Recessively Inherited Deficiency of Secreted WFDC2 (HE4) Causes Nasal Polyposis and Bronchiectasis.

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.

Atmospheric solids analysis probe mass spectrometry: An easy bolt-on for the synthetic undergraduate teaching laboratory.

RATIONALE: High costs and student numbers can often hinder implementation of mass spectrometry (MS) in the undergraduate teaching laboratory, often with technicians running samples on students' behalf, and the implementation of MS only in discrete or isolated experiments. This study explores the use of atmospheric solids analysis probe MS (ASAP-MS) as a relatively low-cost, benchtop instrument, and its potential for application as a 'bolt-on' to existing undergraduate organic chemistry experiments. METHODS: Thirteen products synthesised in undergraduate laboratory experiments were analysed by ASAP-MS, along with their starting materials. Analysis was carried out with a Waters RADIAN ASAP mass spectrometer, at four different cone voltages simultaneously to provide fragmentation information. RESULTS: Out of the 13 undergraduate experiments, ASAP-MS was shown to be complementary in 11 of these, either through simple analysis of the precursor ion or by a more complex analysis of the fragments. CONCLUSIONS: ASAP-MS provided spectra that both complement and enhance intended learning outcomes in existing organic chemistry experiments, showing its versatility as a bolt-on technique. Moving forward, ASAP-MS will be integrated into the University of Surrey's undergraduate teaching laboratory.

Selectivity of Explosives Analysis with Ambient Ionization Single Quadrupole Mass Spectrometry: Implications for Trace Detection.

Ambient ionization (AI) is a rapidly growing field in mass spectrometry (MS). It allows for the direct analysis of samples without any sample preparation, making it a promising technique for the detection of explosives. Previous studies have shown that AI can be used to detect a variety of explosives, but the exact gas-phase reactions that occur during ionization are not fully understood. This is further complicated by differences in mass spectrometers and individual experimental set ups between researchers. This study investigated the gas-phase ion reactions of five different explosives using a variety of AI techniques coupled to a Waters QDa mass spectrometer to identify selective ions for explosive detection and identification based on the applied ambient ionization technique. The results showed that the choice of the ion source can have a significant impact on the number of ions observed. This can affect the sensitivity and selectivity of the data produced. The findings of this study provide new insights into the gas-phase ion reactions of explosives and could lead to the development of more sensitive and selective AI-based methods for their detection.

Assessment of atmospheric solids analysis probe as a tool for the rapid determination of drug purity.

The ability to determine the purity (% controlled compound) of drug-of-abuse samples is necessary for public health and law enforcement. Here, we describe the assessment of atmospheric solids analysis probe (ASAP) for the rapid determination of drug purity for a set of formulated pharmaceuticals, chosen due to their availability, uncontrolled status and consistency. Paracetamol and loratadine were used as models of high and low purity compounds being ~90% and ~10% active ingredient, respectively. Individual tablets were ground up and diluted in an internal standard solution. The resulting samples were analysed by ASAP coupled to a Waters QDa mass spectrometer followed by confirmatory testing by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The inclusion of a non-matched internal standard (quinine) improved linearity and repeatability of drug analysis by ASAP-MS. Levels of drug purity using formulated pharmaceutical tablets were found to be highly comparable with results produced by the 'gold standard' LC-MS/MS technique. Rapid determination of drug purity is therefore possible with ASAP-MS for highly concentrated samples with minimal sample preparation. It may be possible to use this deployable system to determine drug purity outside of a laboratory setting.

Direct analysis in real-time mass spectrometry: Observations of helium, nitrogen and argon as ionisation gas for the detection of small molecules using a single quadrupole instrument.

RATIONALE: Direct analysis in real time is typically performed using helium as the ionisation gas for the detection of analytes by mass spectrometry (MS). Nitrogen and argon are found with abundance in the air and provide a cheaper and greener alternative to the use of helium as ionisation gas. This study explores the use of helium, nitrogen and argon as ionisation gas for the detection of organic compounds. METHODS: Four illicit drugs, two amino acids and five explosives were chosen as target analytes to understand selectivity, sensitivity and linearity when helium, nitrogen or argon was used as the ionisation gas with the direct analysis in real time (DART) source. Analysis was carried out on a Waters Acquity QDa single quadrupole mass spectrometer. RESULTS: Calibration curves over the range of 5-100 ng were produced for each analyte using the different ionisation gases to assess the instrument response. Nitrogen gave a higher response to concentration than helium or argon; however, the lowest limits of detection were observed when helium was used. CONCLUSIONS: All the target analytes were detected using DART-MS with helium, nitrogen or argon as the ionisation gas. Whereas helium provided the highest sensitivity, nitrogen produced reasonable limits of detection and had good linearity across the concentration range explored, suggesting it provides a greener and cheaper alternative to helium.

