Arch Immunol Ther Exp, 2004, 52, 113–120 WWW.AITE–ONLINE .

ORG
PL ISSN 0004-069X Review

Received: 2003.11.10
Accepted: 2003.12.11 Genetic and biochemical background
Published: 2004.04.15
of chronic granulomatous disease
Monika Jurkowska1, 2, Ewa Bernatowska3 and Jerzy Bal1

1
National Research Institute of Mother and Child, Warsaw, Poland
2
Postgraduate School of Molecular Medicine, Medical University of Warsaw, Poland
3
Children’s Memorial Health Institute, Warsaw, Poland

Source of support: self financing

Summary
Chronic granulomatous disease (CGD) is a rare inherited immunodeficency syndrome
caused by a profound defect in the oxygen metabolic burst machinery. Activity of NADPH
oxidase is absent or profoundly diminished, as at least one of its components (gp91phox,
p22phox, p47phox and p67phox) is lacking or non-functional. This review explains the mole-
cular basis of NADPH oxidase dysfunction by the effects of mutations in genes coding for
particular oxidase components. Among the four types of CGD, the most common is X-
-linked CGD (approximately 65%), with defects in the CYBB gene encoding gp91phox. A
wide spectrum of mutations has been described in the CYBB gene with no predominant
genotype. The second most common subtype of CGD caused by NCF1 mutation accounts
for 30% of CGD patients and is inherited in an autosomal recessive manner, with pre-
dominance of a homozygotous ∆GT deletion in the genotype. The other two CGD sub-
types having an autosomal recessive pattern together account for no more than 10% of
CGD cases. A strategy for the molecular diagnostics in CGD patients is proposed and prin-
ciples of genetic counseling are discussed here.
Key words: CGD • NADPH oxidase • genes • molecular diagnostics

Full-text PDF: http://www.aite−online/pdf/vol_52/no_2/5240.pdf

Author’s address: Monika Jurkowska, Department of Medical Genetics, Institute of Mother and Child, Kasprzaka 17a,
01−211 Warsaw, Poland, tel./fax: +48 22 32 77 200, e−mail: mjurkowska@imid.med.pl or mjurkowska@gazeta.pl

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Arch Immunol Ther Exp, 2004, 52, 113–120

CLINICAL FEATURES tochemistry as the absence one or more of its com-

OF CHRONIC GRANULOMATOUS DISEASE ponents, i.e. gp91phox, p22phox, p47phox and p67phox
(Table 1). In rare cases, the functional activity of one
Chronic granulomatous disease (CGD; OMIM of these components is significantly decreased, also
(http://www.ncbi.nlm.nih.gov/Omim/) 306400, 233690, resulting in the CGD phenotype. Generally, the phe-
233700, 233710) is a rare inherited immunodeficien- notype of NADPN oxidase deficiency is quite easy to
cy syndrome seen in approximately 1 in 250 000 indi- detect, although sometimes other host defense sys-
viduals without major regard to ethnic background. tem defects or a mild course of the disease may be
The disease is caused by a profound defect in the oxy- confusing.
gen metabolic burst that normally accompanies
phagocytosis in all myeloid cells: neutrophils, Biochemical differential diagnosis of CGD is based
eosinophils, monocytes and macrophages. on respiratory burst measurement manifested in oxy-
gen consumption, superoxide (O2–) generation in the
Biochemically, CGD is characterized by the inability nitroblue tetrazolium test (NBT), or hydrogen perox-
of phagocytic leukocytes to generate the reactive oxy- ide production. Chemiluminescence and flow cytom-
gen compounds which are needed for the intracellu- etry can also follow these parameters.
lar killing of phagocytized microorganisms9. The oxy-
gen derivatives created by NADPH oxidase during The complex structure of the bust machinery enables
the burst (superoxide, hydrogen peroxide, hypo- numerous possible defect sites. In fact, it is not
halous acids, and hydroxyl radical) play a critical role reflected in the uniform phenotype represented by
in the killing of pathogenic bacteria and fungi. CGD patients. No matter which element fails, com-
Therefore, when the system fails the most common plete lack of or (in rare cases) a profound decrease in
pathogens encountered in CGD patients are cata- oxidase burst is observed with all clinical conse-
lase-positive organisms, because catalase prevents quences.
the CGD phagocytes from using microbial-generated
hydrogen peroxide to kill these pathogens. NADPH oxidase assembly

