Medical Genetics and Genomics guidelines. We assessed regional distribution of implicated genes and genetic variants as well as genotype correlations with laterality defects and FEV1. RESULTS: The study included 1236 individuals carrying 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%, range 47-100%) and laterality defects (mean 42%, range 28-69%) varied widely among 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). Median FEV1 z-scores were significantly lower in CCNO (-3.26), CCDC39 (-2.49) and CCDC40 (-2.96) variant groups, while the FEV1 z-score reductions were significantly milder in DNAH11 (-0.83) and ODAD1 (-0.85) variant groups compared to the whole PCD cohort. CONCLUSION: This unprecedented multinational dataset of DNA variants and information on their distribution across countries facilitates interpretation of the genetic epidemiology of PCD and indicates that the genetic variant can predict diagnostic and phenotypic features such as the course of lung function.

The genetic framework of primary ciliary dyskinesia assessed by soft computing analysis.

BACKGROUND: International guidelines disagree on how best to diagnose primary ciliary dyskinesia (PCD), not least because many tests rely on pattern recognition. We hypothesized that quantitative distribution of ciliary ultrastructural and motion abnormalities would detect most frequent PCD-causing groups of genes by soft computing analysis. METHODS: Archived data on transmission electron microscopy and high-speed video analysis from 212 PCD patients were re-examined to quantitate distribution of ultrastructural (10 parameters) and functional ciliary features (4 beat pattern and 2 frequency parameters). The correlation between ultrastructural and motion features was evaluated by blinded clustering analysis of the first two principal components, obtained from ultrastructural variables for each patient. Soft computing was applied to ultrastructure to predict ciliary beat frequency (CBF) and motion patterns by a regression model. Another model classified the patients into the five most frequent PCD-causing gene groups, from their ultrastructure, CBF and beat patterns. RESULTS: The patients were subdivided into six clusters with similar values to homologous ultrastructural phenotype, motion patterns, and CBF, except for clusters 1 and 4, attributable to normal ultrastructure. The regression model confirmed the ability to predict functional ciliary features from ultrastructural parameters. The genetic classification model identified most of the different groups of genes, starting from all quantitative parameters. CONCLUSIONS: Applying soft computing methodologies to PCD diagnostic tests optimizes their value by moving from pattern recognition to quantification. The approach may also be useful to evaluate atypical PCD, and novel genetic abnormalities of unclear disease-producing potential in the future.

Safety and efficacy of the epithelial sodium channel blocker idrevloride in people with primary ciliary dyskinesia (CLEAN-PCD): a multinational, phase 2, randomised, double-blind, placebo-controlled crossover trial.