A fundamental study on the degradation of paracetamol under single- and dual-frequency ultrasound.

The degradation of paracetamol, a widely found emerging pharmaceutical contaminant, was investigated under a wide range of single-frequency and dual-frequency ultrasonic irradiations. For single-frequency ultrasonic irradiation, plate transducers of 22, 98, 200, 300, 400, 500, 760, 850, 1000, and 2000 kHz were employed and for dual-frequency ultrasonic irradiation, the plate transducers were coupled with a 20 kHz ultrasonic horn in opposing configuration. The sonochemical activity was quantified using two dosimetry methods to measure the yield of HO• and H2O2 separately, as well as sonochemiluminescence measurement. Moreover, the severity of the bubble collapses as well as the spatial and size distribution of the cavitation bubbles were evaluated via sonoluminescence measurement. The paracetamol degradation rate was maximised at 850 kHz, in both single and dual-frequency ultrasonic irradiation. A synergistic index higher than 1 was observed for all degrading frequencies (200 - 1000 kHz) under dual-frequency ultrasound irradiation, showing the capability of dual-frequency system for enhancing pollutant degradation. A comparison of the results of degradation, dosimetry, and sonoluminescence intensity measurement revealed the stronger dependency of the degradation on the yield of HO• for both single and dual-frequency systems, which confirms degradation by HO• as the main removal mechanism. However, an enhanced degradation for frequencies higher than 500 kHz was observed despite a lower HO• yield, which could be attributed to the improved mass transfer of hydrophilic compounds at higher frequencies. The sonoluminescence intensity measurements showed that applying dual-frequency ultrasonic irradiation for 200 and 400 kHz made the bubbles larger and less uniform in size, with a portion of which not contributing to the yield of reactive oxidant species, whereas for the rest of the frequencies, dual-frequency ultrasound irradiation made the cavitation bubbles smaller and more uniform, resulting in a linear correlation between the overall sonoluminescence intensity and the yield of reactive oxidant species.

Applications of ambient ionization mass spectrometry in 2022: An annual review.

The development of ambient ionization mass spectrometry (AIMS) has transformed analytical science, providing the means of performing rapid analysis of samples in their native state, both in and out of the laboratory. The capacity to eliminate sample preparation and pre-MS separation techniques, leading to true real-time analysis, has led to AIMS naturally gaining a broad interest across the scientific community. Since the introduction of the first AIMS techniques in the mid-2000s, the field has exploded with dozens of novel ion sources, an array of intriguing applications, and an evident growing interest across diverse areas of study. As the field continues to surge forward each year, ambient ionization techniques are increasingly becoming commonplace in laboratories around the world. This annual review provides an overview of AIMS techniques and applications throughout 2022, with a specific focus on some of the major fields of research, including forensic science, disease diagnostics, pharmaceuticals and food sciences. New techniques and methods are introduced, demonstrating the unwavering drive of the analytical community to further advance this exciting field and push the boundaries of what analytical chemistry can achieve.

The role of SPAG1 in the assembly of axonemal dyneins in human airway epithelia.

Mutations in SPAG1, a dynein axonemal assembly factor (DNAAF) that facilitates the assembly of dynein arms in the cytoplasm before their transport into the cilium, result in primary ciliary dyskinesia (PCD), a genetically heterogenous disorder characterized by chronic oto-sino-pulmonary disease, infertility and laterality defects. To further elucidate the role of SPAG1 in dynein assembly, we examined its expression, interactions and ciliary defects in control and PCD human airway epithelia. Immunoprecipitations showed that SPAG1 interacts with multiple DNAAFs, dynein chains and canonical components of the R2TP complex. Protein levels of dynein heavy chains (DHCs) and interactions between DHCs and dynein intermediate chains (DICs) were reduced in SPAG1 mutants. We also identified a previously uncharacterized 60 kDa SPAG1 isoform, through examination of PCD subjects with an atypical ultrastructural defect for SPAG1 variants, that can partially compensate for the absence of full-length SPAG1 to assemble a reduced number of outer dynein arms. In summary, our data show that SPAG1 is necessary for axonemal dynein arm assembly by scaffolding R2TP-like complexes composed of several DNAAFs that facilitate the folding and/or binding of the DHCs to the DIC complex.

