Rationale: Bronchiectasis is a pathological dilatation of the bronchi in the respiratory airways associated with environmental or genetic causes (e.g., cystic fibrosis, primary ciliary dyskinesia, and primary immunodeficiency disorders), but most cases remain idiopathic. Objectives: To identify novel genetic defects in unsolved cases of bronchiectasis presenting with severe rhinosinusitis, nasal polyposis, and pulmonary Pseudomonas aeruginosa infection. Methods: DNA was analyzed by next-generation or targeted Sanger sequencing. RNA was analyzed by quantitative PCR and single-cell RNA sequencing. Patient-derived cells, cell cultures, and secretions (mucus, saliva, seminal fluid) were analyzed by Western blotting and immunofluorescence microscopy, and mucociliary activity was measured. Blood serum was analyzed by electrochemiluminescence immunoassay. Protein structure and proteomic analyses were used to assess the impact of a disease-causing founder variant. Measurements and Main Results: We identified biallelic pathogenic variants in WAP four-disulfide core domain 2 (WFDC2) in 11 individuals from 10 unrelated families originating from the United States, Europe, Asia, and Africa. Expression of WFDC2 was detected predominantly in secretory cells of control airway epithelium and also in submucosal glands. We demonstrate that WFDC2 is below the limit of detection in blood serum and hardly detectable in samples of saliva, seminal fluid, and airway surface liquid from WFDC2-deficient individuals. Computer simulations and deglycosylation assays indicate that the disease-causing founder variant p.Cys49Arg structurally hampers glycosylation and, thus, secretion of mature WFDC2. Conclusions: WFDC2 dysfunction defines a novel molecular etiology of bronchiectasis characterized by the deficiency of a secreted component of the airways. A commercially available blood test combined with genetic testing allows its diagnosis.

Background Primary ciliary dyskinesia is a genetic disorder caused by aberrant motile cilia function that results in defective ciliary airway clearance and subsequently leads to recurrent airway infections and bronchiectasis. We aimed to determine: how many functional multiciliated airway cells are sufficient to maintain ciliary airway clearance? Methods To answer this question we exploited the molecular defects of the X-linked recessive primary ciliary dyskinesia variant caused by pathogenic variants in DNAAF6 (PIH1D3), characterised by immotile cilia in affected males. We carefully analysed the clinical phenotype and molecular defect (using immunofluorescence and transmission electron microscopy) and performed in vitro studies ( particle tracking in air-liquid interface cultures) and in vivo studies (radiolabelled tracer studies) to assess ciliary clearance of respiratory cells from female individuals with heterozygous and male individuals with hemizygous pathogenic DNAAF6 variants. Results Primary ciliary dyskinesia male individuals with hemizygous pathogenic DNAAF6 variants displayed exclusively immotile cilia, absence of ciliary clearance and severe primary ciliary dyskinesia symptoms. Owing to random or skewed X-chromosome inactivation in six female carriers with heterozygous pathogenic DNAAF6 variants, 54.3 +/- 10% (range 38-70%) of multiciliated cells were defective. Nevertheless, in vitro and in vivo assessment of the ciliary airway clearance was normal or slightly abnormal. Consistently, heterozygous female individuals showed no or only mild respiratory symptoms. Conclusions Our findings indicate that having 30-62% of multiciliated respiratory cells functioning can generate either normal or slightly reduced ciliary clearance. Because heterozygous female carriers displayed either no or subtle respiratory symptoms, complete correction of 30% of cells by precision medicine could improve ciliary airway clearance in individuals with primary ciliary dyskinesia, as well as clinical symptoms.

