Molecular Characterization of Autosomal Recessive Chronic Granulomatous Disease Caused by a Defect of the Nicotinamide Adenine Dinucleotide Phosphate (Reduced Form) Oxidase Component p67-phox

Chronic granulomatous disease (CGD) is a rare inherited disorder of phagocytes in which defective production of microbicidal oxidants leads to severe recurrent infections. CGD is caused by mutations in any of 4 genes encoding components of nicotinamide adenine dinucleotide phosphate (reduced form; NADPH) oxidase, the multisubunit enzyme that produces the precursor of these oxidants, superoxide. Approximately 5% of CGD patients have an autosomal recessive form of disease caused by a severe deficiency of p67-phox, a 526-amino acid subunit of the oxidase that appears to regulate electron transport within the enzyme. Here we report the biochemical and molecular characterization of 6 unrelated kindreds with p67-phox deficiency. These studies show that, as in gp91-phox and p22-phox deficiencies, the p67-phox CGD patients show a high degree of heterogeneity in the genetic defects that underlie their disease. Five different mutant alleles were identified: (1) a nonsense mutation in exon 4 (C304 → T); (2) a 5-nucleotide (nt) deletion in exon 13 (nts 1169-1173); (3) a splice mutation in the first nucleotide of intron 4 (G → A); (4) a deletion of 1 nt in exon 9 (A728); and (5) a 9-nt in-frame deletion in exon 2 (nts 55-63). The splice mutation was seen in 3 unrelated kindreds, while the 5-nt deletion was seen in 2 apparently unrelated families (both of Palestinian origin). Homozygosity was present in 4 of the kindreds, 2 of which had consanguineous parentage. In the isolated neutrophils of each of the affected patients in the 6 kindreds, there was no measurable respiratory burst activity and no p67-phox protein detected by immunoblot analysis. The level of 67-phox mRNA was less than 10% of normal in the mononuclear leukocytes from 3 of the 4 patients analyzed by Northern blot studies. Thus, this heterogeneous group of mutations in p67-phox all lead to marked instability of mRNA or protein (or both) that results in the complete loss of NADPH oxidase activity.

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Transient Association of the Nicotinamide Adenine Dinucleotide Phosphate Oxidase Subunits p47phox and p67phox With Phagosomes in Neutrophils From Patients With X-Linked Chronic Granulomatous Disease

Blood ◽

10.1182/blood.v93.10.3521.410k21_3521_3530 ◽

1999 ◽

Vol 93 (10) ◽

pp. 3521-3530 ◽

Cited By ~ 67

Author(s):

Lee-Ann H. Allen ◽

Frank R. DeLeo ◽

Annabelle Gallois ◽

Satoshi Toyoshima ◽

Kensuke Suzuki ◽

...

Keyword(s):

Nadph Oxidase ◽

Chronic Granulomatous Disease ◽

Nicotinamide Adenine Dinucleotide ◽

Polymorphonuclear Leukocytes ◽

Nicotinamide Adenine Dinucleotide Phosphate ◽

Actin Binding ◽

Nicotinamide Adenine ◽

Cell Periphery ◽

Granulomatous Disease ◽

Flavocytochrome B

Optimal microbicidal activity of polymorphonuclear leukocytes (PMNs) requires recruitment of a functional nicotinamide adenine dinucleotide phosphate (NADPH) oxidase to the phagosome. In this study, we used a synchronized phagocytosis assay and immunofluorescence microscopy (IFM) to examine the association of cytosolic NADPH oxidase subunits with phagosomes containing opsonized zymosan (OpZ). Ingestion of OpZ began within 30 seconds of particle binding and forming phagosomes were enriched for both F-actin and the actin-binding protein p57. NADPH oxidase subunits p47phox and p67phox were also recruited to forming phagosomes and were retained on mature phagosomes for at least 15 minutes. Colocalization of F-actin, p57, and p47phox on phagosomes was confirmed by immunoblotting. Translocation of p67phox, but not p57, to forming phagosomes was deficient in PMNs lacking p47phox. Surprisingly, we found that in PMNs from six individuals with X-linked chronic granulomatous disease (CGD), p47phox and p67phox accumulated in the periphagosomal area during ingestion of OpZ. However, in marked contrast to normal PMNs, p47phox and p67phox were shed from nascent phagosomes along with F-actin and p57 once OpZ was internalized (≈5 minutes). These data support a model in which flavocytochrome b is required for stable membrane binding of p47phox and p67phox, but not their association with the cytoskeleton or transport to the cell periphery.