Expression of a Truncated Form of ODAD1 Associated with an Unusually Mild Primary Ciliary Dyskinesia Phenotype.

Primary ciliary dyskinesia (PCD) is a rare lung disease caused by mutations that impair the function of motile cilia, resulting in chronic upper and lower respiratory disease, reduced fertility, and a high prevalence of situs abnormalities. The disease is genetically and phenotypically heterogeneous, with causative mutations in > 50 genes identified, and clinical phenotypes ranging from mild to severe. Absence of ODAD1 (CCDC114), a component of the outer dynein arm docking complex, results in a failure to assemble outer dynein arms (ODAs), mostly immotile cilia, and a typical PCD phenotype. We identified a female (now 34 years old) with an unusually mild clinical phenotype who has a homozygous non-canonical splice mutation (c.1502+5G>A) in ODAD1. To investigate the mechanism for the unusual phenotype, we performed molecular and functional studies of cultured nasal epithelial cells. We demonstrate that this splice mutation results in the expression of a truncated protein that is attached to the axoneme, indicating that the mutant protein retains partial function. This allows for the assembly of some ODAs and a significant level of ciliary activity that may result in the atypically mild clinical phenotype. The results also suggest that partial restoration of ciliary function by therapeutic agents could lead to significant improvement of disease symptoms.

Structural insights into the cause of human RSPH4A primary ciliary dyskinesia.

Cilia and flagella are evolutionarily conserved eukaryotic organelles involved in cell motility and signaling. In humans, mutations in Radial Spoke Head Component 4A (RSPH4A) can lead to primary ciliary dyskinesia (PCD), a life-shortening disease characterized by chronic respiratory tract infections, abnormal organ positioning, and infertility. Despite its importance for human health, the location of RSPH4A in human cilia has not been resolved, and the structural basis of RSPH4A-/- PCD remains elusive. Here, we present the native three-dimensional structure of RSPH4A-/- human respiratory cilia using samples collected noninvasively from a PCD patient. Using cryo-electron tomography (cryo-ET) and subtomogram averaging, we compared the structures of control and RSPH4A-/- cilia, revealing primary defects in two of the three radial spokes (RSs) within the axonemal repeat and secondary (heterogeneous) defects in the central pair complex. Similar to RSPH1-/- cilia, the radial spoke heads of RS1 and RS2, but not RS3, were missing in RSPH4A-/- cilia. However, RSPH4A-/- cilia also exhibited defects within the arch domains adjacent to the RS1 and RS2 heads, which were not observed with RSPH1 loss. Our results provide insight into the underlying structural basis for RSPH4A-/- PCD and highlight the benefits of applying cryo-ET directly to patient samples for molecular structure determination.

Induction of ciliary orientation by matrix patterning and characterization of mucociliary transport.

Impaired mucociliary clearance (MCC) is a key feature of many airway diseases, including asthma, bronchiectasis, chronic obstructive pulmonary disease, cystic fibrosis, and primary ciliary dyskinesia. To improve MCC and develop new treatments for these diseases requires a thorough understanding of how mucus concentration, mucus composition, and ciliary activity affect MCC, and how different therapeutics impact this process. Although differentiated cultures of human airway epithelial cells are useful for investigations of MCC, the extent of ciliary coordination in these cultures varies, and the mechanisms controlling ciliary orientation are not completely understood. By introducing a pattern of ridges and grooves into the underlying collagen substrate, we demonstrate for the first time, to our knowledge, that changes in the extracellular matrix can induce ciliary alignment. Remarkably, 90% of human airway epithelial cultures achieved continuous directional mucociliary transport (MCT) when grown on the patterned substrate. These cultures maintain transport for months, allowing carefully controlled investigations of MCC over a wide range of normal and pathological conditions. To characterize the system, we measured the transport of bovine submaxillary gland mucin (BSM) under several conditions. Transport of 5% BSM was significantly reduced compared with that of 2% BSM, and treatment of 5% BSM with the reducing agent tris(2-carboxyethyl)phosphine (TCEP) reduced viscosity and increased the rate of MCT by approximately twofold. Addition of a small amount of high-molecular-weight DNA increased mucus viscosity and reduced MCT by ∼75%, demonstrating that the composition of mucus, as well as the concentration, can have significant effects on MCT. Our results demonstrate that a simple patterning of the collagen substrate results in highly coordinated ciliated cultures that develop directional MCT, and can be used to investigate the mechanisms controlling the regulation of ciliary orientation. Furthermore, the results demonstrate that this method provides an improved system for studying the effects of mucus composition and therapeutic agents on MCC.

