Autosomal Recessive Cytochrome B-Positive Chronic Granulomatous Disease Type I

Abstract 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 lysates of intact neutrophils from ten CGD patients. In the controls and three patients with A+ CGD, both cytochrome subunits were easily detected. Consistent with the previously reported finding in five X- patients, neither subunit could be identified in neutrophils from three additional X- patients. Both subunits were also undetectable in four patients with A- CGD (three females, one male). This latter group of patients most likely bears a normal 91kD gene, since the patients are genetically distinct from the 91kD-defective X- group. The mutation in A- CGD, therefore, probably involves the 22kD gene and the eventual expression of the 22kD subunit. Furthermore, the expression of the 91kD subunit in this group of patients appears to be prevented due to the 22kD mutation in a manner converse to that seen in the X- CGD patients. Based on these studies, we hypothesize that the stable of expression of either of the two cytochrome subunits is dependent upon the other.

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Absence of both the 91kD and 22kD subunits of human neutrophil cytochrome b in two genetic forms of chronic granulomatous disease

Blood ◽

10.1182/blood.v73.6.1416.bloodjournal7361416 ◽

1989 ◽

Vol 73 (6) ◽

pp. 1416-1420 ◽

Cited By ~ 1

Author(s):

CA Parkos ◽

MC Dinauer ◽

AJ Jesaitis ◽

SH Orkin ◽

JT Curnutte

Keyword(s):

Chronic Granulomatous Disease ◽

Cytochrome B ◽

Autosomal Recessive ◽

Polyclonal Antibodies ◽

Granulomatous Disease ◽

Absorbance Spectrum ◽

Phagocytic Cells ◽

Inherited Disorders ◽

Western Blots ◽

Respiratory Burst Oxidase

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 lysates of intact neutrophils from ten CGD patients. In the controls and three patients with A+ CGD, both cytochrome subunits were easily detected. Consistent with the previously reported finding in five X- patients, neither subunit could be identified in neutrophils from three additional X- patients. Both subunits were also undetectable in four patients with A- CGD (three females, one male). This latter group of patients most likely bears a normal 91kD gene, since the patients are genetically distinct from the 91kD-defective X- group. The mutation in A- CGD, therefore, probably involves the 22kD gene and the eventual expression of the 22kD subunit. Furthermore, the expression of the 91kD subunit in this group of patients appears to be prevented due to the 22kD mutation in a manner converse to that seen in the X- CGD patients. Based on these studies, we hypothesize that the stable of expression of either of the two cytochrome subunits is dependent upon the other.

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Reconstitution of defective respiratory burst activity with partially purified human neutrophil cytochrome B in two genetic forms of chronic granulomatous disease: possible role of Rap1A

Blood ◽

10.1182/blood.v79.9.2438.2438 ◽

1992 ◽

Vol 79 (9) ◽

pp. 2438-2445 ◽

Cited By ~ 9

Author(s):

MT Quinn ◽

JT Curnutte ◽

CA Parkos ◽

ML Mullen ◽

PJ Scott ◽

...

Keyword(s):

Chronic Granulomatous Disease ◽

Cytochrome B ◽

Human Neutrophil ◽

Autosomal Recessive ◽

Plasma Membranes ◽

Close Association ◽

Granulomatous Disease ◽

Detergent System

Abstract Neutrophil plasma membranes from patients with the X-linked and autosomal recessive forms of chronic granulomatous disease (CGD) that lack cytochrome b are incapable of generating superoxide anion (O2-) in vivo and in vitro. The O2- generating activity of these defective membranes was reconstituted with the addition of partially purified human neutrophil cytochrome b in a detergent-based, cell-free activation system. Depending on the detergent system used, 50% to 100% of the activity of control membranes was recovered, and this activity was directly dependent on the cytochrome b concentration. However, when cytochrome b was purified to 99% homogeneity, the reconstitutive capacity of the cytochrome was lost, possibly because of subtle denaturation of the cytochrome or the removal of an additional required cofactor. Examination of the latter possibility with respect to a protein known to coassociate with the cytochrome, ie, Rap1A, indicated that this ras-like protein was present in the partially purified cytochrome preparation used to reconstitute activity in CGD membranes, but was missing in the highly purified preparation. However, the finding that Rap1A was present in normal amounts in the neutrophil membranes from all four major types of CGD (including those missing cytochrome b) suggested that the conditions required of the reconstitution assay did not favor the reassociation of the membrane- derived Rap1A with exogenously added cytochrome b or that another unidentified membrane component was lost during the final purification step. The normal expression of Rap1A in CGD cell membranes also indicates that this protein is not responsible for the absence of O2- production in the X-linked and autosomal recessive cytochrome b- negative forms of CGD. Finally, these results show that the expression of Rap1A in the plasma membrane is not dependent on the coordinate expression of cytochrome b, despite the close association shown for these two proteins in the normal cell membrane.