BACKGROUND: Mucociliary clearance is dysfunctional in people with primary ciliary dyskinesia, resulting in the accumulation of dehydrated mucus in the airways that is difficult to clear. We undertook a study to assess the benefit on lung function of treatment with a nebulised epithelial sodium channel (ENaC) blocker, idrevloride, with or without hypertonic saline, in people with primary ciliary dyskinesia. METHODS: The CLEAN-PCD trial was a phase 2, randomised, double-blind, placebo-controlled crossover trial conducted at 32 tertiary adult and paediatric care centres and university hospitals in Canada, Denmark, Germany, Italy, the Netherlands, Poland, the UK, and the USA. People with a confirmed diagnosis of primary ciliary dyskinesia, aged 12 years or older, with a percentage of predicted FEV1 (ppFEV1) in the range of 40% to <90%, were randomly assigned in a 2:2:1:1 ratio (block size=6), stratified by ppFEV1 at screening, to one of four sequences: (1) idrevloride in hypertonic saline in treatment period 1 then hypertonic saline in treatment period 2; (2) hypertonic saline in treatment period 1 then idrevloride in hypertonic saline in treatment period 2; (3) idrevloride in treatment period 1 then placebo in treatment period 2; and (4) placebo in treatment period 1 then idrevloride in treatment period 2. The idrevloride dose was 85 µg and hypertonic saline was 4·2% NaCl. 3 mL of each study treatment was nebulised twice daily for 28 days in treatment periods 1 and 2; the two 28-day treatment periods were separated by a 28-day washout period. The primary endpoint was absolute change from baseline in ppFEV1 after 28 days. Safety assessments and reports of adverse events were made at clinic visits during each treatment period and by a follow-up telephone call 28 days after the last dose of study drug. Additionally, adverse events could be reported at a follow-up telephone call 3 days after the start of dosing and as they arose. Participants who received at least one dose of study drug were included in the safety analyses (safety set), and those who also had spirometry data were included in the efficacy analyses (full analysis set). The completed study is registered (EudraCT 2015-004917-26; ClinicalTrials.govNCT02871778). FINDINGS: Between Sep 14, 2016, and May 31, 2018, 216 patients were screened and 123 were randomly assigned to one of four crossover sequences. Across the two treatment periods, treatment with idrevloride in hypertonic saline was initiated in 80 patients and completed in 78 patients (all 78 had data available and were included in the analysis); hypertonic saline initiated in 81 patients and completed in 76 patients (75 had data available and were included in the analysis); idrevloride initiated in 37 patients and completed in 35 patients (34 had data available and were included in the analysis); and placebo initiated in 36 patients and completed in 34 patients (all 34 had data available and were included in the analysis). Greater absolute increases in ppFEV1 from baseline to 28 days of treatment were seen with idrevloride in hypertonic saline (least-squares mean absolute change from baseline 1·0 percentage points, 95% CI -0·4 to 2·4) than with hypertonic saline alone (least-squares mean absolute change from baseline of -0·5 percentage points, -2·0 to 0·9; difference 1·5 percentage points, 95% CI <0·1 to 3·0; p=0·044). There was no significant difference in ppFEV1 for the parallel comparison of idrevloride in hypertonic saline compared with placebo or the crossover comparison of idrevloride with placebo. Adverse events were similar across treatments (57 to 65% of patients). Cough occurred in a greater proportion of participants during treatments that contained idrevloride or hypertonic saline compared with placebo, and oropharyngeal pain occurred in a greater proportion of participants during idrevloride treatments than during treatment with hypertonic saline alone or placebo, whereas chest discomfort was more common during treatments that included hypertonic saline. INTERPRETATION: In this phase 2 crossover study, idrevloride in hypertonic saline was safe and associated with improved lung function over a 28-day period in people with primary ciliary dyskinesia compared with hypertonic saline alone. Larger, longer clinical studies are warranted to explore the potential benefits of idrevloride in combination with hypertonic saline in people with primary ciliary dyskinesia. FUNDING: Parion Sciences, under agreement with Vertex Pharmaceuticals.

The disease-specific clinical trial network for primary ciliary dyskinesia: PCD-CTN.

Primary ciliary dyskinesia (PCD) is a rare genetic disorder characterised by impaired mucociliary clearance leading to irreversible lung damage. In contrast to other rare lung diseases like cystic fibrosis (CF), there are only few clinical trials and limited evidence-based treatments. Management is mainly based on expert opinions and treatment is challenging due to a wide range of clinical manifestations and disease severity. To improve clinical and translational research and facilitate development of new treatments, the clinical trial network for PCD (PCD-CTN) was founded in 2020 under the framework of the European Reference Network (ERN)-LUNG PCD Core. Applications from European PCD sites interested in participating in the PCD-CTN were requested. Inclusion criteria consisted of patient numbers, membership of ERN-LUNG PCD Core, use of associated standards of care, experience in PCD and/or CF clinical research, resources to run clinical trials, good clinical practice (GCP) certifications and institutional support. So far, applications from 22 trial sites in 18 European countries have been approved, including >1400 adult and >1600 paediatric individuals with PCD. The PCD-CTN is headed by a coordinating centre and consists of a steering and executive committee, a data safety monitoring board and committees for protocol review, training and standardisation. A strong association with patient organisations and industrial companies are further cornerstones. All participating trial sites agreed on a code of conduct. As CTNs from other diseases have demonstrated successfully, this newly formed PCD-CTN operates to establish evidence-based treatments for this orphan disease and to bring new personalised treatment approaches to patients.