Mutation of CFAP57, a protein required for the asymmetric targeting of a subset of inner dynein arms in Chlamydomonas, causes primary ciliary dyskinesia.

Primary ciliary dyskinesia (PCD) is characterized by chronic airway disease, reduced fertility, and randomization of the left/right body axis. It is caused by defects of motile cilia and sperm flagella. We screened a cohort of affected individuals that lack an obvious axonemal defect for pathogenic variants using whole exome capture, next generation sequencing, and bioinformatic analysis assuming an autosomal recessive trait. We identified one subject with an apparently homozygous nonsense variant [(c.1762C>T), p.(Arg588*)] in the uncharacterized CFAP57 gene. Interestingly, the variant results in the skipping of exon 11 (58 amino acids), which may be due to disruption of an exonic splicing enhancer. In normal human nasal epithelial cells, CFAP57 localizes throughout the ciliary axoneme. Nasal cells from the PCD patient express a shorter, mutant version of CFAP57 and the protein is not incorporated into the axoneme. The missing 58 amino acids include portions of WD repeats that may be important for loading onto the intraflagellar transport (IFT) complexes for transport or docking onto the axoneme. A reduced beat frequency and an alteration in ciliary waveform was observed. Knockdown of CFAP57 in human tracheobronchial epithelial cells (hTECs) recapitulates these findings. Phylogenetic analysis showed that CFAP57 is highly conserved in organisms that assemble motile cilia. CFAP57 is allelic with the BOP2/IDA8/FAP57 gene identified previously in Chlamydomonas reinhardtii. Two independent, insertional fap57 Chlamydomonas mutant strains show reduced swimming velocity and altered waveforms. Tandem mass tag (TMT) mass spectroscopy shows that FAP57 is missing, and the "g" inner dyneins (DHC7 and DHC3) and the "d" inner dynein (DHC2) are reduced, but the FAP57 paralog FBB7 is increased. Together, our data identify a homozygous variant in CFAP57 that causes PCD that is likely due to a defect in the inner dynein arm assembly process.

A human ciliopathy reveals essential functions for NEK10 in airway mucociliary clearance.

Mucociliary clearance, the physiological process by which mammalian conducting airways expel pathogens and unwanted surface materials from the respiratory tract, depends on the coordinated function of multiple specialized cell types, including basal stem cells, mucus-secreting goblet cells, motile ciliated cells, cystic fibrosis transmembrane conductance regulator (CFTR)-rich ionocytes, and immune cells1,2. Bronchiectasis, a syndrome of pathological airway dilation associated with impaired mucociliary clearance, may occur sporadically or as a consequence of Mendelian inheritance, for example in cystic fibrosis, primary ciliary dyskinesia (PCD), and select immunodeficiencies3. Previous studies have identified mutations that affect ciliary structure and nucleation in PCD4, but the regulation of mucociliary transport remains incompletely understood, and therapeutic targets for its modulation are lacking. Here we identify a bronchiectasis syndrome caused by mutations that inactivate NIMA-related kinase 10 (NEK10), a protein kinase with previously unknown in vivo functions in mammals. Genetically modified primary human airway cultures establish NEK10 as a ciliated-cell-specific kinase whose activity regulates the motile ciliary proteome to promote ciliary length and mucociliary transport but which is dispensable for normal ciliary number, radial structure, and beat frequency. Together, these data identify a novel and likely targetable signaling axis that controls motile ciliary function in humans and has potential implications for other respiratory disorders that are characterized by impaired mucociliary clearance.

Author Correction: A human ciliopathy reveals essential functions for NEK10 in airway mucociliary clearance.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Identification of genetic variants in CFAP221 as a cause of primary ciliary dyskinesia.

Primary ciliary dyskinesia (PCD) is a rare disorder that affects the biogenesis or function of motile cilia resulting in chronic airway disease. PCD is genetically and phenotypically heterogeneous, with causative mutations identified in over 40 genes; however, the genetic basis of many cases is unknown. Using whole-exome sequencing, we identified three affected siblings with clinical symptoms of PCD but normal ciliary structure, carrying compound heterozygous loss-of-function variants in CFAP221. Computational analysis suggests that these variants are the most damaging alleles shared by all three siblings. Nasal epithelial cells from one of the subjects demonstrated slightly reduced beat frequency (16.5 Hz vs 17.7 Hz, p = 0.16); however, waveform analysis revealed that the CFAP221 defective cilia beat in an aberrant circular pattern. These results show that genetic variants in CFAP221 cause PCD and that CFAP221 should be considered a candidate gene in cases where PCD is suspected but cilia structure and beat frequency appear normal.

