A Novel PCD Phenotype: Inherent Mucus Hypersecretion by Patient-Derived Airway Epithelial Cells.Joseph Castlen1, Benjamin Gaston2, Laura Smith2, Michael D Davis2, Allison Norlander3, Andrew Tilston-Lunel2.1 Division of Pediatric Pulmonology, Allergy & Immunology, and Sleep Medicine, Department of Pediatrics, Indiana University School of Medicine.2 Herman B Wells Center for Pediatric Research, Department of Pediatrics, Indiana University School of Medicine.3 Department of Anatomy, Cell Biology & Physiology, Indiana University School of Medicine.Corresponding author: alunel@iu.eduTo the Editor:Primary ciliary dyskinesia (PCD) is a motile ciliopathy characterized by recurrent pulmonary infections during childhood, laterality defects, and infertility, with more than 54 disease-causing genes identified [1, 2]. In this clinical correspondence, we describe a clinical history for a PCD patient and then correlate her history with a novel in vitro phenotype in which her airway epithelial cells transformed into hypersecretory, mucus-producing cells.AV is a 19-year-old woman with PCD. She was born at 40 weeks and 1 day of gestation following an uncomplicated pregnancy. On day 3 of life, she was noted to be in respiratory distress. She was placed on supplemental oxygen via nasal cannula, underwent an unrevealing workup for sepsis and congenital heart defects, and was ultimately discharged home on day 11 of life after oxygen was weaned. The patient’s mother recalls an early wet cough and rhinorrhea, though the exact age of onset is unclear. These symptoms were present by age three. She was frequently treated for diffuse wheezing presumed to be asthma; however, it was only partially responsive to albuterol and persisted despite prednisone and antibiotics.At the time of her initial PCD diagnosis at 10 years of age, she had low nasal nitric oxide concentrations (using ATS guidelines) of 35, 38, 50, and 60 nL/min on four separate dates. Genetic testing showed one intronic pathological variant (Het; c921+3_921+6delAAGT), and one variant of uncertain significance (Het; c.1934A>G(pTyr645Cys)) later determined to be pathologic in the Radial Spoke Head Component 4A (RSPH4A ) gene. The patient had an older sister with similar symptoms, who had the same RSPH4A findings with an additional variant of uncertain significance in one allele of DNAI1 . Parental testing showed one RSPH4A variant in each parent. Nasal ciliary biopsy and electron microscopy analysis done at age 9 showed normal ciliary ultrastructure, consistent with her genotype. RSPH4A is a clinically relevant gene implicated in the diagnosis of PCD [3]. Both of AV’s mutations have been reported on ClinVar [4] .Bronchoscopies have demonstrated thick secretions, often with mucus plugging (Fig.1A), throughout the airway. Chest CTs have shown bronchiectasis in the right middle and lower lobes along with atelectasis and tree-in-bud opacities throughout the bilateral lungs(Fig.1B). Serial bronchoalveolar lavages (BALs) and sputum cultures over in total have grown ten different bacterial species as well as mold. Her total IgE and eosinophil counts were both normal.Overall, her clinical course was marked by chronic wheezing that showed only partial response to asthma therapies (inhaled and systemic corticosteroids and long-acting beta agonists), though she did meet ATS/ERS criteria for bronchial hyperresponsiveness with an 11% improvement in FEV1 following albuterol administration (Fig. 1C). Although this patient has increased mucus production, as described below, her pre- and post-bronchodilator spirometry indicates that her bronchodilator response is more likely related to true reversal of bronchoconstriction rather than mobilization of secretions secondary to the airway clearance effect of repeated forced exhalation. This is evident in that subsequent attempts at spirometry before receiving albuterol resulted in a decreasing FEV1.For most of AV’s life, she required only two hospital admissions for respiratory exacerbations. Infection with Mycobacterium avium-intracellulare complex (MAI), however, resulted in eight additional hospitalizations and notable disease progression She was started on a minimum 12-month treatment regimen consisting of azithromycin, clofazimine, and rifabutin which was later expanded to include inhaled amikacin and bedaquiline. Her FEV1, which declined to 35% of predicted, improved to 82% of predicted (near her baseline of 88%) during MAI treatment. The patient’s course was further complicated by severe sinus infections requiring surgery, recurrent otitis, medication intolerance, central line infections, an eating disorder, as well as anxiety and depression.Interestingly, as seen in this patient, it has been reported that most children with likely PCD also have a diagnosis code for asthma in their chart [5]. Another study in vitro showed that antigen stasis in the PCD airway epithelial cells promotes an asthma-like inflammatory response [6]. In the literature, cell culture has been used as a technique to correlate genotype to phenotypic findings, including in cases when a patient’s clinical phenotype is discordant from the degree of disease expected based on genetics [7, 8]. As such, given