Longitudinal Nitric Oxide Levels and Infections by Ultrastructure and Genotype in Primary Ciliary Dyskinesia.

BACKGROUND: We hypothesized that differences in nasal nitric oxide (nNO) and fractional exhaled nitric oxide (Feno) relate to prognosis in primary ciliary dyskinesia (PCD). RESEARCH QUESTION: What is the relationship between baseline values and longitudinal evolution of nNO and Feno and ultrastructure, genotype, and respiratory infections in PCD? STUDY DESIGN AND METHODS: Prospective, longitudinal, single-center study in adults and children evaluated biannually for up to 10 years. We compared cross-sectional and longitudinal values of nNO and Feno in ultrastructural (inner dynein arm [IDA] and microtubular disorganization [MTD]) and genetic (CCDC39 and CCDC40) groups known to have worse pulmonary function with patients within the ultrastructural and genetic groups with a better prognosis. Linear mixed-effects models were used to evaluate longitudinal associations. RESULTS: One hundred forty-one patients with PCD underwent 1,014 visits. At enrollment, no differences were found in children in nNO or Feno between the IDA and MTD group and the other ultrastructural groups. In adults, nNO (P = .038) and Feno (P = .032) were significantly lower in the IDA and MTD group than in all other combined ultrastructural groups. Feno values were significantly lower in the CCDC39 and CCDC40 group than in the DNAH5 and DNAH11 combined genotype group (P = .033) and in all other genotypes (P = .032). The IDA and MTD group showed a significant decline in nNO with age (P < .01) compared with other ultrastructural groups who showed stable levels. The CCDC39 and CCDC40 group showed the steepest decline in nNO over time (P < .01) compared with all other genotypes. A higher nNO was associated with lower likelihood of any positive bacterial isolate from the lower respiratory tract (P = .008). Changes in Feno over time did not differ between structural groups or genotypes. INTERPRETATION: Lower nNO in patients with PCD with genetic and ultrastructural changes associated with greater lung function decline may be related to worse prognosis, but whether a low nNO is causal needs further study. If lower nNO directly results in a poorer prognosis, strategies augmenting upper airway nitric oxide production may be worth evaluating.

Prevention of recurrent respiratory infections : Inter-society Consensus.

Recurrent respiratory infections (RRIs) are a common clinical condition in children, in fact about 25% of children under 1 year and 6% of children during the first 6 years of life have RRIs. In most cases, infections occur with mild clinical manifestations and the frequency of episodes tends to decrease over time with a complete resolution by 12 years of age. However, RRIs significantly reduce child and family quality of life and lead to significant medical and social costs.Despite the importance of this condition, there is currently no agreed definition of the term RRIs in the literature, especially concerning the frequency and type of infectious episodes to be considered. The aim of this consensus document is to propose an updated definition and provide recommendations with the intent of guiding the physician in the complex process of diagnosis, management and prevention of RRIs.

COVID-19 Pandemic and Reduced Physical Activity: Is There an Impact on Healthy and Asthmatic Children?