BackgroundPrimary ciliary dyskinesia is a rare genetic disorder caused by insufficient mucociliary clearance leading to chronic airway infections. The diagnostic guideline of the European Respiratory Society primarily recommends an evaluation of the clinical history (e.g.by the PICADAR prediction tool), nasal nitric oxide production rate measurements, high-speed videomicroscopy analysis of ciliary beating and an assessment of ciliary axonemesviatransmission electron microscopy. Genetic testing can be implemented as a last step.AimsIn this study, we aimed to characterise primary ciliary dyskinesia with a defective C1d projection of the ciliary central apparatus and we evaluated the applicability of the European Respiratory Society diagnostic guideline to this primary ciliary dyskinesia type.MethodsUsing a high-throughput sequencing approach of genes encoding C1d components, we identified pathogenic variants in the novel primary ciliary dyskinesia genesCFAP46andCFAP54, and the known primary ciliary dyskinesia geneCFAP221. To fully assess this primary ciliary dyskinesia type, we also analysed individuals with pathogenic variants inCFAP74.ResultsCareful evaluation revealed that C1d-defective primary ciliary dyskinesia is associated with normal situs composition, normal nasal nitric oxide production rates, normal ciliary ultrastructure by transmission electron microscopy and normal ciliary beating by high-speed videomicroscopy analysis. Despite chronic respiratory disease, PICADAR does not reliably detect this primary ciliary dyskinesia type. However, we could show byin vitrociliary transport assays that affected individuals exhibit insufficient ciliary clearance.ConclusionsOverall, this study extends the spectrum of primary ciliary dyskinesia genes and highlights that individuals with C1d-defective primary ciliary dyskinesia elude diagnosis when using the current diagnostic algorithm. To enable diagnosis, genetic testing should be prioritised in future diagnostic guidelines.

Hintergrund Der Begriff Primäre Ciliäre Dyskinesie (PCD) umfasst eine Gruppe seltener, genetisch bedingter Erkrankungen, die durch eine Dysfunktion motiler Zilien charakterisiert ist. Sie ist klinisch und genetisch heterogen, aktuell sind mehr als 50 Gene bekannt, die mit einer PCD assoziiert sind. Typischerweise kommt es durch eine gestörte mukoziliäre Reinigung zu einer Sekretretention und zu rezidivierenden Infektionen der oberen und unteren Atemwege, die eine dauerhafte Lungenschädigung zur Folge haben.

Disease-causing bi-allelic DNA variants in CCDC39 and CCDC40 are frequent causes of the hereditary disorder of primary ciliary dyskinesia (PCD). The encoded proteins form a molecular ruler complex, crucial for maintaining the 96 nm repeat units along the ciliary axonemes. Defects of those proteins cause a stiff, rapid, and flickery ciliary beating pattern, recurrent respiratory infections, axonemal disorganization, and abnormal assembly of GAS8, CCDC39, and DNALI1. We performed molecular characterization of the defects in the 96 nm axonemal ruler due to disease-causing variants in CCDC39 and CCDC40 and analyzed the effect on additional axonemal components. We identified a cohort of 51 individuals with disease-causing variants in CCDC39 and CCDC40 via next-generation sequencing techniques and demonstrated that the IDA heavy chains DNAH1, DNAH6, and DNAH7 are conspicuously absent within the respiratory ciliary axonemes by immunofluorescence analyses. Hence, we show for the first time that the centrin2 (CETN2) containing IDAs are also affected. These findings underscore the crucial role of CCDC39 and CCDC40 in the assembly and function of IDAs in human respiratory cilia. Thus, our data improve the diagnostics of axonemal ruler defects by further characterizing the associated molecular IDA defects.

Primary Ciliary Dyskinesia (PCD) is a rare genetic disorder affecting the function of motile cilia in several organ systems. In PCD, male infertility is caused by defective sperm flagella composition or deficient motile cilia function in the efferent ducts of the male reproductive system. Different PCD-associated genes encoding axonemal components involved in the regulation of ciliary and flagellar beating are also reported to cause infertility due to multiple morphological abnormalities of the sperm flagella (MMAF). Here, we performed genetic testing by next generation sequencing techniques, PCD diagnostics including immunofluorescence-, transmission electron-, and high-speed video microscopy on sperm flagella and andrological work up including semen analyses. We identified ten infertile male individuals with pathogenic variants in CCDC39 (one) and CCDC40 (two) encoding ruler proteins, RSPH1 (two) and RSPH9 (one) encoding radial spoke head proteins, and HYDIN (two) and SPEF2 (two) encoding CP-associated proteins, respectively. We demonstrate for the first time that pathogenic variants in RSPH1 and RSPH9 cause male infertility due to sperm cell dysmotility and abnormal flagellar RSPH1 and RSPH9 composition. We also provide novel evidence for MMAF in HYDIN- and RSPH1-mutant individuals. We show absence or severe reduction of CCDC39 and SPEF2 in sperm flagella of CCDC39- and CCDC40-mutant individuals and HYDIN- and SPEF2-mutant individuals, respectively. Thereby, we reveal interactions between CCDC39 and CCDC40 as well as HYDIN and SPEF2 in sperm flagella. Our findings demonstrate that immunofluorescence microscopy in sperm cells is a valuable tool to identify flagellar defects related to the axonemal ruler, radial spoke head and the central pair apparatus, thus aiding the diagnosis of male infertility. This is of particular importance to classify the pathogenicity of genetic defects, especially in cases of missense variants of unknown significance, or to interpret HYDIN variants that are confounded by the presence of the almost identical pseudogene HYDIN2.