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Disturbed interaction of p21-rac with mutated p67-phox causes chronic granulomatous disease.

Journal of Experimental Medicine ◽

10.1084/jem.184.4.1243 ◽

1996 ◽

Vol 184 (4) ◽

pp. 1243-1249 ◽

Cited By ~ 57

Author(s):

J H Leusen ◽

A de Klein ◽

P M Hilarius ◽

A Ahlin ◽

J Palmblad ◽

...

Keyword(s):

Nadph Oxidase ◽

Chronic Granulomatous Disease ◽

Autosomal Recessive ◽

Cell Activation ◽

Nicotinamide Adenine Dinucleotide Phosphate ◽

Granulomatous Disease ◽

Related Protein ◽

Intracellular Killing ◽

First Case ◽

Recessive Form

Chronic granulomatous disease (CGD) is characterized by the failure of phagocytic leukocytes to generate superoxide, needed for the intracellular killing of microorganisms. This is caused by mutations in any one of the four subunits of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. In a rare, autosomal recessive form of CGD, a 67-kD cytosolic component of this enzyme (p67-phox) is missing. We here report on a patient with a mutation in the p67-phox gene that leads to expression of a nonfunctional p67-phox protein. The purified granulocytes of this patient failed to produce superoxide and contained about half of the normal amount of p67-phox. Analysis of the cDNA and genomic DNA of this patient showed that the patient is a compound heterozygote for a triplet nucleotide deletion in the p67-phox gene, predicting an in-frame deletion of lysine 58 in the p67-phox protein and a larger deletion of 11-13 kb in the other allele. Interestingly, the 58Lys deletion in p67-phox disrupts the interaction with p21-rac1, a ras-related protein involved in the activation of the NADPH oxidase. In contrast to normal neutrophils, in which p47-phox and p67-phox translocate to the plasma membrane upon cell activation, the cells of the patient did not show this translocation, indicating that an interaction between p67-phox and p21-rac1 is essential for translocation of these cytosolic proteins and activation of the NADPH oxidase. Moreover, this CGD patient represents the first case of disease caused by a disturbed binding of a ras-related protein to its target protein.

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Undiagnosed Chronic Granulomatous Disease,Burkholderia cepacia complexPneumonia, and Acquired Hemophagocytic Lymphohistiocytosis: A Deadly Association

Case Reports in Pulmonology ◽

10.1155/2013/874197 ◽

2013 ◽

Vol 2013 ◽

pp. 1-5 ◽

Cited By ~ 1

Author(s):

Maxime Maignan ◽

Colin Verdant ◽

Guillaume F. Bouvet ◽

Michael Van Spall ◽

Yves Berthiaume

Keyword(s):

