Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) is a multimeric complex composed of enzymes from the NOX family. NOX plays a pivotal role in the formation of superoxide anion (O-) expending NADPH in the process. The formation of superoxide anion is critical in killing microorganisms in phagocytic leukocytes.
First identified in the 1950s, chronic granulomatous disease (CGD) is a rare heterogeneous condition described as a series of recurrent life-threatening infections. Defective phagocyte NADPH oxidase causes the disease. The ultimate result is the inability of phagocytes such as neutrophils, monocytes, and macrophages to destroy certain microbes. This article describes updates to the clinical biochemistry and management of this phagocyte disorder.
The NOX family (NOX1-5) has conserved structural properties, which allow for catalytic activity. Each homolog is comprised of 6 to 7 transmembrane domains, with 2 heme units in the N-terminal region containing histidine residues and an NADPH binding site in the cytoplasmic C-terminal. The isoforms make a multimeric complex, characterized by a core catalytic subunit with 5 regulatory subunits. The NOX2 isoform is most commonly implicated in CGD.
Normally, phagocytes synthesize reactive oxygen species (ROS) by NADPH oxidase. Superoxide is a potent ROS and is generated within the phagosome that has engulfed a microorganism. NADPH oxidase comprises 5 subunits, 3 cytosolic (p47phox, p67phox and p40phox), and 2 membranous units (gp91phox and p22phox). On activation, the cytosolic components migrate after recruiting Rac1/2 to gp91phox. Once all NADPH oxidase subunits and Rac 1/2 combine, they constitute the fully formed enzyme and become active.
The gp91phox subunit plays a pivotal role in transferring electrons from NADPH and coupling them to molecular oxygen inside the phagosome, via heme and flavin adenine dinucleotide (FAD). This forms an untimely superoxide anion which can then give rise to other ROS, including hydrogen peroxide via superoxide dismutase and hypochlorous acid (via myeloperoxidase), hydroxyl radicals, and secondary amines which can efficiently kill microorganisms. The rapid oxygen consumption and production of superoxide and its metabolites are described as the respiratory burst.
The external membrane of the phagocytic cell merging with bacteria leads to the formation of an intracellular vesicle, where superoxide and oxygen-derived products enable potassium influx and an increased pH within the phagosome. After that, activation of granule proteases such as elastase, proteinase 3, and cathepsin G, lead to the destruction of ingested microorganisms. ROS can give rise to toxic intermediates with added reactivity. The reactive compounds and enzymes collectively form a highly toxic environment that can kill almost all pathogens. Consequentially, patients with CGD having defective ROS production cannot directly or indirectly kill certain pathogens effectively.
There have been studies reporting that CGD patients are unable to enhance neutrophil extracellular traps (NETs), which are involved in entangling and killing bacteria and fungi, due to the reduced hydrogen peroxide formation. Other research suggests that CGD results in reduced efferocytosis, a process where phagocytes remove apoptotic inflammatory cells, and, consequentially, these defects may contribute to the granulomatous inflammation often observed in CGD.
CGD arises from mutations resulting in loss or functional inactivation of the NADPH oxidase complex subunits. There are various genes associated with the 5 components of NADPH oxidase, and mutations to gp91phox, p22phox, p47phox, p67phox, or p40phox genes, which make up the NADPH oxidase complex, account for most of the CGD phenotypes. Most mutations for CGD are autosomal recessive, but only the NOX2 variant is X-linked recessive.
The NOX2 (gp91phox) gene is encoded by CYBB (cytochrome b (-245), beta subunit), which is located on chromosome X. Approximately 70 percent of the CGD patients have the CYBB variant, which accounts for the greater prevalence of CGD in males. The p47phox gene, encoded by NCF1, accounts for 25% of cases due to a guanine-thymine deletion in exon 2. The other mutations account for the remaining 10% of cases.