Introduction Primary Ciliary Dyskinesia (PCD) is characterized by impaired mucociliary clearance and progressive airway damage due to dysfunctional motile cilia in the airways and more than 50 genes are associated with PCD. Clinical care is symptomatic and no causal treatment is available. We studied the feasibility of mRNA therapy to correct the molecular defect in human respiratory epithelial cells (hRECs) from PCD individuals with bi-allelic disease-causing variants in Coiled-Coil Domain Containing 40 (CCDC40). In CCDC40 deficient cells, ciliary beating is hampered due to microtubular disorganization, and loss of CCDC40 binding partners Coiled-Coil Domain Containing 39 (CCDC39), Growth Arrest Specific 8 (GAS8) and Dynein Axonemal Light Intermediate Chain 1 (DNALI1). Here, we provide proof-of-concept (PoC) that Stabilized Non-Immunogenic mRNA (SNIM RNA) rescues ciliary defects.

Hintergrund Die Primäre Ciliäre Dyskinesie (PCD) ist eine genetisch und klinisch heterogene Erkrankung, die durch eine Dysfunktion motiler Zilien gekennzeichnet ist. Die Diagnose der PCD basiert auf verschiedenen Tests, inklusive der Messung von nasalem Stickstoffmonoxid (nNO), Hochgeschwindigkeits-Videomikroskopie (HSVM), Transmissionselektronenmikroskopie (TEM), Immunfluoreszenzmikroskopie (IF) und genetischer Analyse.

Since 2000, caffeine is used as citrate salt of caffeine for treatment of apnea of prematurity. However, it remained elusive if caffeine citrate has a direct function in the respiratory epithelium, potentially as modulator for ciliary beat frequency (CBF) regulation in human respiratory cells (hRECs). Here, we studied the mechanism of action of caffeine citrate on the respiratory epithelium on hRECs derived from healthy donors and individuals with cystic fibrosis (CF) cultured under air-liquid-interface (ALI) conditions. CBF analyses of hRECs were performed by high-speed video microscopy. In the presence of caffeine, hRECs show a significant increase in CBF over ciliary base frequency (CBFb), demonstrating a direct influence of caffeine on ciliary function. We also demonstrate a specific action of caffeine at ryanodine receptor (RYR) channels, mediating calcium efflux from intracellular stores to increase CBF. We describe a novel pharmacological effect of caffeine citrate - direct increase of CBF of primary hRECs cultured at ALI. Caffeine citrate has certain advantages over other methylxanthines: it was shown to have stable plasma concentration, longer half-life, and is better tolerated than e.g. theophylline. We conclude that caffeine citrate therapy might be suitable strategy to treat respiratory diseases with compromised mucociliary clearance.

Background and objectives: Primary ciliary dyskinesia (PCD, ORPHA:244) is a group of rare genetic disorders characterized by dysfunction of motile cilia. It is phenotypically and genetically heterogeneous, with more than 50 genes involved. Thanks to genetic, clinical, and functional characterization, immense progress has been made in the understanding and diagnosis of PCD. Nevertheless, it is underdiagnosed due to the heterogeneous phenotype and complexity of diagnosis. This review aims to help clinicians navigate this heterogeneous group of diseases. Here, we describe the broad spectrum of phenotypes associated with PCD and address pitfalls and difficult-to-interpret findings to avoid misinterpretation.Method: Review of literatureConclusion: PCD diagnosis is complex and requires integration of history, clinical picture, imaging, functional and structural analysis of motile cilia and, if available, genetic analysis to make a definitive diagnosis. It is critical that we continue to expand our knowledge of this group of rare disorders to improve the identification of PCD patients and to develop evidence-based therapeutic approaches.(c) 2023 Elsevier Masson SAS. All rights reserved.

