CGD results from mutations in any of the five genes encoding sub units of the NADPH oxidase or a gene encoding a chaperone protein (see Fig. 1; Table 1). These genes are expressed primarily in myeloid cells and in B and perhaps T lymphocytes, although here its function is not well understood. The biochemical and genetic analysis of CGD has been instrumental in characterizing this com plex enzyme. The oxidase subunits are referred to by their apparent molecular mass (kDa) and have been given the designation phox, for phagocyte oxidase. A b-type cytochrome known as flavocytochrome b558 , a membrane-bound heterodimer composed of gp91phox and p22phox, is the redox center of the oxidase. In North American, Latin American, and European registries, approximately two-thirds of CGD cases result from defects in the X-linked gene encoding the gp91phox subunit, which contains both the flavoprotein and heme binding domains responsible for electron transport. X-linked recessive CGD accounts for a smaller percentage of CGD in areas with higher rates of consanguinity, where autosomal recessive (AR) forms are more frequent. One AR form of CGD is caused by mutations in the gene encoding p22phox, the smaller subunit of flavocytochrome b558 , which provides a critical docking site for a regulatory subunit, p47phox. Defects in EROS, an endoplasmic reticulum protein that facilitates flavocytochrome assembly, are found in a rare new sub group of AR CGD. Other cases of AR CGD involve genetic defects in p47phox, p67phox, or p40phox, three regulatory proteins associated with each other in the cytosol of unstimulated cells but which rapidly move to the membrane to activate flavocytochrome b558 and super oxide formation upon cell activation by inflammatory or phagocytic stimuli. The p40phox subunit plays a selective role in stimulating high level superoxide production within phagosomes or endosomes via membrane-bound phosphatidylinositol-3-phosphate. Formation of the active NADPH oxidase complex also involves the activation of the small guanosine triphosphate (GTP)-binding protein Rac, which then binds to the plasma membrane and p67phox.3 No cases of CGD have been identified resulting from genetic defects in Rac, although a mutation in the blood cell–specific Rac2 isoform was found in an infant with recurrent infections and abnormal neutrophil adhesion, motility, and partial NADPH oxidase defects.
Fig1. NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE (NADPH) OXIDASE AND MOLECULAR GENETICS OF CHRONIC GRANULOMATOUS DISEASE. The leukocyte NADPH oxidase enzyme complex is composed of membrane and cytosolic subunits, referred to by their molecular mass (kDa) and the designation “phox,” for phagocyte oxidase. Chronic granulomatous disease (CGD) results from inactivating recessive mutations in any one of the five phox subunits or CYBC1, as indicated with the approximate incidence, gene, and chromosomal location. CYBB and CYBA refer to cytochrome b-245 β chain and cytochrome b-245 α chain, the large and small subunits of flavocytochrome b558 , whereas NCF refers to neutrophil cytosolic factor, used to designate the cytosolic regulatory subunits of the oxidase. Flavocytochrome b558 is the electron transferase and is located in plasma, specific granule (in neutrophils), and phagosome and some endosome membranes. This heterodimer is composed of gp91phox and p22phox, which are affected in X-linked and an autosomal recessive (AR) form of CGD, respectively. The gp91phox subunit, which contains the flavine adenine dinucleotide (FAD) and heme redox centers, is sometimes referred to as NOX2 (NADPH oxidase 2). CYBC1, also known as EROS (essential for reactive oxygen species), is an endoplasmic reticulum protein important for expression of the flavocytochrome b558 heterodimer and is affected in a rare AR form of CGD. Mutations in the genes encoding p47phox, p67phox, and p40phox account for other AR subgroups. These soluble regulatory proteins form a complex in the cytosol, and upon leukocyte activation, phosphorylation induced conformational changes lead to their binding to flavocytochrome b558 and, for p40phox, to phosphatidylinositol 3-phosphate on phagosome and endosome membranes. The small GTPase, Rac, is also essential for NADPH oxidase enzymatic activity, which in its active GTP-bound state, becomes membrane-bound and activates p67phox. Mutations in Rac do not result in a CGD phenotype. Together, these regulatory proteins activate flavocytochrome b558-mediated transfer of electrons from cytosolic NADPH across the membrane to molecular oxygen, thereby forming superoxide in the extracellular space or within phagosomes or endosomes. Superoxide is converted into H2 O2 , which can diffuse across membranes, and other reactive oxygen species (ROS). (Originally published in Blood. Dinauer MC. Inflammatory consequences of inherited disorders affecting neutrophil function. Blood. 2019;133(20):2130–2139. © the American Society of Hematology.)