In some cases, the mutations still render the NADPH oxidase partially intact, and consequentially, the disease phenotype is less severe. However, this depends largely on the gene mutated, the type of mutation, and the position of the mutation within the gene. In general, the NCF1 gene mutations lead to milder forms of the disease.
CGD occurs in 1 in every 200,000 live births in the United States. Due to the X-chromosome linked gene mutation, approximately 80% of CGD patients are males. The rates of this disorder are nearly identical across ethnic and racial groups, and approximately one-third of the X-linked mutations occur de-novo. In cultures where consanguineous marriage is common, the autosomal recessive subtype of the disease is more common than the X-linked recessive forms, and the overall incidence rates are higher. Children with the X-linked variant of CGD tend to have an earlier onset, and suffer a more severe disorder, than the autosomal recessive form.
CGD is due to a failure of the patient’s phagocytic leucocytes to kill a variety of pathogens due to defective NADPH oxidase. This ultimately leads to the poor formation of ROS, which are key molecules in the destruction of microbes. Most commonly, patients with CGD present with pneumonia typically due to catalase-positive organisms. Catalase is an enzyme that can inactivate the hydrogen peroxide that is produced by some bacteria and fungi. It is believed that CGD patients can use hydrogen peroxide produced by catalase-negative microbes to form reactive oxidants, and, consequentially, bypass the intrinsic CGD defect. However, catalase-positive organisms have a greater propensity to infect CGD patients since phagocytes cannot hijack their hydrogen peroxide.
Children with CGD suffer from numerous recurrent bacterial and fungal infections. Symptoms typically begin within the first 2 years of life, however some experience symptoms later in life. The median age at diagnosis is 2.5 to 3 years of age. In patients with a mild case of the disease, diagnosis may not be made until later childhood or adulthood.
The infections often occur in organs that are exposed to the outside environment including lungs, gastrointestinal tract, and skin, as well as the lymph nodes that drain these areas. Many other organs may also be affected by contiguous or hematogenous spread including the liver, bones, kidney, and brain.
Patients also experience other systemic problems in organ systems that are involved in protecting the body from microorganism entry from the outside environment. The most common include skin, gingival, lungs, lymph nodes, gastrointestinal tract, liver, and spleen. Majority of patients experience their first symptoms of CGD during their first year of life, including infections, dermatitis, gastrointestinal complications (obstruction or intermittent bloody diarrhea from colitis), and failure to thrive. Specific complications include portal venopathy, liver abscesses, hepatomegaly, urethral strictures, urinary tract infections, altered renal function, keratitis, periodontitis, gingivitis, gingival hypertrophy, discoid lupus, photosensitivity, vasculitis, chronic respiratory disease, immune thrombocytopenia, juvenile idiopathic arthritis, and growth retardation. There may also be an association with X-linked CGD and McLeod syndrome due to deletion of genes of the Kell erythrocyte antigens leading to hemolytic anemia, neuroacanthocytosis, elevated creatinine phosphokinases, and late-onset peripheral and central nervous system manifestations. Clinical presentation is highly variable, with some suffering multiple complications and others experiencing few symptoms. The importance of this issue is that patients with X–linked CGD need to be evaluated for their Kell phenotype to avoid serious transfusion reactions.
Infections in patients with CGD are typically from catalase positive organisms, commonly Staphylococcus aureus, Burkholderia (Pseudomonas) cepacia complex, Serratia marcescens, and Nocardia species. Aspergillus species are the most common fungal infectious agents. Bacillus Calmette-Guerin (BCG) infections are an issue for patients who received BCG vaccinations and living in endemic tuberculosis areas. Cutaneous abscesses and lymphadenitis are the next most common infection types, but some patients also experience cellulitis, impetigo, osteomyelitis, bacteremia, and adenitis. Other common microbial types include Escherichia coli, Klebsiella species, and Candida species.