Physical activity (PA) has been seen to improve asthma symptoms, lung function, and quality of life, as well as to reduce airway inflammation and bronchial responsiveness. As a consequence of the COVID-19 pandemic, the minimal amount of PA recommended by the World Health Organization-i.e., about 60 min/day of moderate-to-high intensity-is difficult to achieve for many children, particularly those living in urban areas. Short-term changes in PA because of the COVID-19 pandemic may become habitual, increasing the risk of adverse asthma outcomes in children. Indeed, prolonged home confinement during the COVID-19 pandemic reduces PA levels and increases sedentary behaviors, possibly impairing immune system function and increasing susceptibility to inflammatory diseases. However, there is limited evidence regarding the effects of lockdown due to COVID-19 on PA and sedentary behaviors in asthmatic children. Given that children stay longer indoors, indoor air pollution represents a major issue to consider during home confinement. This narrative review aims to summarize the available evidence about the impact of decreased PA and increased sedentary behaviors on children with asthma during the COVID-19 pandemic. In addition, strategies for supporting PA in children with asthma during the COVID-19 pandemic are suggested, also looking at the issue of indoor air quality.

Longitudinal Lung Volume Changes by Ultrastructure and Genotype in Primary Ciliary Dyskinesia.

Rationale: Genotype-phenotype relationships are emerging in primary ciliary dyskinesia (PCD), but little is known about lung volume changes over time. Objectives: To investigate the evolution of static lung volumes with ultrastructural defects, gene mutations, body mass index, and specific infections in PCD. Methods: Prospective, longitudinal, single-center study in children and adults evaluated twice yearly for up to 10 years. Linear mixed-effects models were used to assess associations between ciliary morphology, genetic mutations, and clinical features. Results: A total of 122 patients had 1,096 visits. At enrollment, almost all spirometric and, especially in adults, plethysmographic parameters were significantly worse in absent inner dynein arms (IDAs), central apparatus (CA) defects, and microtubular disorganization (MTD) (IDA/CA/MTD) compared with patients with normal electron microscopy (EM) results. The mean trend increase with time for residual volume (RV) was significantly higher in IDA/CA/MTD group compared with groups with outer dynein arm defect and normal EM results. The mean trend of RV/total lung capacity in the IDA/CA/MTD group was significantly worse than in all other groups. The steepest rise in lung volumes was in CCDC39 and CCDC40, whereas hyperinflation increased less in DNAH5 and DNAH11 groups. RV/total lung capacity showed a significantly steeper rise in patients with Pseudomonas aeruginosa compared with patients with other infections or patients without infection. Conclusions: Patients with IDA/CA/MTD defects or CCDC39 and CCDC40 mutations had the greatest increase in hyperinflation, whereas those with outer dynein arm defect and normal EM results or DNAH11 and DNAH5 mutations had less severe changes. We have robustly confirmed the worse prognosis for some genetic and ultrastructural defects, which association hitherto rested solely on spirometry.

Impact of a supervised training course on spirometry competency for primary care pediatricians.

OBJECTIVE: Spirometry is the most commonly performed lung function test, and performance, adherence to acceptability and repeatability criteria, and accurate interpretation of results help optimize the test's usefulness. This study aimed to measure the effects of spirometry training courses supported by the Italian Pediatric Respiratory Society (IPRS) on primary care pediatricians' (PCP) knowledge of spirometry test quality, ability to interpret results, and overall degree of satisfaction with the course. METHODS: Of the six face-to-face courses, four lasted two days and two lasted one day: mean duration of theoretical lessons was five and four hours respectively; and practical sessions lasted eight and six hours, respectively. At the end of each course, participants took a learning test consisting of evaluating six flow-volume curves. Degree of satisfaction was assessed by asking participants to rank the relevance, quality, and usefulness of the course. RESULTS: 261 PCPs were involved, with most (67.43%) taking two-day courses. Nearly all participants correctly identified normal and restrictive patterns. Intrathoracic large-airway obstruction was the pattern most difficult to identify correctly (70.5% overall), whereas > 80% of the participants correctly classified artifacts, obstructive-restrictive, and obstructive patterns. Participants in longer courses reported significantly higher values on the learning score. The overall degree of satisfaction average ranged between "good" and "excellent". CONCLUSIONS: This pilot study showed the greater impact of two-day courses than one-day courses for training PCPs to properly interpret spirometry, confirming that a practical module lasting at least six hours is sufficient to deliver adequate training on spirometry for healthcare professionals.