Einführung Die Primäre Ciliäre Dyskinesie (PCD) ist eine heterogene Multisystemerkrankung, bei welcher es durch eine Dysfunktion motiler Zilien u.a. zu einer destruktiven Lungenerkrankung kommt. Der diagnostische Algorithmus der American Thoracic (ATS) und der European Respiratory Society (ERS) beinhaltet die Messung von nasalem Stickstoffmonoxid (nNO), die Transmissionselektronenmikroskopie, die Evaluation des Zilienschlages und die genetische Analyse, um eine PCD zu diagnostizieren.

Rationale: Primary ciliary dyskinesia (PCD) is a heterogeneous, multisystem disorder characterized by defective ciliary beating. Diagnostic guidelines of the American Thoracic Society and European Respiratory Society recommend measurement of nasal nitric oxide (nNO) for PCD diagnosis. Several studies demonstrated low nNO production rates in PCD individuals, but underlying causes remain elusive. Objectives: To determine nNO production rates in a well-characterized PCD cohort, including subgroup analyses with regard to ultrastructural and ciliary beating phenotypes. Methods: This study included 301 individuals assessed according to European Respiratory Society guidelines. Diagnostic cutoffs for nNO production rates for this study cohort and subgroups with normal and abnormal ultrastructure were determined. Diagnostic accuracy was also tested for the widely used 77 nl/min cutoff in this study cohort. The relationship between nNO production rates and ciliary beat frequencies (CBFs) was evaluated. Results: The study cohort comprised 180 individuals with definite PCD diagnosis, including 160 individuals with genetic diagnosis, 16 individuals with probable PCD diagnosis, and 105 disease controls. The 77 nl/min nNO cutoff showed a test sensitivity of 0.92 and specificity of 0.86. Test sensitivity was lower (0.85) in the subgroup of 47 PCD individuals with normal ultrastructure compared with 133 PCD individuals with abnormal ultrastructure (0.95). The optimal diagnostic cutoff for the nNO production rate for the whole study cohort was 69.8 nl/min (sensitivity, 0.92; specificity, 0.89); however, it was 107.8 nl/min (sensitivity, 0.89; specificity, 0.78) for the subgroup of PCD with normal ultrastructure. PCD individuals with normal ultrastructure compared with abnormal ultrastructure showed higher ciliary motility. Consistently, PCD individuals with higher CBFs showed higher nNO production rates. In addition, laterality defects occurred less frequently in PCD with normal ultrastructure. Conclusions: Measurements of nNO below the widely used 77 nl/min cutoff are less sensitive in detecting PCD individuals with normal ultrastructure. Our findings indicate that higher nNO production in this subgroup with a higher cutoff for the nNO production rate (107.8 nl/min) and higher residual ciliary motility is dependent on the underlying molecular PCD defect. Higher nNO production rates, higher residual CBFs, and the lower prevalence of laterality defects hamper diagnosis of PCD with normal ultrastructure. Adjusting the cutoff of nNO production rate to 107.8 nl/min might promote diagnosing PCD with normal ultrastructure.