In some cases, patients with CGD do not develop overt pyogenic infections instead chronic inflammation develops. Consequentially, granulomas form, which is a hallmark of this disorder, causing symptoms of obstruction (biliary tract, bladder, gastrointestinal, uteropelvic, or bronchial obstruction). The gastrointestinal and genitourinary tracts are the most problematic, but the retina, liver, lungs, and bone are also affected by granulomata. The exact reason for granuloma formation in CGD is unknown, but the general belief is that CGD cells are incapable of disarming chemotactic and inflammatory signals, and, as a result, exuberant inflammation persists.
At the cellular level, CGD can be diagnosed by measuring the capacity of phagocytic leucocytes to form superoxide or hydrogen peroxide. Neutrophilic granulocytes are usually used to detect NADPH oxidase activation in the cells. Additionally, NADPH oxidase assays include cytochrome c reduction assay and the nitroblue tetrazolium slide assay, both of which measure superoxide. Other assays exist to measure hydrogen peroxide including the dihydrorhodamine-123 (DHR) assay and the Amplex Red assay. While clinical history may help indicate the patient’s genetic inheritance pattern, genetic testing is available to identify genetic mutations.
When this disorder was first identified, affected children were certain to die, but now CGD can be managed and has a high survival rate. Management of CGD is based on 3 principles: 1) lifelong antibacterial and antifungal prophylaxis, 2) early diagnosis of infection, and 3) aggressive management of infectious complications. Globally, therapy involves trimethoprim-sulfamethoxazole and itraconazole. Some countries also add interferon gamma therapy. However, this therapy is not accepted globally. This combination of treatment can reduce the rate of severe infections from one per patient per year to almost one per patient per ten years. Live bacterial vaccines are best avoided.
Treatment of acute infections should start early, and include determination of the exact complicating infectious agent and selection of antibiotics or anti-fungal therapies. Early and aggressive therapy is essential to preventing the spread of infection. For infections that fail to respond to therapy within 24 to 48 hours, additional diagnostic procedures should be used to identify the microorganism. For fungal infection, anti-fungal therapy should be started before a diagnosis is confirmed. Surgical removal of refractory fungal infections may be necessary as well. Oral glucocorticoids are commonly prescribed for inflammatory manifestations of CGD. The use of tumor necrosis factor-alpha inhibitors in patients with CGD can be associated with high-risk infections and is not generally recommended.
Hematopoietic cell transplantation (HCT) is the only established curative treatment for CGD. Making a rapid diagnosis is important to identify if HCT is possible. In patients who have undergone HCT, the success rate is highest in young and disease-free individuals. Ultimately, the decision to undergo HCT depends on the prognosis, donor availability, access to transplantation, and patient preference.
Patients without an HLA compatible donor for HCT may be candidates for gene therapy. Gene therapy may also be suited for patients with CGD since the disease often results from single genetic defects. This process includes the transfer of autologous hematopoietic stem and progenitor cells (HSPCs) by retroviral vectors and semi-random integration into the genome. Retroviral vectors provide normal genes to reconstruct the NADPH oxidase activity in deficient cells. Until now, gene therapy success has been limited. Some patients have experienced severe complications, including death, due to abnormal clonal hematopoiesis caused by vector integration. As gene repair technology becomes more advanced, DNA editing using short palindromic repeat/CRISPR associated 9 (CRISPR/Cas9) may be used to repair defective genes in X-linked recessive CGD cases. This method of gene therapy has been shown to restore NADPH oxidase in vitro.
Clinicians evaluating patients with suspected CGD, often consider other disorders such as cystic fibrosis (CF), hyperimmunoglobulin E syndrome, Glucose-6-phosphate dehydrogenase (G6PD) deficiency, Glutathione synthase (GS) deficiency, and Crohn’s disease.