Sweat chloride assay by inductively coupled plasma mass spectrometry: a confirmation test for cystic fibrosis diagnosis.

The current guidelines for sweat chloride analysis identify the procedures for sweat collection, but not for chloride assay, which is usually performed by methods originally not aiming at the low concentrations of chloride found in sweat. To overcome this limitation, we set up, characterized, and adopted an original inductively coupled plasma mass spectrometry (ICP-MS) method for sweat chloride determination, which was designed for its easy use in a clinical laboratory. The method was linear in the range 8.5E-3 to 272.0E-3 mM, precision exhibited a relative standard deviation < 6%, and accuracy was in the range 99.7-103.8%. Limit of blank, limit of detection, and limit of quantitation were 2.1 mM, 3.2 mM, and 7.0 mM, respectively, which correspond to real concentrations injected into the mass spectrometer of 3.9E-3 mM for LOD and 8.5E-3 mM for LOQ. At first, the method was tested on 50 healthy volunteers who exhibited a mean chloride concentration of 15.7 mM (25-75th percentile 10.1-19.3 mM, range 2.8-37.4 mM); then, it was used to investigate two patients with suspected cystic fibrosis, who exhibited sweat chloride values of 65.6 mM and 81.2 mM, respectively. Moreover, the method was cross-validated by assaying 50 samples with chloride concentration values in the range 10-131 mM, by both ICP-MS and coulometric titration, which is the technology officially used in Tuscany for cystic fibrosis newborn screening. The reference analytical performances and the relatively low cost of ICP-MS, accompanied by the advantageous cost of a single sweat chloride assay, make this technology the best candidate to provide a top reference method for the quantification of chloride in sweat. The method that we propose was optimized and validated for sweat samples ≥ 75 mg, which is the minimum amount requested by the international protocols. However, the method sensitivity and, in addition, the possibility to reduce the sample dilution factor, make possible the quantification of chloride even in samples weighting < 75 mg that are discarded according to the current guidelines. Graphical abstract.

Italian pediatric respiratory society recommendations on pediatric pulmonary function testing during COVID-19 pandemic.

BACKGROUND: Effective prevention and control strategies are mandatory to prevent SARS-CoV-2 infection. MAIN TEXT: The Italian Pediatric Respiratory Society promotes a series of new recommendations that should be followed in pulmonary function testing laboratories during the COVID-19 pandemic. CONCLUSION: Pulmonary function testing should be performed in children with chronic lung disease only if it is needed to guide management and limited to the necessary tests, namely spirometry. When performed, strict infection control measures should be followed due to the potential risk of transmitting SARS-CoV-2.

Targeting Pseudomonas aeruginosa in the Sputum of Primary Ciliary Dyskinesia Patients with a Combinatorial Strategy Having Antibacterial and Anti-Virulence Potential.

In primary ciliary dyskinesia (PCD) patients, Pseudomonas aeruginosa is a major opportunistic pathogen, frequently involved in chronic infections of the lower airways. Infections by this bacterial species correlates with a worsening clinical prognosis and recalcitrance to currently available therapeutics. The antimicrobial peptide, lin-SB056-1, in combination with the cation chelator ethylenediaminetetraacetic acid (EDTA), was previously demonstrated to be bactericidal against P. aeruginosa in an artificial sputum medium. The purpose of this study was to validate the anti-P. aeruginosa activity of such a combination in PCD sputum and to evaluate the in vitro anti-virulence effects of EDTA. In combination with EDTA, lin-SB056-1 was able to significantly reduce the load of endogenous P. aeruginosa ex vivo in the sputum of PCD patients. In addition, EDTA markedly reduced the production of relevant bacterial virulence factors (e.g., pyocyanin, proteases, LasA) in vitro by two representative mucoid strains of P. aeruginosa isolated from the sputum of PCD patients. These results indicate that the lin-SB056-1/EDTA combination may exert a dual antimicrobial and anti-virulence action against P. aeruginosa, suggesting a therapeutic potential against chronic airway infections sustained by this bacterium.

