A study of 25 patients with chronic granulomatous disease: A new classification by correlating respiratory burst, cytochrome b, and flavoprotein (original) (raw)
Blood, 1983
A patient with an X-linked genetic disease resembling chronic granulomatous disease (CGD) but differing in several aspects from previously studied cases is described. The oxidase enzyme of the patient's granulocytes was normally activated, but had reduced activity as shown by an increased Michaelis constant and decreased maximum velocity of NADPH-dependent superoxide production. Cytochrome-b was undetectable in dithionite difference spectra. This CGD-like disease further implicates cytochrome-b as an important component of the microbicidal NADPH oxidase system and provides insight into its role in the enzyme complex.
The Journal of Infectious Diseases, 2003
Chronic granulomatous disease (CGD) is a rare congenital syndrome that results in severe, recurrent bacterial and fungal infections. The most common form is caused by defects in the CYBB gene, leading to the absence of gp91phox associated with totally abolished NADPH oxidase activity (X91 0 CGD). We report 3 brothers with atypical cases of X-linked CGD, characterized by low levels of expression of gp91phox (X91 Ϫ CGD). A point mutation (T-55C) identified in the CYBB gene's promoter region appears to prevent the full expression of this gene in neutrophils. This results in low levels of expression of gp91phox protein that are correlated with residual oxidase activity in the whole population of neutrophils. The total O 2 Ϫ production in these cells was ∼5% of normal. Despite this oxidase activity, the patients experienced severe and life-threatening infections. It was concluded that the O 2 Ϫ production in the neutrophils of these patients was not sufficient to protect them against infections, and this X91 Ϫ CGD phenotype must be considered to be a severe clinical form of CGD. Chronic granulomatous disease (CGD) is an immunodeficiency syndrome clinically characterized by severe, recurrent bacterial and fungal infections [1, 2]. CGD is a rare inherited disorder in which phagocytic cells are unable to kill engulfed microorganisms and is caused by a defect in the NADPH oxidase system [3-5]. This enzyme catalyzes the formation of superoxide anions, which are precursors of the generation of potent oxidants involved in the first step of phagocytic-cell microbicidal activity. The NADPH oxidase system consists of membrane-bound flavocytochrome b 558 (i.e., the redox center of the oxidase) and 3 cytosolic compo
European Journal of Clinical Investigation, 1992
Chronic Granulomatous Disease (CGD) manifests as a predisposition to infection as a result of defective function of the NADPH oxidase of phagocytic cells. Proteins identified as part of this system include two subunits of a cytochrome b (cytochrome b-245) and two cytosolic factors. The affected oxidase component was determined in 63 CGD patients from 57 families, by Western blotting of extracts of their neutrophils with antibodies to those proteins. 38 (67%) of the families were X-linked with a defect of the subunit of the cytochrome. 13 (23%) lacked p47-phox, 3 (5%) p67-phox, and 3 (5%) the a subunit of the cytochrome.
Journal of Leukocyte Biology, 1992
Five male patients from four different families presented with a clinical record of chronic granulomatous disease (CGD): recurrent infections of the skin and/or respiratory tract with catalase-positive microorganisms, sometimes in combination with granulomata and/or abscesses in various organs. These patients differed from "classical" forms of the disease in that their neutrophils, although deficient in killing in vitro of Staphylococcus aureus, contained a decreased but measurable amount of cytochrome b358 (10-60% of normal on a heme basis), causing weak staining in the nitroblue tetrazolium dye test and a depressed respiratory burst after contact of the cells with fluid or particulate activators of the NADPH:02 oxidoreductase. In the cell-free activation system, the defect in the patients' cells was localized in the membrane fraction. In each of the four families, the cellular abnormalities showed an X-linked inheritance. Fusion experiments performed with the monocytes from these patients and those from patients with classical Xlinked, cytochrome b558-negative (Xb#{176}) or autosomal, cytochrome b558-positive (Ab) CGD showed complementation of NADPH:02 oxidoreductase activity in the latter but not in the former combination. Thus, the unusual CGD patients represent variant forms of XbO CGD, with mutations in the gene coding for the beta subunit of cytochrome b558 that do not cause complete loss of this protein.J. Leukoc. Biol. 51: 164-171; 1992.