Nadph Oxidase ◽

Chronic Granulomatous Disease ◽

Hemophagocytic Lymphohistiocytosis ◽

Burkholderia Cepacia ◽

Autosomal Recessive ◽

Dna Analysis ◽

Nicotinamide Adenine Dinucleotide Phosphate ◽

Multiple Organ ◽

Burkholderia Cepacia Complex ◽

Granulomatous Disease

Background. Chronic granulomatous disease is a rare inherited disorder of the phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. The clinical course of the disease is marked by recurrent infections, includingBurkholderia cepacia complexinfection.Case Report. Here we report the case of a 21-year-old male hospitalized for aBurkholderia cepacia complexpneumonia. Despite the broad spectrum antibiotic treatment, fever continued and patient’s condition worsened. Anemia and thrombocytopenia developed together with hypofibrinogenemia. The patient died of multiple organ dysfunction 17 days after his admission. Autopsy revealed hemophagocytosis, suggesting the diagnosis of acquired hemophagocytic lymphohistiocytosis. DNA analysis showed a deletion in the p47phox gene, confirming the diagnosis of autosomal recessive chronic granulomatous disease.Discussion. In addition to chronic granulomatous disease, recent findings have demonstrated thatBurkholderia cepacia complexcan decrease activity of the NADPH oxidase. Interestingly, hemophagocytic lymphohistiocytosis is characterized by an impaired function of the T-cell mediated inflammation which is partly regulated by the NADPH oxidase. Physicians should therefore pay particular attention to this deadly association.

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NADPH oxidase correction by mRNA transfection of apheresis granulocytes in chronic granulomatous disease

Blood Advances ◽

10.1182/bloodadvances.2020003224 ◽

2020 ◽

Vol 4 (23) ◽

pp. 5976-5987

Author(s):

Suk See De Ravin ◽

Julie Brault ◽

Ronald J. Meis ◽

Linhong Li ◽

Narda Theobald ◽

...

Keyword(s):

Nadph Oxidase ◽

Chronic Granulomatous Disease ◽

Messenger Rna ◽

Nicotinamide Adenine Dinucleotide Phosphate ◽

Functional Restoration ◽

In Vivo Studies ◽

Allogeneic Bone ◽

Granulomatous Disease ◽

Primate Model

Abstract Granulocytes from patients with chronic granulomatous disease (CGD) have dysfunctional phagocyte reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase that fails to generate sufficient antimicrobial reactive oxidative species. CGD patients with severe persistent fungal or bacterial infection who do not respond to antibiotic therapy may be given apheresis-derived allogeneic granulocyte transfusions from healthy volunteers to improve clearance of intractable infections. Allogeneic granulocyte donors are not HLA matched, so patients who receive the donor granulocyte products may develop anti-HLA alloimmunity. This not only precludes future use of allogeneic granulocytes in an alloimmunized CGD recipient, but increases the risk of graft failure of those recipients who go on to need an allogeneic bone marrow transplant. Here, we provide the first demonstration of efficient functional restoration of CGD patient apheresis granulocytes by messenger RNA (mRNA) electroporation using a scalable, Good Manufacturing Practice–compliant system to restore protein expression and NADPH oxidase function. Dose-escalating clinical-scale in vivo studies in a nonhuman primate model verify the feasibility, safety, and persistence in peripheral blood of infusions of mRNA-transfected autologous granulocyte-enriched apheresis cells, supporting this novel therapeutic approach as a potential nonalloimmunizing adjunct treatment of intractable infections in CGD patients.

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A new genetic subgroup of chronic granulomatous disease with autosomal recessive mutations in p40phox and selective defects in neutrophil NADPH oxidase activity

Blood ◽

10.1182/blood-2009-07-231498 ◽

2009 ◽

Vol 114 (15) ◽

pp. 3309-3315 ◽

Cited By ~ 287

Author(s):

Juan D. Matute ◽

Andres A. Arias ◽

Nicola A. M. Wright ◽

Iwona Wrobel ◽

Christopher C. M. Waterhouse ◽

...