Does lung microbiome play a causal or casual role in asthma?

Asthma is the most common chronic disease in childhood. The pathogenesis of asthma is multifactorial and is thought to include environmental factors interacting with genetics during pregnancy and in the first years of life. In the last decades, a possible role of gut microbiota in allergic disease pathogenesis has been demonstrated. Next generation sequencing techniques have allowed the identification of a distinct microbiome in the healthy lungs. The lung microbiome is characterized by the prevalence of bacteria belonging to the phylum Bacteroidetes (mostly Prevotella and Veilonella spp) in healthy subjects and to the phylum Proteobacteria in asthmatics (mostly Haemophilus, Moraxella, and Neisseria spp). In asthma, as well as in other diseases, the lung microbiome composition changes due to a disruption of the delicate balance between immigration and elimination of bacteria. The lung microbiome can interact with the immune system, thus influencing inflammation. Early infections with viruses, such as respiratory syncytial virus, may alter lung microbiome composition favoring the emergence of Proteobacteria, a phylum which is also linked to severity of asthma and bronchial hyperreactivity. Lastly, antibiotics may alter the gut and lung microbiota and potentially disturb the relationship between microbiota and host. Therefore, antibiotics should be prescribed with increasing awareness of their potential harmful effect on the microbiota in young children with and without asthma. The potential effects of probiotics and prebiotics on lung microbiome are unknown.

The Virome and Its Major Component, Anellovirus, a Convoluted System Molding Human Immune Defenses and Possibly Affecting the Development of Asthma and Respiratory Diseases in Childhood.

The microbiome, a thriving and complex microbial community colonizing the human body, has a broad impact on human health. Colonization is a continuous process that starts very early in life and occurs thanks to shrewd strategies microbes have evolved to tackle a convoluted array of anatomical, physiological, and functional barriers of the human body. Cumulative evidence shows that viruses are part of the microbiome. This part, called virome, has a dynamic composition that reflects what we eat, how and where we live, what we do, our genetic background, and other unpredictable variables. Thus, the virome plays a chief role in shaping innate and adaptive host immune defenses. Imbalance of normal microbial flora is thought to trigger or exacerbate many acute and chronic disorders. A compelling example can be found in the respiratory apparatus, where early-life viral infections are major determinants for the development of allergic diseases, like asthma, and other non-transmissible diseases. In this review, we focus on the virome and, particularly, on Anelloviridae, a recently discovered virus family. Anelloviruses are major components of the virome, present in most, if not all, human beings, where they are acquired early in life and replicate persistently without causing apparent disease. We will discuss how modulation of innate and adaptive immune systems by Anelloviruses can influence the development of respiratory diseases in childhood and provide evidence for the use of Anelloviruses as useful and practical molecular markers to monitor inflammatory processes and immune system competence.

Olfactory dysfunction is worse in primary ciliary dyskinesia compared with other causes of chronic sinusitis in children.

Cilia have multiple functions including olfaction. We hypothesised that olfactory function could be impaired in primary ciliary dyskinesia (PCD). Olfaction, nasal nitric oxide (nNO) and sinus CT were assessed in patients with PCD and non-PCD sinus disease, and healthy controls (no CT scan). PCD and non-PCD patients had similar severity of sinus disease. Despite this, defective olfaction was more common in patients with PCD (P<0.0001) and more severe in patients with PCD with major Transmission Electron Microscopy (TEM) abnormalities. Only in classical PCD did olfaction inversely correlate with sinusitis and nNO. We speculate that defective olfaction in PCD is primary in nature.

Matrix metalloproteinases and airway remodeling and function in primary ciliary dyskinesia.