that the severity of this patient’s respiratory exacerbations was greater than expected given her degree of bronchiectasis, in vitro studies were begun to further investigate her underlying pathophysiology.Airway basal cells obtained from nasal brushings were differentiated at air-liquid interface (ALI) for 35 days. Her resulting cultures showed a low proportion of multiciliated cells (Fig. 2A), with most cells being mucus-producing (Fig. 2B). The increased number of goblet cells in these airway epithelial cultures is a notable finding, particularly given the association between PCD and asthma. Additionally, defects in the actin cytoskeleton and the confluency of her differentiated cultures were observed. To add to this, the height of her ALI cultures was severely compromised when compared to healthy controls (Fig. 2C). Notably, the localization of RSPH4A was localized to motile cilia in healthy patient controls, while mutant RSPH4A was localized to the nuclei in the PCD patient cells (Fig. 2D, 2E). These cellular defects might explain the patient’s bronchiectasis. Further studies are being conducted to better characterize this finding, which may account for her more severe clinical course.In summary, this 19-year-old woman with PCD has a novel phenotype with transformation of airway epithelial cells into hypersecretory, mucus-producing cells. Clinically, she experienced a decline in lung function that was out of proportion with her degree of bronchiectasis.In vitro studies have correlated well with her clinical history, and these findings warrant further investigation. She had bronchodilator responsiveness on spirometry but was incompletely responsive to asthma treatment. Taken together, clinical implications could include the use of biologic agents such as dupilumab to target potential sources of mucus hypersecretion and airway inflammation. In vitro studies are ongoing.Funding statement: The project described was supported by Award Number T32HL091816 and 5P01HL158507-05 from the National Heart, Lung, and Blood Institute. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Heart, Lung, and Blood Institute or the National Institutes of Health.Ethics Approval: Ethical approval for this study was obtained from the Indiana University Institutional Review Board (IRB: #11042). The study was conducted per recognized ethical standards, including the Declaration of Helsinki and the U.S. Federal Policy for the Protection of Human Subjects. Written informed consent was obtained for anonymized patient information to be published in this article.References1. Hannah, W.B., et al., The global prevalence and ethnic heterogeneity of primary ciliary dyskinesia gene variants: a genetic database analysis. Lancet Respir Med, 2022. 10 (5): p. 459-468.2. Despotes, K.A., et al., Primary Ciliary Dyskinesia: A Clinical Review. Cells, 2024. 13 (11).3. De Jesus-Rojas, W., et al., The RSPH4A Gene in Primary Ciliary Dyskinesia. Int J Mol Sci, 2023. 24 (3).4. Landrum, M.J., et al., ClinVar: updates to support classifications of both germline and somatic variants. Nucleic Acids Res, 2025. 53 (D1): p. D1313-D1321.5. Zein, J., et al., Asthma Among Children With Primary Ciliary Dyskinesia. JAMA Netw Open, 2024. 7 (12): p. e2449795.6. Gaston, B., et al., Antigen stasis and airway nitrosative stress in human primary ciliary dyskinesia. Am J Physiol Lung Cell Mol Physiol, 2024. 326 (4): p. L468-L476.7. Chivukula, R.R., et al.,Author Correction: A human ciliopathy reveals essential functions for NEK10 in airway mucociliary clearance. Nat Med, 2020.26 (2): p. 300.8. Suryadinata, R., et al., Heterozygous cis HYDIN mutations cause primary ciliary dyskinesia. Med, 2025.6 (1): p. 100508.Figure Legends:Figure 1. Bronchoscopic, Radiographic, and Functional Assessment of Airway Abnormalities. A) Images taken from the same bronchoscopy showing a large mucus plug being actively suctioned in the right middle lobe (left) and copious, thick mucus in the distal portion of the left mainstem bronchus (right). B) Bronchiectasis is visible in the airways of the medial right middle lobe. C) The patient demonstrated bronchodilator-responsiveness as seen in this graph of FEV1 attempts pre- and post-bronchodilator (BD). This represents an 11% improvement in FEV1 from her baseline.Figure 2. Defective Differentiation Programs in PCD Patient-Derived Airway Cultures. A) Ciliated cell differentiation is markedly impaired compared to healthy controls. Acetylated tubulin is high enriched in axonemes of motile cilia. B) Airway cultures from the PCD patient are predominantly composed of Goblet cells. MUC5AC was used to label goblet cells. C) X-Z profiles reveal an aberrant differentiation program, evidenced by multiple layers of nuclei. Keratin 8 (KRT8) is a pan-luminal cells marker, Actin was stained with Phalloidin, D) Maximum intensity projections of healthy control and PCD patient cells highlight defects in RSPH4A localization. E) X-Z profiles demonstrate proper RSPH4A localization to motile cilia in healthy control cells (yellow arrows), whereas in the PCD patient cells, mutant RSPH4A is localized to the nucleus (white dashed circles). All images were acquired on a Zeiss LSM 800 and have a 20µm scale bar.