Typical infections are pneumonia, lymphadenitis, In resting, non-phagocytizing leukocytes, NADPH

cutaneous, hepatic and perirectal abscesses, osteo- oxidase lacks activity and the enzyme components are
myelitis, sepsis caused by Staphylococcus aureus, localized in different parts of the cell. Phagocyte acti-
Aspergillus spp., Candida albicans, Escherichia coli, vation, e.g. by the binding of opsonized microorgan-
Salmonella spp., Brukholderia cepacia (Pseudomonas isms to cell-surface receptors, leads to assembly of
cepacia), and other Gram-negative bacteria. In addi- the active enzyme complex bound together with Src
tion, CGD patients suffer from diffuse granulomas of homology 3 (SH3) domains that interact with pro-
the esophagus, stomach, biliary system, brain, line-rich regions (Fig. 1). Oxidase activation needs at
ureters, or urinary bladder, presumably caused by least five different protein factors. In fact, during the
microbes. Granulomas are an important cause of course of neutrophil stimulation, the cytosolic factors
chronic complications in CGD. Most CGD patients p47phox, p67phox/p40phox, and GTPase Rac1/2 translo-
develop first symptoms during early childhood; for cate to the plasma membrane and associate with
some, residual respiratory burst activity CGD can cytochrome b55811. Cytochrome b558 is a flavohemo-
present fully in adolescence18. protein consisting of p22phox and heavily glycosylated
gp91phox subunits: this is the redox oxidase center that
MOLECULAR BACKGROUND OF THE CGD PHENOTYPE catalyzes the electron transfer from NADPH to oxy-
gen (NADPH + 2O2 → NADP+ + 2O2– + H+)35. It
The heterogeneous group of CGD patients lacks the binds two non-identical hemes with two pairs of his-
activity of NADPH oxidase, defined by immunohis- tidines8 and at least one flavin-adenine-dinucleotide

Table 1. Characteristic of NADPH oxidase components

Gene Protein Cellular location Assembling domains Aberrant in CGD (%)

CYBB gp91 phox
membrane − ~ 65
NCF1 p47phox cytosol two proline-rich, two SH3 motifs ~ 25
NCF2 p67phox cytosol one proline-rich, two SH3 motifs ~5
CYBA p22phox membrane one proline-rich motif ~5
NCF4 p40phox cytosol one SH3 motif −

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 M. Jurkowska et al. – Genetics and biochemistry of CGD

secretory vesicle
or specific granules
phagosome activation rap1A
O2
gp91
Ĵ
cytosol O2 • p22
rap1A
gp91 Rac1/2

p22
p67
p47
p40
p47
p67

p40

Figure 1. Hypothetical assembly of the NADPH oxidase in phagocytic cells32.

(FAD). The identity of the cytosolic component(s) tein constituents and their genes have been identified
responsible for causing the conformational change in and cloned.
gp91phox is a still an unsettled issue, as is the question
of whether this is the consequence of direct interac- The CYBB gene (GenBank see http://www.ncbi.nlm.
tion of the cytosolic component(s) with gp91phox or an nih.gov/Entrez/) accession number X04011), which
event secondary to its binding to the p22phox subunit. encodes gp91phox, was one of the first to be identified
by positional cloning34 following chromosomal local-
In resting neutrophils, this flavocytochrome is local- ization to Xp21.11. Other human genes encoding the
ized together with rap1A protein in the membranes major components of NADPH oxidase are mapped
of specific granules or a secretory vesicle. During cell each to a different chromosome: NCF1 (p47phox) to
activation, fusion of these organelles with the plasma 7q11.23, NCF2 (p67phox) to 1q25, CYBA (p22phox) to
membrane leads to re-allocation of the oxidase. At 16q24, and NCF4 encoding p40phox to 22q13.1. Up to
the same time, the p47-p67-p40 complex translocates now, mutations in four genes of the NADPH oxidase
to the plasma membrane and forms a complex with complex (except in NCF4) have been associated with
the cytochrome b558. p47phox and p67phox are phospho- CGD. A single missense mutation in Rac2 has
rylated upon activation of the enzyme complex17. In recently been reported to cause a CGD-like condi-
addition, one or more cytosolic GTP-binding pro- tion in one individual. The patient was heterozygous
teins appear to be required for oxidase activity16. For for the mutation but severely affected, suggesting
example, Rac-GTP not only binds to the regulatory that the amino acid substitution acts in a dominant,
p67phox, but also interacts directly with the oxidase negative fashion37.
flavocytochrome and can initiate a signaling pathway
leading to the translocation of cytosolic subunits and gp91phox
assemblage of the enzyme complex15. Binding of
p67phox to the cytochrome b permits electron flow Several studies have probed indirectly the structure
from NADPH to FAD, whereas p47phox-binding is of the gp91phox glycoprotein molecule and its molecu-
necessary for electron flow from FAD via the hemes lar interactions, but the locations of its functional
groups to the oxygen5. domains have been inferred mostly by sequence
homology rather than by direct demonstration.
The NADPH oxidase enzyme system forms a small
transmembrane electron-transport system that gp91phox functions as a flavodehydrogenase, and from
results in the oxidation of NADPH on the cytoplas- sequence comparison between the C-terminal half of
mic surface and the generation of superoxide on the gp91phox and the ferredoxin-NADP1 reductase
outer surface of the membrane, which in turn flavoenzyme family the putative location of the FAD-
becomes the inner surface of the phagosome when -binding and NADPH-binding domains within
invagination occurs during phagocytosis. gp91phox have been deduced33. A recent report, show-
ing that inward H+ currents are absent in cells from a
Analysis of the defects responsible for CGD has gp91phox-lacking patient, suggested that gp91phox
helped to define many of the biochemical and mole- behaves as an unusual H+ channel that allows H+
cular features of this complex system. Individual pro- influx and cytosolic acidification2. Expression of full-