BACKGROUND: The balance between matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) is important in the regulation of airway damage. OBJECTIVE: To evaluate whether they are important in the pathophysiology of primary and secondary ciliary dyskinesia (PCD, SCD). METHODS: We measured sputum bacteriology, lung CT changes, MMPs, TIMPs and lung function in 86 patients (51 PCD, 35 SCD) in a cross-sectional study; the 10 controls studied did not have HRCT or sputum cultures. MMPs, TIMPs and lung function were evaluated longitudinally for up to one year in 38 PCD patients. RESULTS: At baseline, there were no differences in MMPs, TIMPs and MMPs/TIMPs, between PCD and SCD but lower levels were found in controls. There was an association between poorer lung function with increasing levels of MMPs in PCD, while in SCD only MMP-9/TIMP-1 values correlated with FRC z-scores. Levels of MMPs and TIMPs significantly correlated with severity HRCT changes. Longitudinally, there were significant correlations between slope of changes in spirometric parameters and slope of change in sputum MMPs in PCD patients. CONCLUSIONS: In conclusion, we report for the first time that increased MMPs are associated with worse airway damage in PCD and SCD, and thus are potential therapeutic targets.

Gene editing of DNAH11 restores normal cilia motility in primary ciliary dyskinesia.

BACKGROUND: Primary ciliary dyskinesia (PCD) is a rare autosomal recessive genetic disorder characterised by dysfunction of motile cilia. Ciliary dysmotility causes poor mucociliary clearance and leads to impairment of pulmonary function and severe respiratory infections. PCD has no specific therapy. With the aim to permanently restore gene function and normalise ciliary motility, we used gene editing to replace mutated with wild-type sequence in defective cells. METHODS: The target gene was dynein heavy chain 11 (DNAH11), an essential component of ciliary structure. Airway ciliated cells were collected from two patients with PCD with DNAH11 nonsense mutations and altered ciliary beating and pattern. Repair of the genetic defect was performed ex vivo by site-specific recombination using transcription activator-like effector nucleases (TALENs). RESULTS: In an epithelial cell line engineered to contain the DNAH11 target site, TALENs cleaved over 80% of the mutated DNAH11 sequence and replaced the mutated sequence with wild-type sequence in about 50% of cells. In airway ciliated cells of patients with PCD, site-specific recombination and normalisation of ciliary beating and pattern occurred in 33% and 29% of cells, respectively. CONCLUSION: This study demonstrates that gene editing can rescue ciliary beating ex vivo, opening up new avenues for treating PCD.

Mannose-binding lectin 2 gene polymorphism and lung damage in primary ciliary dyskinesia.

BACKGROUND: Mannose-binding lectin (MBL) plays an important role in innate immunity and has been reported to be associated with the age-related decline in lung function in cystic fibrosis. HYPOTHESIS: MBL polymorphisms are associated with lung function decline in Primary Ciliary Dyskinesia (PCD). METHODS: We performed sputum microbiology, spirometry pre- and post-administration of salbutamol, ciliary motion analysis, ultrastructural assessment of cilia, ciliogenesis in culture, and chest high resolution computed tomography in children with a clinical history of respiratory tract infections and/or presence of bronchiectasis suggestive of PCD or secondary ciliary dyskinesia (SCD). All subjects were evaluated for single nucleotide polymorphisms in the gene encoding MBL-2. RESULTS: The diagnosis of PCD was established in 45 subjects, while in the remaining 53 the diagnosis was SCD. A significant bronchodilator response was observed only in PCD associated with the MBL2-3 genotype, which is known to be associated with low/undetectable MBL serum levels. Also, bronchiectasis severity was significantly greater in subjects with MBL2-3 in both PCD and SCD. No other association was found between MBL genotypes and clinical findings. CONCLUSIONS: MBL plays a relatively minor role as a disease modifier in PCD. A similar finding in SCD supports the likely significance of this result.