Modeling TCIRG1 Neutropenia by Utilizing Patient Derived Induced Pluripotent Stem Cells

Congenital neutropenia is characterized by a reduced neutrophil count, decreased innate immunity and increased susceptibility to recurrent infections. While congenital neutropenia has various genetic causes, recent studies have linked <i>TCIRG1</i> mutations to this condition. <i>TCIRG1</i>, a key component of the vacuolar ATPase (V-ATPase) complex, is essential for osteoclast function, but its role in hematopoiesis remains unclear. We previously identified heterozygous <i>TCIRG1</i> mutations, including R736S, R736C, R736P, and E722D, in individuals with congenital neutropenia. However, the mechanism by which these mutations lead to impaired granulopoiesis remains unknown. To investigate the functional consequences of <i>TCIRG1</i> mutations, we generated induced pluripotent stem cells (iPSCs) from affected individuals and healthy controls. Using <i>in vitro</i> differentiation protocols, we assessed hematopoietic progenitor formation, proliferation, survival, and neutrophil differentiation. We observed significant defects in myeloid differentiation and increased cell death in patient-derived iPSC lines. CRISPR/Cas9-mediated correction of the R736C mutation restored normal neutrophil differentiation, confirming its pathogenic role. Immunofluorescence analysis revealed reduced expression and altered intracellular localization of the <i>TCIRG1</i> protein, characterized by a more diffuse cytosolic distribution in the mutant cell lines. Our findings suggest that <i>TCIRG1</i> mutations impair neutrophil development, likely through structural and functional disruption of the V-ATPase complex. This study provides new insights into the molecular basis of <i>TCIRG1</i>-associated neutropenia and highlights potential avenues for therapeutic intervention.