Table1. Classification of Chronic Granulomatous Disease
The gene for gp91phox, termed CYBB, spans approximately 30 kb in the Xp21.1 region of the X chromosome. More than 600 distinct mutations have been identified in the gp91phox gene in X-linked CGD (MIM306400), which include deletions, frameshifts, splice site, non sense, and missense mutations that are distributed throughout the gene (Table 2). Approximately 10% to 15% of cases of X-linked CGD are caused by new germline mutations. In most X-linked CGD, gp91phox is completely absent, and there is no measurable flavocytochrome b or superoxide production (the X91° subtype). In about 5% of X-linked cases, gp91phox can be present in normal levels but be nonfunctional (X91+), mutated in such a way that gp91phox is poorly functional (X91−), or expressed in only a small fraction of phagocytes (X91−). The first two “variant” forms of X-linked CGD result from coding sequence mutations, and the latter are caused by mutations in the regulatory portion of the gp91phox gene. Some X-linked CGD patients have large deletions that affect not only CYBB but also portions of or all the flanking gene loci for McLeod hemolytic anemia syndrome (absence of the Kell erythrocyte antigen, Kx), Duchenne muscular dystrophy, and X-linked retinitis pigmentosa. Rare missense point mutations in CYBB lead to markedly impaired flavocytochrome b expression and activity in macrophages, with much less effects in neutrophils. Affected patients were susceptible to mycobacterial infections but did not have other bacterial and fungal infections characteristic of CGD, highlighting the importance of macrophages for controlling mycobacteria.
Table2. Summary of Mutations in the CYBB Gene Encoding gp91pbox in 261 Kindreds With X-linked Chronic Granulomatous Disease
AR CGD involving p22phox (MIM 233690) occurs in approximately 5% of CGD patients and usually involves the complete absence of cytochrome b (A22°), encoded by CYBA. Mutations in A22 CGD are heterogeneous and range from large interstitial gene deletions to point mutations associated with missense, frameshift, or RNA splicing defects. Because the full expression of flavocytochrome b in the membrane requires the production of both subunits, a primary deficiency of either component leads to a secondary loss of the other. Thus neither subunit can be detected on immunoblot analysis in either X91° or A22° CGD. A patient with A22+ CGD has been described with a missense mutation disrupting the binding site for p47phox.
AR patients with p47phox-deficient CGD (MIM 233700) account for approximately one-fourth of cases in the United States and Europe, but only approximately 7% of cases in Japan. The p47phox subunit is encoded by the NCF1 gene. A limited number of mutations have been identified in NCF1. Virtually all patients are either homozygotes or compound heterozygotes for a mutant allele with a GT deletion at the beginning of exon 2 that predicts a premature stop codon following the amino acid residue and results in absence of the p47phox protein. The high frequency of the p47phox GT deletion mutation appears to reflect the existence of at least one closely linked highly conserved p47phox pseudogene(s) that contains this GT deletion. This close physical proximity leads to recombination events between the wild-type gene and pseudogene(s).
A heterogeneous group of mutations in the p67phox gene, NCF2, are responsible for A67 CGD, a rare AR form of CGD (MIM233710) accounting for approximately 5% of cases overall. Almost all mutations identified to date in A67 CGD lead to absent expression of the p67phox protein. However, one A67+ patient has been reported in which a nonfunctional form of p67phox with an amino acid deletion is expressed but is unable to translocate to the membrane or bind to Rac.
