Murine Modeling of Human Severe Congenital Neutropenia and Non-Immune Chronic Idopathic Neutropenia.

Severe Congenital Neutropenia (SCN) is a rare hematological disease manifested by a complete block in the maturation of neutrophils. The lack of neutrophils leads to recurrent bacterial and fungal infections. Most SCN patients have mutations in Ela2, the gene encoding neutrophil elastase; however, introduction of SCN-patient mutations into murine Ela2 or disruption of murine Ela2 does not recapitulate neutropenia in mice. Thus, there are no mouse models of SCN. Gfi1 is a transcriptional repressor leukemia oncoprotein involved in HSC maintenance as well as proper lymphoid and myeloid development. Mice lacking Gfi1 have profound neutropenia. We recently described four patients in which Ela2 was intact but mutant for Gfi1. The heterozygous single aminoacid substitutions were found in zinc finger 5 (N382S) and zinc finger 6 (K403R) correlating to either SCN or Non-immune Chronic Idiopathic Neutropenia of Adults (NICINA) respectively. Given that Gfi1 null mice lack neutrophils, and that people with Gfi1 mutations display SCN or NICINA, we asked whether the human mutations when introduced into murine Gfi1 are sufficient to model neutropenia in mice. To this end, we constructed murine Gfi1 cDNAs containing the SCN and NICINA patient mutations, retrovirally overexpressed them in murine Lin- bone marrow cells, then analyzed them by in vitro differentiation assays. Strikingly, cells overexpressing Gfi1-N382S produced no granulocytic colonies (similar to the profound phenotype of SCN). Whereas, the expression of Gfi1-K403R mutation resulted in a moderate reduction in granulocytic colonies (corresponding to the mild phenotype of NICINA). We have constructed a virtual model of Gfi1 zinc finger/DNA interaction to explore the molecular defect engendered by the N382S mutation. At the meeting, we will discuss the molecular basis of the profound phenotype engendered by Gfi1-N382S expression. These data demonstrate for the first time that human neutropenia can be modeled in murine cultures through the introduction of mutations in Gfi1 found in SCN and NICINA patients.