Neutrophil extracellular traps formation driven by novel ELANE mutations in cyclic neutropenia patients

Cyclic neutropenia (CyN) is a rare haematological disorder most often caused by mutations in the neutrophil elastase gene (ELANE). We describe three novel ELANE mutations in CyN patients and link them to impaired neutrophil extracellular trap (NET) formation. ELANE sequencing was performed using the Sanger method, and AlphaFold2 was used to visualize the impact of mutations on the structure of neutrophil elastase (NE). NET formation was assessed by colocalization of MPO, NE, and DNA using fluorescence microscopy, and free DNA was quantified fluorometrically. We identified two missense substitutions (M66K, V133G) and one deletion (G192-G196del). These mutations did not significantly alter the overall NE structure but exhibited functional effects: M66K may disrupt substrate binding, V133G increases protein mobility, and the Deletion of G192-G196 may limit the opening of the catalytic pocket. NETosis was impaired in CyN patients compared with controls. Their NETs appeared less expanded, and DNA release into supernatants was reduced, reaching the lowest levels during neutropenia, even in patients with normal neutrophil counts. These abnormalities were accompanied by reduced NE activity. In sum, novel ELANE variants may contribute to altered NE function and defective NET formation in CyN, suggesting that impaired NETosis is partly independent of cyclic changes in neutrophil numbers.