A goal of this laboratory's efforts is to employ molecular genetics to understand the structure, regulation, and function of specific human genes that are expressed in hematopoietic cells and relevant to disease. An important host defense system of phagocytes is a plasma membrane associated NADPH oxidase that reduces molecular oxygen to superoxide. The components of the oxidase system, their correspondence with specific genetic loci, and the primary protein defect(s) in chronic granulomatous disease (CGD) (a spectrum of genetic disorders that incapacitates this enzyme system) have been largely unresolved. Recently, the gene and cDNA for the product lacking in X-linked CGD, the classical inherited disease in the system, was cloned by """"""""reverse genetics"""""""" and shown to encode a 90 kd membrane glycoprotein subunit of the phagocyte b-cytochrome. In the proposed research the structure, expression, and function of the 90 kd X-CGD protein will be pursued from a molecular perspective. Attention will be directed toward delineating the cis-acting DNA elements that regulate lineage specific (phagocytic) expression of the gene by gene transfer of promoter/enhancer fusions and toward identification and characterization of nuclear DNA-binding proteins that interact with the critical DNA regions. To study the X-CGD protein and its function antibodies to specific domains will be generated to determine its membrane orientation and to modulate the activity of the system form the extracellular space. A functional assay for the protein will be established by transfer of full-length cDNA into heterologous phagocytic and non-phagocytic cell lines, and into deficient myelomonocytic cells. With this assay and in vitro mutagenesis of the cDNA the role of specific domains of the protein and interaction with a 22 kd component of the phagocyte b-cytochrome will be approached directly. To study the interaction of the 90 kd and 22 kd components, cDNA for the 22 kd will be isolated and used in coexpression experiments. Finally, the genetics of specific X-CGD mutations will be investigated, especially in variant patients with residual b-cytochrome activity, and RFLP probe suitable for improved prenatal diagnosis of X- CGD by DNA analysis will be developed.

Project Start
1983-07-01
Project End
1993-06-30
Budget Start
1990-07-01
Budget End
1991-06-30
Support Year
8
Fiscal Year
1990
Total Cost
Indirect Cost
Name
Children's Hospital Boston
Department
Type
DUNS #
076593722
City
Boston
State
MA
Country
United States
Zip Code
02115
Dinauer, M C; Orkin, S H (1992) Chronic granulomatous disease. Annu Rev Med 43:117-24
Dinauer, M C; Pierce, E A; Erickson, R W et al. (1991) Point mutation in the cytoplasmic domain of the neutrophil p22-phox cytochrome b subunit is associated with a nonfunctional NADPH oxidase and chronic granulomatous disease. Proc Natl Acad Sci U S A 88:11231-5
Skalnik, D G; Dorfman, D M; Perkins, A S et al. (1991) Targeting of transgene expression to monocyte/macrophages by the gp91-phox promoter and consequent histiocytic malignancies. Proc Natl Acad Sci U S A 88:8505-9
Skalnik, D G; Strauss, E C; Orkin, S H (1991) CCAAT displacement protein as a repressor of the myelomonocytic-specific gp91-phox gene promoter. J Biol Chem 266:16736-44
Dinauer, M C; Pierce, E A; Bruns, G A et al. (1990) Human neutrophil cytochrome b light chain (p22-phox). Gene structure, chromosomal location, and mutations in cytochrome-negative autosomal recessive chronic granulomatous disease. J Clin Invest 86:1729-37
Ezekowitz, R A; Sieff, C A; Dinauer, M C et al. (1990) Restoration of phagocyte function by interferon-gamma in X-linked chronic granulomatous disease occurs at the level of a progenitor cell. Blood 76:2443-8
Dinauer, M C; Curnutte, J T; Rosen, H et al. (1989) A missense mutation in the neutrophil cytochrome b heavy chain in cytochrome-positive X-linked chronic granulomatous disease. J Clin Invest 84:2012-6
Hirschhorn, R; Tzall, S; Ellenbogen, A et al. (1989) Identification of a point mutation resulting in a heat-labile adenosine deaminase (ADA) in two unrelated children with partial ADA deficiency. J Clin Invest 83:497-501
Newburger, P E; Ezekowitz, R A; Whitney, C et al. (1988) Induction of phagocyte cytochrome b heavy chain gene expression by interferon gamma. Proc Natl Acad Sci U S A 85:5215-9
Ezekowitz, R A; Dinauer, M C; Jaffe, H S et al. (1988) Partial correction of the phagocyte defect in patients with X-linked chronic granulomatous disease by subcutaneous interferon gamma. N Engl J Med 319:146-51

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