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Arch Immunol Ther Exp, 2004, 52, 113–120

Table 2. Properties of genes involved in CGD

Gene Location Size (kb) Exons mRNA size (kb) Amino acids Modifications
CYBB Xp21.1 30 13 4.7 570 glycosylated
NCF1 7q11.23 18 9 1.4 390 phosphorylated during activation
NCF2 1q25 40 16 2.4 526 phosphorylated during activation
CYBA 16q24 8.5 6 0.8 195 –

-length or N-truncated gp91phox and mutagenesis cytochrome b558 have been identified (X91+). Some
experiments were consistent with gp91phox being the of these have provided interesting information about
H+ channel itself13. the NADPH oxidase structure and activation mecha-
nisms. Rare cases describing X91+ CGD patients
Among the four types of CGD, the most common is have either mutations leading to substitutions in the
X-linked CGD (approximately 65%), with defects in N-terminal part of gp91phox, which affect heme bind-
the CYBB gene encoding gp91phox. The CYBB gene ing and stable interaction with p22phox 6, or bear muta-
encompasses 13 exons spanning 30 kb of genomic tions in the cytosolic C-terminal part of gp91phox. This
DNA (Table 2). A wide spectrum of mutations (sub- region of gp91phox is important for FAD- and
stitutions, deletions, insertions, and splice-site)32 has NADPH-binding and is also involved in the recruit-
been described in the CYBB gene, representing more ment of cytosolic phox proteins27 (Fig. 2). One case
than 200 different changes (http://www.uta.fi/imt/ was reported where cytochrome b558 displayed abnor-
/bioinfo/CYBBbase/). mal distribution within the leukocytes: it was present
in the lysate but not on the surface of the cells. The
The heterogeneity of mutations and the lack of any mutation responsible for that phenotype was
predominant genotype indicate that the worldwide located within a probable FAD-binding sequence
incidence of the disease represents many different (His338Tyr)26.
mutational events, with no evidence for a founder
effect. Such a pattern is expected for a disorder with Several mutations were identified in the promoter
the phenotype of a severe immune-system defect and region of the CYBB gene, one leading to the interest-
recurrent infections. Mutations lead to complete lack ing phenomenon of oxidase activity restricted to
of cytochrome (gp910) or partial loss of CYBB expres- eosinophils with a mild course of CGD36. This indi-
sion (gp91–). In a very few rare cases, missense muta- cates the different mechanism of gp91phox expression
tions resulting in normal but nonfunctional levels of regulation in eosinophils.

156

outside
54

57

inside NH2

537 309
415
339 335 325
403
546 500

369 360
gp91Ǧ phenotype

gp91+ phenotype
COOH
NADPH-binding site
FAD-binding site

Figure 2. Schematic representation of gp91phox unit of cytochrome b558 29 (modified).

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 M. Jurkowska et al. – Genetics and biochemistry of CGD

