Abstract
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 TCIRG1 mutations to this condition. TCIRG1, 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 TCIRG1 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 TCIRG1 mutations, we generated induced pluripotent stem cells (iPSCs) from affected individuals and healthy controls. Using in vitro 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 TCIRG1 protein, characterized by a more diffuse cytosolic distribution in the mutant cell lines.
Our findings suggest that TCIRG1 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 TCIRG1-associated neutropenia and highlights potential avenues for therapeutic intervention.
Keywords
TCIRG1, Neutropenia, Granulopoiesis, Neutrophils, iPSCs, CRISPR/Cas9, V-ATPase