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Human neutrophil cytochrome b light chain (p22-phox). Gene structure, chromosomal location, and mutations in cytochrome-negative autosomal recessive chronic granulomatous disease.

Journal of Clinical Investigation ◽

10.1172/jci114898 ◽

1990 ◽

Vol 86 (5) ◽

pp. 1729-1737 ◽

Cited By ~ 225

Author(s):

M C Dinauer ◽

E A Pierce ◽

G A Bruns ◽

J T Curnutte ◽

S H Orkin

Keyword(s):

Chronic Granulomatous Disease ◽

Cytochrome B ◽

Gene Structure ◽

Light Chain ◽

Human Neutrophil ◽

Autosomal Recessive ◽

Chromosomal Location ◽

Granulomatous Disease ◽

P22 Phox

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Reconstitution of defective respiratory burst activity with partially purified human neutrophil cytochrome B in two genetic forms of chronic granulomatous disease: possible role of Rap1A

Blood ◽

10.1182/blood.v79.9.2438.bloodjournal7992438 ◽

1992 ◽

Vol 79 (9) ◽

pp. 2438-2445 ◽

Cited By ~ 1

Author(s):

MT Quinn ◽

JT Curnutte ◽

CA Parkos ◽

ML Mullen ◽

PJ Scott ◽

...

Keyword(s):

Chronic Granulomatous Disease ◽

Cytochrome B ◽

Human Neutrophil ◽

Autosomal Recessive ◽

Plasma Membranes ◽

Close Association ◽

Granulomatous Disease ◽

Detergent System

Neutrophil plasma membranes from patients with the X-linked and autosomal recessive forms of chronic granulomatous disease (CGD) that lack cytochrome b are incapable of generating superoxide anion (O2-) in vivo and in vitro. The O2- generating activity of these defective membranes was reconstituted with the addition of partially purified human neutrophil cytochrome b in a detergent-based, cell-free activation system. Depending on the detergent system used, 50% to 100% of the activity of control membranes was recovered, and this activity was directly dependent on the cytochrome b concentration. However, when cytochrome b was purified to 99% homogeneity, the reconstitutive capacity of the cytochrome was lost, possibly because of subtle denaturation of the cytochrome or the removal of an additional required cofactor. Examination of the latter possibility with respect to a protein known to coassociate with the cytochrome, ie, Rap1A, indicated that this ras-like protein was present in the partially purified cytochrome preparation used to reconstitute activity in CGD membranes, but was missing in the highly purified preparation. However, the finding that Rap1A was present in normal amounts in the neutrophil membranes from all four major types of CGD (including those missing cytochrome b) suggested that the conditions required of the reconstitution assay did not favor the reassociation of the membrane- derived Rap1A with exogenously added cytochrome b or that another unidentified membrane component was lost during the final purification step. The normal expression of Rap1A in CGD cell membranes also indicates that this protein is not responsible for the absence of O2- production in the X-linked and autosomal recessive cytochrome b- negative forms of CGD. Finally, these results show that the expression of Rap1A in the plasma membrane is not dependent on the coordinate expression of cytochrome b, despite the close association shown for these two proteins in the normal cell membrane.

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Reevaluation of cytochrome b and flavin adenine dinucleotide in neutrophils from patients with chronic granulomatous disease and description of a family with probable autosomal recessive inheritance of cytochrome b deficiency

Blood ◽

10.1182/blood.v67.4.1132.1132 ◽

1986 ◽

Vol 67 (4) ◽

pp. 1132-1138 ◽

Cited By ~ 43

Author(s):

Y Ohno ◽

ES Buescher ◽

R Roberts ◽

JA Metcalf ◽

JI Gallin

Keyword(s):

