Septin family proteins belong to the RAS-like GTPase superclass and are considered as a structural element of the cytoskeleton. They are evolutionarily conserved and are important in cytokinesis, polarity, cell cycle, vesicle trafficking, and exocytosis. They are implicated in T and B cell lymphomas; and acute myeloid leukemias, where they frequently act as fusion partners with the Mixed-Lineage Leukemia (MLL) gene. Previously, we described two non-syndromic patients with severe neutropenia which progressed to myelodysplastic syndrome (MDS) (Renella et al. AJH, 2022, Mohamad et al. ASH, 2022). Next-generation sequencing revealed both patients (P1 and P2) to have novel X-linked germline mutations in codon 428 of the SEPTIN6gene (P1: SEPTIN6 c.1282T>C; P2: SEPTIN6 c.1282T>A). One of these patients (P1) had an additional somatic stop-gain mutation (P1: SEPTIN6 c.43C>T) of unclear function. Both patient germline mutations were predicted to add 9 amino acids to the C-terminal end creating a novel mutated SEPTIN6 protein. Since SEPTIN6 is located on the X-chromosome and expression of these mutated proteins occurred in males, no wild-type protein was present in the patient cells. To further explore the biological relevance of these mutations, single base-pair gene editing was used to introduce the (P1: T>C) mutation into normal male donor CD34+ cells. Morphological studies demonstrated that SEPTIN6edited cells had enlarged, multi-nucleated and dysplastic nuclei; and reduced clonogenic activity.