Keyword(s):

Nadph Oxidase ◽

Chronic Granulomatous Disease ◽

Oxidase Activity ◽

Autosomal Recessive ◽

Premature Stop Codon ◽

Superoxide Production ◽

Human Neutrophils ◽

Granulomatous Disease ◽

Recessive Mutations ◽

Px Domain

Abstract Chronic granulomatous disease (CGD), an immunodeficiency with recurrent pyogenic infections and granulomatous inflammation, results from loss of phagocyte superoxide production by recessive mutations in any 1 of 4 genes encoding subunits of the phagocyte NADPH oxidase. These include gp91phox and p22phox, which form the membrane-integrated flavocytochrome b, and cytosolic subunits p47phox and p67phox. A fifth subunit, p40phox, plays an important role in phagocytosis-induced superoxide production via a phox homology (PX) domain that binds to phosphatidylinositol 3-phosphate (PtdIns(3)P). We report the first case of autosomal recessive mutations in NCF4, the gene encoding p40phox, in a boy who presented with granulomatous colitis. His neutrophils showed a substantial defect in intracellular superoxide production during phagocytosis, whereas extracellular release of superoxide elicited by phorbol ester or formyl-methionyl-leucyl-phenylalanine (fMLF) was unaffected. Genetic analysis of NCF4 showed compound heterozygosity for a frameshift mutation with premature stop codon and a missense mutation predicting a R105Q substitution in the PX domain. Parents and a sibling were healthy heterozygous carriers. p40phoxR105Q lacked binding to PtdIns(3)P and failed to reconstitute phagocytosis-induced oxidase activity in p40phox-deficient granulocytes, with premature loss of p40phoxR105Q from phagosomes. Thus, p40phox binding to PtdIns(3)P is essential for phagocytosis-induced oxidant production in human neutrophils and its absence can be associated with disease.

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Nox2 NADPH oxidase is dispensable for platelet activation or arterial thrombosis in mice

Blood Advances ◽

10.1182/bloodadvances.2018025569 ◽

2019 ◽

Vol 3 (8) ◽

pp. 1272-1284 ◽

Cited By ~ 13

Author(s):

Vijay K. Sonkar ◽

Rahul Kumar ◽

Melissa Jensen ◽

Brett A. Wagner ◽

Anjali A. Sharathkumar ◽

...

Keyword(s):

Carotid Artery ◽

Nadph Oxidase ◽

Chronic Granulomatous Disease ◽

Platelet Activation ◽

Arterial Thrombosis ◽

High Performance ◽

Nicotinamide Adenine Dinucleotide Phosphate ◽

Granulomatous Disease ◽

Female Mice ◽

Chromatography Analysis

Abstract Deficiency of the Nox2 (gp91phox) catalytic subunit of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is a genetic cause of X-linked chronic granulomatous disease, a condition in which patients are prone to infection resulting from the loss of oxidant production by neutrophils. Some studies have suggested a role for superoxide derived from Nox2 NADPH oxidase in platelet activation and thrombosis, but data are conflicting. Using a rigorous and comprehensive approach, we tested the hypothesis that genetic deficiency of Nox2 attenuates platelet activation and arterial thrombosis. Our study was designed to test the genotype differences within male and female mice. Using chloromethyl-dichlorodihydrofluorescein diacetate, a fluorescent dye, as well as high-performance liquid chromatography analysis with dihydroethidium as a probe to detect intracellular reactive oxygen species (ROS), we observed no genotype differences in ROS levels in platelets. Similarly, there were no genotype-dependent differences in levels of mitochondrial ROS. In addition, we did not observe any genotype-associated differences in platelet activation, adhesion, secretion, or aggregation in male or female mice. Platelets from chronic granulomatous disease patients exhibited similar adhesion and aggregation responses as platelets from healthy subjects. Susceptibility to carotid artery thrombosis in a photochemical injury model was similar in wild-type and Nox2-deficient male or female mice. Our findings indicate that Nox2 NADPH oxidase is not an essential source of platelet ROS or a mediator of platelet activation or arterial thrombosis in large vessels, such as the carotid artery.

