This is the first competing renewal for a grant from a PI who had been a new investigator and that was extended through the Presidential Early Career Award for Scientists and Engineers (PECASE) and was successful for the discovery, molecular characterization, and animal modeling of genes responsible for hereditary neutropenia. Neutrophils are the most abundant of the white blood cells and act as an innate defense against bacterial and fungal sepsis. Neutropenia refers to a deficiency in the number of neutrophils, and there are two main hereditary forms: autosomal dominant cyclic hematopoiesis (also known as """"""""cyclic neutropenia"""""""") and the genetically heterogeneous severe congenital neutropenia (SCN, also known as """"""""Kostmann syndrome""""""""). Individuals with cyclic hematopoiesis suffer from neutrophil counts that oscillate with three week periodicity, alternating between near normal values and zero and leaving them vulnerable to infection during the nadir of the cycle. SCN consists of continuous neutropenia and a predisposition to leukemia. A genome wide screen for linkage with positional cloning showed that heterozygous mutation of ELA2, encoding the neutrophil granule serine protease, neutrophil elastase (NE), causes cyclic hematopoiesis, and candidate gene analysis found it also to be the most common cause of SCN. Because of their severity, and attendantly reduced genetic fitness, many cases arise sporadically from new dominant mutations. Candidate gene investigation identified rare cases of SCN attributable to mutations in the transcriptional represser oncogene Gfi1. Genetic analysis of canine cyclic hematopoiesis found it to result from mutations in AP3B1, encoding a subunit of the APS complex involved in subcellular trafficking. Gfi1 represses the expression of NE, which, in turn, is trafficked by APS from the trans-Golgi network to neutrophil granules, and is hypothesized to function, along with other factors identified through yeast two- hybrid and human mutational screens, as a negative feedback regulator of hematopoiesis whose interruption accounts for the cyclic phenomenon.
Three Specific Aims are proposed within the framework of a test of the feedback hypothesis: 1. Develop ELA2 cellular and mouse models of hereditary neutropenia. 2. Test molecular interactions between nodal points (PFAAP5, SOCS3, and Notch family proteins) of the proposed feedback circuit. 3. Evaluate the genes encoding nodal points of the proposed feedback circuit as candidates for unaccounted cases of neutropenia.
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