Lymphocyte Cytosolic Protein 1 I232F Mutation Impairs Granulocytic Proliferation with a G2/M Block in Severe Neutropenia

▪ Introduction: As critical effectors of innate immune response, neutrophils migrate from the vasculature into inflamed tissue, where they engage in phagocytosis and clearance of pathogens and apoptotic cells. Migration, phagocytosis, and granule release require multiple well-coordinated events involving remodeling of the actin cytoskeleton. While mutations of some regulators of actin polymerization, such as Wiskott-Aldrich Syndrome gene (WAS) result in Severe Congenital Neutropenia (SCN), the role of actin cytoskeleton in regulating neutrophil ontogeny and functions remain poorly understood. Lymphocyte Cytosolic Protein 1 (LCP1) is an actin bundling protein encoded by LCP1 gene. A 7-year-old girl presented at age of 2 years with chronic severe neutropenia and frequent infections. Next generation sequencing did not reveal a variant in genes known to cause neutropenia. Whole exome sequencing identified a novel LCP1 p.I232F variant of unknown significance, which was not present in either parent. Bone marrow examination revealed a granulocytic hypoplasia with significant dysmorphic giant granulocytes with complex hypersegmentation and increased apoptosis. (Figure 1A) Based on these observations, we hypothesized that missense mutation in LCP1 (I232F) impairs granulocytic proliferation, and causes neutropenia. Aims: To investigate the role of LCP1 I232F, discovered in a child with severe symptomatic neutropenia, in granulopoiesis. Methods: in vitro experiments involving the expression of wild type and mutant LCP1 I232F in murine myeloblast cell (32D cells) and human cervical cancer HeLa cell. We deployed various biologic and functional studies to investigate the role of LCP1 I232F in granulocyte proliferation, differentiation, survival, and function. Results: To determine the pathophysiologic significance of LCP1 I232F, we stably transduced interleukin 3-dependent murine myeloblast 32D and human cervical cancer HeLa cell lines with a doxycycline-inducible lentiviral constructs containing the cDNA for either wild type LCP1 or LCP1 I232F. The mutant LCP1 I232F expressing 32D cells showed impaired proliferation with the appearance of dysplastic granulocytic cells. (Figure 1A, B) However, we did not observe block in differentiation. A cell cycle arrest at G2/M phase occurred only in the LCP1 I232F expressing cells (Figure 1B) and was associated with an upregulation of markers of cell cycle arrest (Cdkn1a and Tp53). LCP1 I232F overexpression did not lead to increase