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Radiographic features of patients with chronic granulomatous disease

LymphoSign Journal ◽

10.14785/lymphosign-2020-0002 ◽

2020 ◽

Vol 7 (2) ◽

pp. 66-80

Author(s):

Paria Kashani ◽

Lara Farras Roca ◽

David Manson

Keyword(s):

Nadph Oxidase ◽

Chronic Granulomatous Disease ◽

Nicotinamide Adenine Dinucleotide Phosphate ◽

Pulmonary Nodules ◽

Severe Infection ◽

Pathogenic Mutation ◽

Lung Abscess ◽

Granuloma Formation ◽

Granulomatous Disease ◽

Radiographic Findings

Introduction: Chronic granulomatous disease (CGD) is one of the most common primary immunodeficiencies of childhood, and is caused by defects in the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex. Alongside neutrophil dysfunction, dysregulation of the immune system predisposes patients to recurrent life-threatening infections as well as granuloma formation, hyperinflammation, and autoimmunity. Examination by imaging (radiography, ultrasound, computed tomography, magnetic resonance) in conjunction with biopsy and tissue or fluid cultures are essential to identify the extent and severity of infections as well as the microorganisms responsible. These modalities also help to guide the management of inflammatory complications. Aim: We highlight the common radiographic findings in 10 pediatric CGD patients followed at our centre over a period of 10 years. Methods: Medical records of patients with confirmed CGD diagnosis were reviewed retrospectively. All had low neutrophil oxidative burst index (NOBI) and pathogenic mutation in 1 of the 5 subunits of the NADPH oxidase. Three patients had autosomal recessive CGD and 7 had X-linked recessive CGD. All but 1 are male. Results: The most common radiographic presentation was hilar lymphadenopathy and pulmonary nodules. Other lung complications include cavitating lesions, lung abscess, pulmonary nodule, and pleuritic nodules. Lymphatic tissue and lymph nodes were involved in 50% of our cohort of patients, while gastrointestinal manifestations were noted in approximately 35% of our patients. These include the presence of pigmented macrophages, multiple granulomas, liver abscess, or detection of Aspergillus in tissue or fluid culture. Discussion: It is essential for clinicians to keep primary immunodeficiency as one of the differential diagnoses in patients who present with severe infection or inflammation. We encourage physicians to consider CGD in patients with above described findings and consider measuring NOBI in patients with early onset infection, inflammation, or granuloma formation. Statement of novelty: We describe the radiographic findings of a pediatric cohort of patients with CGD.

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Histopathological features of patients with chronic granulomatous disease

LymphoSign Journal ◽

10.14785/lymphosign-2019-0009 ◽

2019 ◽

Vol 6 (3) ◽

pp. 107-112

Author(s):

Paria Kashani ◽

Haiying Chen

Keyword(s):

Nadph Oxidase ◽

Chronic Granulomatous Disease ◽

Primary Immunodeficiency ◽

Tissue Injury ◽

Nicotinamide Adenine Dinucleotide Phosphate ◽

Granulomatous Inflammation ◽

Severe Infection ◽

Granulomatous Disease ◽

Histopathological Features ◽

Paediatric Cohort

Introduction: Chronic granulomatous disease (CGD) is a primary immunodeficiency caused by defects in the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex. Affected patients suffer recurrent life-threatening infections due to phagocyte dysfunction and dysregulation of the immune system. Histopathological assessment is important to help identify the extent and severity of infection and tissue injury. Aim: We present pathological findings in 5 patients with CGD who were followed at our centre. Methods: Patient information was reviewed retrospectively in accordance with local institutional guidelines. All patients had confirmed diagnosis of CGD with mutation in one of the 5 subunits of the NADPH oxidase. Results: Histopathological features of the gastrointestinal tract, liver, and spleen are noted, and include the presence of granulomatous inflammation and pigmented macrophages. Discussion: It is essential for clinicians to keep primary immunodeficiency as one of the differential diagnoses in patients with severe infection or inflammation, whether in the absence or presence of granuloma formation. The detection of PAS-positive macrophages, diffuse granulomatous inflammation, and hepatic abscesses should raise strong suspicion of CGD. Statement of novelty: We describe the histopathological findings of a paediatric cohort of patients with CGD.

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