Nanoextraction Coupled to Liquid Chromatography Mass Spectrometry Delivers Improved Spatially Resolved Analysis.

Direct analyte-probed nanoextraction (DAPNe) is a technique that allows extraction of drug and endogenous compounds from a discrete location on a tissue sample using a nano capillary filled with solvent. Samples can be extracted from spot diameters as low as 6 µm. Studies previously undertaken by our group have shown that the technique can provide good precision (5%) for analyzing drug molecules in 150 µm diameter areas of homogenized tissue, provided an internal standard is sprayed on to the tissue prior to analysis. However, without an isotopically labeled standard, the repeatability is poor, even after normalization to the spot area or matrix compounds. By application to tissue homogenates spiked with drug compounds, we can demonstrate that it is possible to significantly improve the repeatability of the technique by incorporating a liquid chromatography separation step. Liquid chromatography is a technique for separating compounds prior to mass spectrometry (LC-MS) which enables separation of isomeric compounds that cannot be discriminated using mass spectrometry alone, as well as reducing matrix interferences. Conventionally, LC-MS is carried out on bulk or homogenized samples, which means analysis is essentially an average of the sample and does not take into account discrete areas. This work opens a new opportunity for spatially resolved liquid chromatography mass spectrometry with precision better than 20%.

Paper spray screening and liquid chromatography/mass spectrometry confirmation for medication adherence testing: A two-step process.

RATIONALE: Paper spray offers a rapid screening test without the need for sample preparation. The incomplete extraction of paper spray allows for further testing using more robust, selective and sensitive techniques such as liquid chromatography/mass spectrometry (LC/MS). Here we develop a two-step process of paper spray followed by LC/MS to (1) rapidly screen a large number of samples and (2) confirm any disputed results. This demonstrates the applicability for testing medication adherence from a fingerprint. METHODS: Following paper spray analysis, drugs of abuse samples were analysed using LC/MS. All analyses were completed using a Q Exactive™ Plus Orbitrap™ mass spectrometer. This two-step procedure was applied to fingerprints collected from patients on a maintained dose of the antipsychotic drug quetiapine. RESULTS: The extraction efficiency of paper spray for two drugs of abuse and metabolites was found to be between 15 and 35% (analyte dependent). For short acquisition times, the extraction efficiency was found to vary between replicates by less than 30%, enabling subsequent analysis by LC/MS. This two-step process was then applied to fingerprints collected from two patients taking the antipsychotic drug quetiapine, which demonstrates how a negative screening result from paper spray can be resolved using LC/MS. CONCLUSIONS: We have shown for the first time the sequential analysis of the same sample using paper spray and LC/MS, as well as the detection of an antipsychotic drug from a fingerprint. We propose that this workflow may also be applied to any type of sample compatible with paper spray, and will be especially convenient where only one sample is available for analysis.

Mice with a Deletion of Rsph1 Exhibit a Low Level of Mucociliary Clearance and Develop a Primary Ciliary Dyskinesia Phenotype.

Primary ciliary dyskinesia (PCD) is a genetically and phenotypically heterogeneous disease caused by mutations in over 40 different genes. Individuals with PCD caused by mutations in RSPH1 (radial spoke head 1 homolog) have been reported to have a milder phenotype than other individuals with PCD, as evidenced by a lower incidence of neonatal respiratory distress, higher nasal nitric oxide concentrations, and better lung function. To better understand genotype-phenotype relationships in PCD, we have characterized a mutant mouse model with a deletion of Rsph1. Approximately 50% of cilia from Rsph1-/- cells appeared normal by transmission EM, whereas the remaining cilia revealed a range of defects, primarily transpositions or a missing central pair. Ciliary beat frequency in Rsph1-/- cells was significantly lower than in control cells (20.2 ± 0.8 vs. 25.0 ± 0.9 Hz), and the cilia exhibited an aberrant rotational waveform. Young Rsph1-/- animals demonstrated a low rate of mucociliary clearance in the nasopharynx that was reduced to zero by about 1 month of age. Rsph1-/- animals accumulated mucus in the nasal cavity but had a lower bacterial burden than animals with a deletion of dynein axonemal intermediate chain 1 (Dnaic1-/-). Thus, Rsph1-/- mice display a PCD phenotype similar to but less severe than that observed in Dnaic1-/- mice, similar to what has been observed in humans. The results suggest that some individuals with PCD may not have a complete loss of mucociliary clearance and further suggest that early diagnosis and intervention may be important to maintain this low amount of clearance.

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.