…, 1994
Chronic granulomatous disease (CGD) is characterized by the failure of phagocytic leukocytes to kill certain bacteria and fungi. This is caused by deficiencies in one of the components of NADPH oxidase, the enzyme in phagocytic leukocytes that generates superoxide. In a rare, autosomal recessive form of CGD, a 67-kD cytosolic component of NADPH oxidase (p67-phox) is missing. Until now, mutations in the gene coding for this protein have not been identified. We now report on a 1 0-year-old girl with lymph node and liver abscesses who was recognized as an A67' CGD patient by lack of NADPH oxidase activity in her granulocytes, a cytosolic defect in a cell-free oxidase system, and lack of immunoreactive material with an antiserum against the p67-phox protein. mRNA for this protein was present HRONIC GRANULOMATOUS disease (CGD) is a rare syndrome clinically characterized by recurrent, life-threatening pyogenic infections, with bacteria and fungi, of subcutaneous tissues, upper airways, lungs, bones, spleen, liver and lymph nodes.' This disease is caused by the inability of the patients' phagocytes (neutrophilic granulocytes, eosinophilic granulocytes, monocytes, and macrophages) to generate superoxide, a precursor of reactive oxygen metabolites essential for the killing of various microorganisms. In normal phagocytes, the enzyme NADPH oxidase starts to produce superoxide when the cells are activated, eg, by phagocytosis of microorganisms. This enzyme is composed of several subunits, some of which are localized in the plasma membrane and others (in resting cells) in the cytosol.* During phagocytosis, the cytosolic components translocate to the plasma membrane and integrate with the membrane-bound components into an enzymatically active complex. Defects in any of the four NADPH oxidase components cause inactivity of the enzyme, thus leading to CGD.3
Journal of Experimental Medicine, 1993
The respiratory burst oxidase of phagocytes and B lymphocytes is a multicomponent enzyme that catalyzes the one-electron reduction of oxygen by NADPH. It is responsible for the O2-production that occurs when these cells are exposed to phorbol 12-myristate 13-acetate or physiologic stimuli, such as phagocytosis in phagocytes or cross-linking of surface immunoglobulin in B lymphocytes. The activity of this enzyme is greatly diminished or absent in patients with chronic granulomatous disease (CGD), an inherited disorder characterized by a severe defect in host defense against bacteria and fungi. In every CGD patient studied so far, an abnormality has been found in a gene encoding one of the four components of the respiratory burst oxidase: the membrane proteins p22phox or gp91phox which together form the cytochrome b558 protein, or the cytosolic proteins p47phox or p67phox. Autosomal recessive cytochrome-negative CGD (A22(0) CGD) is associated with mutations in the gene coding for p22p...
Journal of Clinical Investigation, 1984
The NADPH-dependent 02-generating oxidase in subcellular fractions from the neutrophils ofthree male patients with chronic granulomatous disease was compared with the corresponding preparations from normal neutrophils. The oxidase from normal neutrophils contained flavin adenine dinucleotide in an -0.9:1 molar ratio with cytochrome b559. Each of the three chronic granulomatous disease patients had decreased amounts of the flavoprotein component of the oxidase fraction. The oxidase from two chronic granulomatous disease patients had undetectable amounts of cytochrome b559 whereas the third patient had a normal content of cytochrome b559, which was spectrally indistinguishable from the normal. The intrinsic cytochrome b559 in the oxidase fraction from stimulated neutrophils of the latter chronic granulomatous disease patient was not reduced by NADPH under anaerobic conditions, in distinction with the previously reported reduction of the normal cytochrome b559 under identical conditions. We conclude that the flavoprotein component of the oxidase may mediate transfer of electrons from NADPH to the cytochrome b559 in normal neutrophils, and that deficiency of this flavoprotein is associated with the chronic granulomatous disease phenotype in the three patients studied.
