Multiomics analysis of a DNAH5-mutated PCD organoid model revealed the key role of the TGF-ß/BMP and Notch pathways in epithelial differentiation and the immune response in DNAH5-mutated patients

Dynein axonemal heavy chain 5 (DNAH5) is the most mutated gene in primary ciliary dyskinesia (PCD), leading to abnormal cilia ultrastructure and function. Few studies have revealed the genetic characteristics and pathogenetic mechanisms of PCD caused by DNAH5 mutation. Here, we established a child PCD airway organoid directly from the bronchoscopic biopsy of a patient with DNAH5 mutation. We found abnormal ciliary function and a decreased immune response caused by DNAH5 mutation through single-cell RNA sequencing (scRNA-seq). Overall design: The child PCD airway organoids, directly from the bronchoscopic biopsy of a patient with DNAH5 mutation, were constructed according to the airway organoid culture protocol. The normal airway organoids were derived from the suspected patient who excluded the PCD in follow-up examinations,The airway organoid with DNAH5 mutation and normal airway organoid pellets, derived from the biopsy, were resuspended with 1 mL of TrypLE 1× (Gibco, A1217701), separately, incubated for 15-30 min at 37 °C, and mechanically sheared with 1-ml pipettes. After washing twice with AdDF+++, the cell pellets were filtered with a 40-µm strainer. Cellular suspensions (~6000 cells) were loaded on a Chromium Single Cell Instrument (10X Genomics) to generate single-cell GEMs. Sample processing and single-cell RNA-seq library preparation were performed using Chromium Single Cell 3' v2 reagents. Sequencing was performed on an Illumina NextSeq500 following the manufacturer's instructions.