CF patients develop complex infections. However, these infections are typically limited to the lungs with significant bronchiectasis, which is not common in CGD. Patients with hyperimmunoglobulin E syndrome develop Aspergillus lung infections only when lung cysts are present, which is typically unseen in CGD patients. These patients also have elevated IgE levels, whereas CGD patients do not. G6PD deficiency and GS deficiency affect neutrophil respiratory burst and increase susceptibility to bacterial infections. However, G6PD deficiency is associated with hemolytic anemia, and GS deficiency is characterized by hemolytic anemia, increased 5-oxoprolinuria, metabolic acidosis, mental retardation, and other neurological manifestations which are not seen in CGD. Both G6PDand GS deficiencies can be confirmed by showing deficiencies of the respective enzymes in cells. Crohn’s disease presents similarly to CGD colitis. However, Crohn’s is not associated with severe infections and presents without lipid-laden macrophages, which are highly characteristic of CGD colitis. Additionally, since the CGD phenotype can be variable, CGD can be mistaken for pyloric stenosis, food allergies, or iron-deficiency anemia.
The prognosis of CGD is improving with advancements in treatment. Patients can prevent infection with good skin hygiene, antifungals, and antibiotics. Autosomal recessive forms of CGD have a better prognosis compared to X-linked CGD. On average, CGD patients survive at least 40 years especially with the use of long-term prophylactic antimicrobials. Often a severe fungal or bacterial infection can be fatal. Aspergillus is the most common fungal respiratory infection and is the most common cause of death in CGD. Mortality and morbidity will continue to decrease as advances are made in prophylactic methods, HSCT, and other immunomodulatory therapy.
Genome-wide association studies (GWAS) revealed a connection between genes encoding oxidase subunits with autoimmune and auto-inflammatory disorders. Variations of the NCF2 encoding p67phox and NCF4 encoding p40phox have been associated with systemic lupus erythematosus (SLE) and Crohn’s disease.
A substantial fraction of CGD patients experiences a form of the inflammatory disease that greatly resembles inflammatory bowel disease (IBD), especially Crohn’s disease. Symptoms can range from mild to bloody diarrhea and malabsorption. Some patients also describe other chronic inflammatory symptoms including non-infectious arthritis, gingivitis, chorioretinitis or uveitis, glomerulonephritis, and rarely white matter lesions in the brain. The CGD genotype seems to accentuate the standard genetic risk associated with IBD. Granulomata in CGD colitis have sharply defined histiocyte aggregates with surrounding lymphocytic inflammation, unlike the poorly formed granulomata often seen in Crohn’s. When staining for macrophage marker, CD68, CGD bowel disease had significantly lower levels compared to typical Crohn’s disease. While patients with CGD granulomatous colitis respond well to tumor necrosis factor-alpha inhibition, such as infliximab, they often have significant infectious complications, sometimes fatal, than typical patients with Crohn’s disease. Clinically it is important to note the relationship between Crohn’s disease and CGD since this can affect patient diagnosis, morbidity, and treatment options.
There have also been reports of an association of discoid lupus erythematosus (DLE) with female carriers of X-linked CGD. It is believed that autoantibodies are formed from the recurrent antigenic stimulation. Affected women can either have normal or impaired oxidative activity due to lyonization. Typically 15% to 20% of normal oxidase activity is enough to handle infections. However, females with less than 20% of normal oxidase activity can present with a severe CGD phenotype. Common findings include photosensitive skin rashes, oral ulcers, and joint pain. IBD has also been described in women with skewed X-inactivation. Their cutaneous lesions closely resemble DLE, even though serologic markers for SLE are often negative. Female carries with skin lesions and chronic diarrhea reportedly had a lower neutrophil respiratory oxidative burst than unaffected carriers. It is recommended that females with DLE who have experienced recurrent infections especially suppurative or have a family history of early childhood deaths to be screened for CGD using the nitroblue tetrazolium test. Despite the immune-compromised state in patients with CGD, immunosuppressive drugs such as prednisone are often necessary for these autoimmune conditions. Further studies are necessary to define the roles of X inactivation in the pathology of autoimmune and inflammatory manifestations in CGD carriers since the degree of lyonization can change over time.