Deficiency of NADPH oxidase activity in chronic granulomatous disease
Journal of Pediatrics, 1977
Deficiency of NADPH granulomatous disease oxidase activity in chronic NADPH oxidase activity was examined in paired 27,000 • g granule fractions isolated from normal polymorphonuclear leukocytes and leukocytes from patients with chronic granulomatous disease. A t O. 17 mM NADPH, the oxidase activity was not measurable in normal resting cells but was activated by phagocytosis. This activation was absent in CGD cells. At higher levels o[ NADPH, activity was present in cells from patients with CGD, although it was lower than normal, and no difference in activity was found between resting and phagocytizing cells. Granule fractions from phagocytizing normal cells exhibited higher activity than granule fractions from resting normal cells at all levels of NADPH. These results suggest that NA DPH oxidase activity is defective in chronic granulomatous disease, and further that the defect is not the absence of the enzyme but rather a faihtre to activate it.
Two X-Linked Chronic Granulomatous Disease Patients with Unusual NADPH Oxidase Properties
Journal of Clinical Immunology, 2011
Background Chronic granulomatous disease (CGD) is an immune deficiency syndrome caused by defects in the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, the enzyme that generates reactive oxygen species (ROS) in phagocytizing leukocytes. This study evaluates the NADPH oxidase capacity in two X-linked CGD patients with mutations in gp91 phox that alter the regions in this membrane-bound NADPH oxidase component involved in docking of the cytosolic component p47 phox .
American journal of …, 1992
Chronic granulomatous disease (CGD) is characterized by the failure of activated phagocytes to generate superoxide. Defects in at least four different genes lead to CGD. Patients with the X-linked form of CGD have mutations in the gene for the beta-subunit of cytochrome b558 (gp9l-phox). Patients with a rare autosomal recessive form of CGD have mutations in the gene for the alpha-subunit of this cytochrome (p22-phox). Usually, this leads to the absence of cytochrome b558 in the phagocytes (A220 CGD). We studied the molecular defect in five European patients from three unrelated families with this type of CGD. P22-phox mRNA was reverse-transcribed, and the coding region was amplified by PCR in one fragment and sequenced. Three patients from one family, with parents that were first cousins, were homozygous for a single base substitution (G-297-'A) resulting in a nonconservative amino acid change (Arg-90->Gln). This mutation was previously found in a compound heterozygote A22°CGD patient. Another patient, also from first-cousin parents, was homozygous for an A-309--G mutation in the open reading frame that predicts a nonconservative amino acid replacement (His-94--'Arg). The fifth patient was also born from a first-cousin marriage and was shown to be homozygous for the absence of exon 4 from the cDNA. In this patient, a G-A substitution was found at position 1 of intron 4 in the genomic DNA. Therefore, the absence of exon 4 in the cDNA of this patient is due to a splicing error. Two additional polymorphisms were also identified -one silent mutation in the open reading frame (G-508*--*A) and one A-640 ---G mutation in the 3' untranslated region of the p22-phox mRNA. This last mutation destroys a DraIII recognition site and is therefore potentially useful for RFLP analysis of CGD families.
American journal of human genetics, 1992
Chronic granulomatous disease (CGD) is characterized by the failure of activated phagocytes to generate superoxide. Defects in at least four different genes lead to CGD. Patients with the X-linked form of CGD have mutations in the gene for the beta-subunit of cytochrome b558 (gp91-phox). Patients with a rare autosomal recessive form of CGD have mutations in the gene for the alpha-subunit of this cytochrome (p22-phox). Usually, this leads to the absence of cytochrome b558 in the phagocytes (A22(0) CGD). We studied the molecular defect in five European patients from three unrelated families with this type of CGD. P22-phox mRNA was reverse-transcribed, and the coding region was amplified by PCR in one fragment and sequenced. Three patients from one family, with parents that were first cousins, were homozygous for a single base substitution (G-297-->A) resulting in a nonconservative amino acid change (Arg-90-->Gln). This mutation was previously found in a compound heterozygote A22...
