Chronic Granulomatous Disease

Etiology

The NOX family (NOX1-5) has conserved structural properties, allowing catalytic activity. Each homolog comprises 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.[4][5][6]

Normally, phagocytes synthesize reactive oxygen species (ROS) by NADPH oxidase. Superoxide is a potent ROS 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 is pivotal 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 respiratory bursts.

The external membrane of the phagocytic cell merging with bacteria leads to an intracellular vesicle formation, 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 leads 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.

Studies report that CGD patients cannot enhance neutrophil extracellular traps (NETs), which are involved in entangling and killing bacteria and fungi, due to reduced hydrogen peroxide formation. Other research suggests that CGD results in reduced efferocytosis, a process where phagocytes remove apoptotic inflammatory cells. These defects may, consequently, 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. Various genes are associated with the 5 components of NADPH oxidase, and mutations to the gp91phox, p22phox, p47phox, p67phox, or p40phox genes 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. The NCF1 gene mutations generally lead to milder forms of the disease.

Epidemiology

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 patients with CGD are males. The incidence rates are nearly identical across ethnic and racial groups, and approximately one-third of the X-linked mutations occur in 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.[7][8][9][10]

Pathophysiology

CGD is due to a failure of the patient’s phagocytic leucocytes to kill various pathogens due to defective NADPH oxidase. This ultimately leads to poor ROS formation, a key molecule 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 produced by some bacteria and fungi. It is believed that patients with CGD can use hydrogen peroxide produced by catalase-negative microbes to form reactive oxidants and, consequentially, bypass the intrinsic CGD defect. However, catalase-positive organisms are more likely to infect patients with CGD since phagocytes cannot hijack microbial hydrogen peroxide production.

History and Physical

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. Diagnosis may not be made until later childhood or adulthood in patients with mild disease.

The infections often occur in organs exposed to the outside environment, including the lungs, gastrointestinal tract, and skin, as well as the lymph nodes that drain these areas. Contiguous or hematogenous spread may also affect many other organs, including the liver, bones, kidney, and brain.

Patients also experience other systemic problems in organ systems that protect the body from microorganism entry from the outside environment. The most common include skin, gingiva, lungs, lymph nodes, gastrointestinal tract, liver, and spleen. The majority of patients experience their first CGD symptoms 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 the Kell erythrocyte antigens' genes 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. This issue is important because 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 live 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, and Candida.

In some cases, patients with CGD do not develop overt pyogenic infections; instead, chronic inflammation develops. Consequentially, granulomas form, a hallmark of this disorder, causing symptoms of obstruction of the biliary tract, bladder, gastrointestinal, uteropelvic, or bronchus. 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. Still, the general belief is that CGD cells are incapable of disarming chemotactic and inflammatory signals, and, as a result, exuberant inflammation persists.

Evaluation

CGD can be diagnosed at the cellular level by measuring phagocytic leucocytes' capacity to form superoxide or hydrogen peroxide. Neutrophilic granulocytes are usually used to detect NADPH oxidase activation in the cells. NADPH oxidase assays include the cytochrome c reduction assay and the nitroblue tetrazolium slide assay, 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 can identify genetic mutations.

Treatment / Management

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 universally. This combination of treatments can reduce severe infections from 1 per patient per year to almost 1 per patient per 10 years. Live bacterial vaccines are best avoided.

Treatment of acute infections should start early, including determining the exact complicating infectious agent and selecting antibiotics or anti-fungal therapies. Early and aggressive therapy is essential in preventing the spread of infection. Additional diagnostic procedures should be used to identify the microorganism for infections that fail to respond to therapy within 24 to 48 hours. Antifungal therapy should be started before a diagnosis of fungal infections 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. The success rate in patients who have undergone HCT 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 CGD patients 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's 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 advances, 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 gene therapy method has been shown to restore NADPH oxidase in vitro.

Differential Diagnosis

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 disease.  Patients with CF develop complex infections. However, these infections are typically limited to the lungs with significant bronchiectasis, which is uncommon in CGD. Patients with hyperimmunoglobulin E syndrome develop Aspergillus lung infections only when lung cysts are present, typically unseen in patients with CGD. 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 G6PD and 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 is 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.

Prognosis

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 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 continue to decrease as advances are made in prophylactic methods, HSCT, and other immunomodulatory therapies.