Chronic Granulomatous Disease ◽

Cytochrome B ◽

Polymorphonuclear Leukocytes ◽

Autosomal Recessive ◽

Flavin Adenine Dinucleotide ◽

Autosomal Recessive Inheritance ◽

Granulomatous Disease ◽

Recessive Inheritance ◽

Generating System ◽

Normal Adults

Abstract Chronic granulomatous disease (CGD) is a genetically heterogeneous syndrome characterized by a microbial killing defect of polymorphonuclear leukocytes (PMNs) due to lack of superoxide O2-. 2 generation. Recent studies indicate that the neutrophil O2-.-generating system consists of at least two components, flavoprotein--flavin adenine dinucleotide (FAD)--and cytochrome b. We evaluate the cytochrome b and FAD content in PMN from 30 CGD patients. The method for quantitating cytochrome b was modified by using PMN sonicates incubated with azide plus hydrogen peroxide. With this approach, several absorption peaks corresponding to myeloperoxidase and eosinophil peroxidase, which overlap with peaks of cytochrome b, were obliterated from reduced-minus-oxidized spectra, whereas the peaks of cytochrome b were not and could be readily quantitated. Cytochrome b was detected in PMNs from all 24 normal adults (47.4 +/- 2.9 pmol/7.5 X 10(6) cells), was absent in PMNs from 11 male CGD patients and one female CGD patient but was present in normal amounts in PMNs from nine male and nine female CGD patients. Stimulated nitroblue tetrazolium (NBT) tests performed on PMNs from mothers of CGD patients indicated that cytochrome b deficiency was associated with X-linked inheritance, except in one case in which probable autosomal recessive inheritance was demonstrated. The PMN NBT test of the mother of another male patient without cytochrome b deficiency suggested an X-linked form of inheritance. In related studies, the FAD content in PMN particulate fractions was reduced in 4 of 28 CGD patients studied. All four CGD patients with reduced FAD lacked cytochrome b. However, three patients with cytochrome b deficiency had normal FAD. Thus, the results indicate that PMN cytochrome b deficiency is observed in most X-linked and in some autosomal recessive CGD, that cytochrome b deficiency may be associated with FAD deficiency, and that cytochrome b and FAD are normal in most patients with non-X-linked CGD.

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Reevaluation of cytochrome b and flavin adenine dinucleotide in neutrophils from patients with chronic granulomatous disease and description of a family with probable autosomal recessive inheritance of cytochrome b deficiency

Blood ◽

10.1182/blood.v67.4.1132.bloodjournal6741132 ◽

1986 ◽

Vol 67 (4) ◽

pp. 1132-1138

Author(s):

Y Ohno ◽

ES Buescher ◽

R Roberts ◽

JA Metcalf ◽

JI Gallin

Keyword(s):

Chronic Granulomatous Disease ◽

Cytochrome B ◽

Polymorphonuclear Leukocytes ◽

Autosomal Recessive ◽

Flavin Adenine Dinucleotide ◽

Autosomal Recessive Inheritance ◽

Granulomatous Disease ◽

Recessive Inheritance ◽

Generating System ◽

Normal Adults

Chronic granulomatous disease (CGD) is a genetically heterogeneous syndrome characterized by a microbial killing defect of polymorphonuclear leukocytes (PMNs) due to lack of superoxide O2-. 2 generation. Recent studies indicate that the neutrophil O2-.-generating system consists of at least two components, flavoprotein--flavin adenine dinucleotide (FAD)--and cytochrome b. We evaluate the cytochrome b and FAD content in PMN from 30 CGD patients. The method for quantitating cytochrome b was modified by using PMN sonicates incubated with azide plus hydrogen peroxide. With this approach, several absorption peaks corresponding to myeloperoxidase and eosinophil peroxidase, which overlap with peaks of cytochrome b, were obliterated from reduced-minus-oxidized spectra, whereas the peaks of cytochrome b were not and could be readily quantitated. Cytochrome b was detected in PMNs from all 24 normal adults (47.4 +/- 2.9 pmol/7.5 X 10(6) cells), was absent in PMNs from 11 male CGD patients and one female CGD patient but was present in normal amounts in PMNs from nine male and nine female CGD patients. Stimulated nitroblue tetrazolium (NBT) tests performed on PMNs from mothers of CGD patients indicated that cytochrome b deficiency was associated with X-linked inheritance, except in one case in which probable autosomal recessive inheritance was demonstrated. The PMN NBT test of the mother of another male patient without cytochrome b deficiency suggested an X-linked form of inheritance. In related studies, the FAD content in PMN particulate fractions was reduced in 4 of 28 CGD patients studied. All four CGD patients with reduced FAD lacked cytochrome b. However, three patients with cytochrome b deficiency had normal FAD. Thus, the results indicate that PMN cytochrome b deficiency is observed in most X-linked and in some autosomal recessive CGD, that cytochrome b deficiency may be associated with FAD deficiency, and that cytochrome b and FAD are normal in most patients with non-X-linked CGD.

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