Two additional forms of AR CGD, both with partial loss of oxidase activity, have been described. Affected patients have a different phenotype than “classic” CGD, with more inflammatory manifestations, including granulomatous gastrointestinal disease and lupus-like skin lesions and few if any severe deep-seated infections. Following an initial report in a boy with mutations in NCF4, which encodes p40phox, other patients with defects in NCF4 (MIM613960) were identified in another dozen families. Although NADPH oxidase activity on the plasma membrane is typically normal in p40phox-deficient CGD, intracellular oxidant production is markedly impaired. Another uncommon form of AR CGD results from AR mutations in CYBC1 (MIM 618935), which encodes the endoplasmic reticulum resident protein EROS (essential for reactive oxygen species [ROS]) that helps mediate assembly of the flavocytochrome b558 heterodimer. Most patients were from Iceland and homozygous for the same founder mutation. CYBC1 defects seem to affect flavocytochrome b558 expression in monocytes and macrophages much more substantially than in neutrophils. Although several patients with CYBC1 defects developed mycobacterial disease, invasive bacterial and opportunistic fungal infections have not been reported.
Even though more than 90% of patients with CGD have respiratory burst defects that result in undetectable levels of O2− production, there is a surprising heterogeneity in the clinical manifestations of the disease. At one end of the spectrum are patients who begin to have severe bacterial and fungal infections during infancy and who rarely have more than 4 to 12 months between such serious infections. At the other end of the spectrum are patients who are well for many years and then unexpectedly develop a serious infection typical of CGD, such as a staphylococcal hepatic abscess or Aspergillus pneumonia. After their first major infection, some of these patients may be relatively healthy again for another 3 to 10 years before the next severe infection occurs. As a group, patients with X-linked CGD, A22 CGD, and A67 CGD seem to have a more severe clinical course compared with patients with A47 CGD, who have a small amount of detectable oxidant production even in the complete absence of this subunit (Fig. 2G). Individuals with partial respiratory burst activity but less than 10% of normal (most X91− patients; see Table 1) also tend to have disease of intermediate severity. Polymorphisms in oxygen-independent antimicrobial systems or other components regulating the innate immune response are also likely to play an important role in modifying disease severity. Specific polymorphisms in the MPO, mannose-binding lectin, and FcγRIIa genes are associated with a higher risk for granulomatous or autoimmune or rheumatologic complications. Finally, as already mentioned, in contrast to “classic” CGD, the clinical spectrum of disease in patients with mutations involving p40phox or CYBC1 resemble an atypical form of CGD, with inflammatory manifestations much more prominent than infections. Because of this heterogeneity, the diagnosis of CGD should be entertained, not only in young children with recurrent severe infections but also in adolescents and young adults who experience exceptionally severe or unusual infections or granulomatous intestinal inflammation.
Fig2. ANALYSIS OF NEUTROPHIL NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE OXIDASE ACTIVITY FOR THE DIAGNOSIS OF CHRONIC GRANULOMATOUS DISEASE. (A–C) Nitroblue tetrazolium (NBT) slide test. Peripheral blood neutrophils and monocytes from a drop of fresh whole blood were made adherent to glass slides and stimulated with phorbol myristate acetate. (A) Normal neutrophils and monocytes, all of which are NBT-positive. (B) Neutrophils and monocytes from an X-linked chronic granulomatous disease (CGD) patient, which are all NBT-negative. (C) A mixture of NBT-positive and NBT-negative neutrophils from the X-linked carrier mother of the patient in (B). (D–G) DHR 123 flow cytometry test. Nonfluorescent DHR 123 is taken up by neutrophils, which become fluorescent after reaction with reactive oxygen species produced in the respiratory burst. (D) Normal neutrophils. (E) Neutrophils from an X-linked CGD patient, which do not fluoresce after stimulation. (F) A mixture of nonfluorescent and fluorescent neutrophils from an X-linked CGD carrier. (G) Neutrophils from a p47phox-deficient patient, which show weak fluorescence after stimulation. DHR, Dihydrorhodamine; PMA, phorbol myristate acetate.