Genetic and biochemical background of chronic granulomatous disease
Archivum immunologiae et therapiae experimentalis
Chronic granulomatous disease (CGD) is a rare inherited immunodeficiency syndrome caused by a profound defect in the oxygen metabolic burst machinery. Activity of NADPH oxidase is absent or profoundly diminished, as at least one of its components (gp91(phox), p22(phox), p47(phox) and p67(phox)) is lacking or non-functional. This review explains the molecular basis of NADPH oxidase dysfunction by the effects of mutations in genes coding for particular oxidase components. Among the four types of CGD, the most common is X-linked CGD (approximately 65%), with defects in the CYBB gene encoding gp91(phox). A wide spectrum of mutations has been described in the CYBB gene with no predominant genotype. The second most common subtype of CGD caused by NCF1 mutation accounts for 30% of CGD patients and is inherited in an autosomal recessive manner, with predominance of a homozygotous deltaGT deletion in the genotype. The other two CGD subtypes having an autosomal recessive pattern together acco...
Blood, 1989
Chronic granulomatous disease (CGD) is a group of inherited disorders in which phagocytic cells fail to generate antimicrobial oxidants. The various forms of CGD can be classified in terms of the mode of inheritance (either X-linked or autosomal recessive), and whether the neutrophils display the absorbance spectrum of a unique b-type cytochrome important for the function of the respiratory burst oxidase. The finding that purified neutrophil cytochrome b is a heterodimer consisting of a 91kD glycosylated and a 22kD nonglycosylated polypeptide has raised the question of which subunits are absent (or defective) in the various types of CGD. To address this question we have studied the expression of the cytochrome b subunits in three genetically distinct forms of CGD: X-linked/cytochrome b-negative (X-), autosomal recessive/cytochrome b-negative (A-), and autosomal recessive/cytochrome b-positive (A+). Using polyclonal antibodies to each of the two subunits, we prepared Western blots of...
Complementation of NADPH oxidase in p67-phox-deficient CGD patients
European Journal of Biochemistry, 2000
Chronic granulomatous disease (CGD) is due to a functional defect of the O 2 2 generating NADPH oxidase of phagocytes. Epstein±Barr-virus-immortalized B lymphocytes express all the constituents of oxidase with activity 100 times less than that of neutrophils. As in neutrophils, oxidase activity of Epstein±Barr-virus-immortalized B lymphocytes was shown to be defective in the different forms of CGD; these cells were used as a model for the complementation studies of two p67-phox-deficient CGD patients. Reconstitution of oxidase activity was performed in vitro by using a heterologous cell-free assay consisting of membrane-suspended or solubilized and purified cytochrome b 558 that was associated with cytosol or with the isolated cytosolicactivating factors (p67-phox, p47-phox, p40-phox) from healthy or CGD patients. In p67-phox-deficient CGD patients, two cytosolic factors are deficient or missing: p67-phox and p40-phox. Not more than 20% of oxidase activity was recovered by complementing the cytosol of p67-phox-deficient patients with recombinant p67-phox. On the contrary, a complete restoration of oxidase activity was observed when, instead of cytosol, the cytosolic factors were added in the cell-free assay after isolation in combination with cytochrome b 558 purified from neutrophil membrane. Moreover, the simultaneous addition of recombinant p67-phox and recombinant p40-phox reversed the previous complementation in a p40-phox dose-dependent process. These results suggest that in the reconstitution of oxidase activity, p67-phox is the limiting factor; the efficiency of complementation depends on the membrane tissue and the cytosolic environment. In vitro, the transition from the resting to the activated state of oxidase, which results from assembling, requires the dissociation of p40-phox from p67-phox for efficient oxidase activity. In the process, p40-phox could function as a negative regulatory factor and stabilize the resting state.