Introduction: Primary ciliary dyskinesia (PCD) is a heterogeneous, multisystem disorder characterized by dysfunctional motile cilia. Diagnostic approaches of the American Thoracic (ATS) and European Respiratory Society (ERS) include nasal nitric oxide (nNO) measurement, transmission electron microscopy, evaluation of ciliary beating and genetic analyses for PCD diagnosis. Methods: All included subjects were evaluated according to ERS diagnostic guidelines. PCD individuals were categorized in subgroups with normal and abnormal ciliary ultrastructure. Diagnostic cutoffs for nNO-production rates as well as ciliary beat frequencies (CBFs) and laterality status were determined. Results: The study cohort comprised 180 PCD individuals (160 with genetic diagnosis) and 105 disease controls. The optimal diagnostic cutoff for the nNO-production rate for the whole PCD cohort was 69.8 nl/min (sensitivity 0.92, specificity 0.89), while it was 107.8 nl/min (sensitivity 0.89, specificity 0.78) for the subgroup of PCD with normal ultrastructure. This subgroup also showed higher ciliary motility and less laterality defects. Conclusions: Higher nNO-production rates, higher residual ciliary motility and lower prevalence of laterality defects make diagnosis of PCD with normal ultrastructure difficult. In distinct cases, only genetic analyses lead to PCD diagnosis. A frequent use of genetic testing and adjusting the cutoff for the nNO-production rate to 107.8 nl/min will promote diagnosing PCD with normal ultrastructure. We thank the PCD-affected families and the German support group. This work was supported by grants from the “Deutsche Forschungsgemeinschaft” (DFG) (e.g. CRU326 (RA3522/1-1; OM6/11) and "IZKF" Münster.

Motile cilia line the efferent ducts of the mammalian male reproductive tract. Several recent mouse studies have demonstrated that a reduced generation of multiple motile cilia in efferent ducts is associated with obstructive oligozoospermia and fertility issues. However, the sole impact of efferent duct cilia dysmotility on male infertility has not been studied so far either in mice or human. Using video microscopy, histological- and ultrastructural analyses, we examined male reproductive tracts of mice deficient for the axonemal motor protein DNAH5: this defect exclusively disrupts the outer dynein arm (ODA) composition of motile cilia but not the ODA composition and motility of sperm flagella. These mice have immotile efferent duct cilia that lack ODAs, which are essential for ciliary beat generation. Furthermore, they show accumulation of sperm in the efferent duct. Notably, the ultrastructure and motility of sperm from these males are unaffected. Likewise, human individuals with loss-of-function DNAH5 mutations present with reduced sperm count in the ejaculate (oligozoospermia) and dilatations of the epididymal head but normal sperm motility, similar to DNAH5 deficient mice. The findings of this translational study demonstrate, in both mice and men, that efferent duct ciliary motility is important for male reproductive fitness and uncovers a novel pathomechanism distinct from primary defects of sperm motility (asthenozoospermia). If future work can identify environmental factors or defects in genes other than DNAH5 that cause efferent duct cilia dysmotility, this will help unravel other causes of oligozoospermia and may influence future practices in genetic and fertility counseling as well as ART.

Primary ciliary dyskinesia (PCD) is a heterogeneous group of disorders affecting mainly the respiratory system, the male and female reproductive system as well as the establishment of left-right body asymmetry. PCD is diagnosed by nasal nitric oxide measurements, analysis of ciliary beating, transmission electron microscopy and genetic testing. To date, more than 40 genes have been published to cause PCD when mutated. In our center, we aim to decipher the genetic causes of PCD in human individuals. By using Sanger sequencing, panel and whole exome sequencing, we here report the genetic results in 1072 PCD-suspected individuals based on clinical symptoms out of a cohort of 4005 PCD-suspected individuals recruited mainly in Europe. 671 individuals carried mutations in PCD-associated genes. The most frequent mutated gene is DNAH5 with 151 individuals, followed by DNAH11 and CCDC40 with 62 individuals each, CCNO with 42 individuals and DNAI1 with 40 individuals. The least mutated genes are RSPH3, FOXJ1, CFAP53 and ENKUR (2 each) and SPEF2, DNAJB13 and C21orf59 with 1 individual each. Interestingly, we identified hotspot and/or founder mutations in 20 genes: DNAH5, DNAI1, CCDC39, CCDC40, C11orf70, DNAAF4, LRRC6, ZMYND10, SPAG1, CCDC114, CCDC151, ARMC4, CCDC103, CCDC164, CCNO, MCIDAS, RSPH1, RSPH9, RSPH4A and HYDIN. Screening for these mutations first will ease and reduce the cost of the genetic diagnosis of PCD. To confirm the identified loss-of-function mutations, we perform immunofluorescence analysis (IF) with antibodies targeting the protein encoded by the mutated gene and prove its absence from the ciliary axoneme. Genetic combined with IF analysis is a powerful combination to diagnose PCD in the majority of cases.