p47phox Because of the redundancy in large blocs of the

sequence, a physical map of the locus has been diffi-
The second most common subtype of CGD is caused cult to construct. Currently, published mapping stud-
by p45phox deficiency and is inherited in an autosomal ies suggest that the wild-type gene is telomeric, and
recessive manner. Interestingly, it accounts for 30% two or more pseudogenes are at a maximum of
of CGD patients, whereas the other two CGD sub- 1.5–2.0 cM in the direction of the centromere, which
types that have an autosomal recessive pattern is close by genomic standards22. Within the 15 kb
together account for no more than 10% of CGD gene there are 21 Alu sequences and nine Chi-like
cases. sequences. Interestingly, nine Alu sequences are con-
tained within the flanking introns 1 and 2 and all are
p47phox protein contains two SH3 motifs and at least orientated in the 3’ to 5’ direction. The high density
one proline-rich region, both involved in oxidase of Alu sequences, particularly orientated in one
assembly. In resting cells it forms a cytosolic complex direction, has been associated with recombination
with p40phox and p67phox, also proved to be essential mutational events in several disorders. Unlike the
for Rac2 translocation during oxidase activation. NCF1 gene, conversions there were detected in a
minority of patients, whereas they are almost exclu-
The NCF1 gene is around 15 kb long and consists of sively detected in p47phox-deficient CGD34. Only
11 exons varying from 55 to 165 bp in length. The approximately 10% of NCF1 mutations are of de
product of translation is 390 amino acids. The NCF1 novo origin.
promoter lacks TATA and CCAAT boxes, but sever-
al possible binding-sites for PU.1, SP-1, AP-1 and p22phox
Oct1 transcription factors have been identified in the
promoting region4. Despite the fact that the p22phox subunit does not con-
tain redox centers, its presence is essential for
The genomic region of 7q11.23, which contains both NADPH oxidase assembly. In myeloid cells, the
the gene and several pseudogenes, is notable for sev- absence of p22phox protein due to genetic defects also
eral closely spaced, duplicated segments of DNA12. results in the loss of gp91phox expression and vice
In fact, 50.37% of the p47 gene is a repetitive versa, indicating that each of these proteins requires
sequence. Pseudogenes of high homology (>98%) the other for mutual stability. p22phox might serve as a
differ in only a few sites20, including the absence of linker protein between cytosolic components and
the GT sequence (∆GT) at the beginning of exon 2, gp91phox. The primary structure of p22phox suggests it
the commonest mutation in p47phox-deficient contains 4 membrane-spanning domains in the N-ter-
patients. This mutation predicts a frameshift and pre- minal two-thirds of the molecule and a proline-rich
mature stop codon at amino acid 51 3. Few addition- domain in the C-terminal cytoplasmic tail (Fig. 3). In
al pathogenic substitutions were identified in A47 fact, the proline-rich domain of p22phox binds the N-
CGD patients, and the large number of polymorphic -terminal SH3 domain of p47phox 23, and this interac-
sites suggests little dependence of p47phox on a strict tion is believed to play a dominant role in promoting
conformation. the association of the cytosolic complex.

Figure 3. The membrane topology of p22phox subunit, as based on “peptide walking” approach10 (modified).

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Arch Immunol Ther Exp, 2004, 52, 113–120

Missense mutations, resulting in complete lack of As in gp91phox and p22phox deficiencies, p67phox CGD
protein, cause amino acid substitutions within the patients show a high degree of heterogeneity in the
transmembrane regions, whereas mutations that do genetic defects that underlie their disease. The muta-
not affect the synthesis of p22phox, as well as polymor- tions in the p67phox gene identified in the patients
phisms, involve residues located outside the mem- reported lead to marked instability of the p67phox
brane30. Of the 10 different missense mutations mRNA or protein (or both). That results in unde-
known by the year 2000, only 1 results in the expres- tectable levels of p67phox, a profound loss of respira-
sion of stable protein (the A22+ phenotype)7. In this tory burst activity, and a relatively severe clinical phe-
case, the patient was homozygous for the substitution notype, despite the fact that the presence of other
of proline 156 with glutamine14. This particular sub- cytochrome b subunits remains intact28.
stitution was very informative functionally, as bio-
chemical analysis showed that it resulted in the fail- These findings are in good agreement with the pro-
ure of p47phox to translocate to the membrane. posal that p67phox is the protein directly responsible
Proline 156 is within a short proline-rich region in the for the induction of a conformational change in
cytoplasmic tail of p22phox (amino acids 151-160) and gp91phox and with the identification of an “activation
the profound effect of its alteration to glutamine domain” in p67phox responsible for interaction with
highlights the importance of this region of the p22phox cytochrome b558 27. It was implied that p67phox inter-
molecule in interactions with an SH3 domain in acts directly with the gp91phox subunit and exclusively
p47phox. transports Rac1 to the complex.

p67phox p40phox resides in a complex with p67phox in the cytosol

of resting neutrophils. Although it is not required for
p67phox deficiency is the rarest form of the disease, oxidase activity in a cell-free assay, it is thought to
accounting for fewer than 6% of cases. The NCF2 play a role in stabilizing p67phox as well as p47phox in
gene spans approximately 40 kb and contains 16 intact cells. The level of p40phox is decreased in p67-
exons21. Translation results in a 526-amino-acid pro- -deficient patients.
tein. p67phox contains two SH3 domains: one was
found to interact with a proline-rich domain in the C MOLECULAR DIAGNOSTICS OF CGD
terminus of p47phox, and the other was possibly
engaged in an intramolecular bond with a proline- CGD is an example of a genetic disorder where
-rich domain at the center of the molecule. (except for X-CGD) only the discovery of a particu-
lar lesion guarantees precise molecular diagnosis and

∆ ∆ ∆
∆

Figure 4. Proposed system of genetic testing in CGD.

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 M. Jurkowska et al. – Genetics and biochemistry of CGD

enables establishment of the carrier status. This is way of testing for such carriers is by molecular genet-
performed by molecular biology techniques such as ic analysis.
polymerase chain reaction, sequencing, or allele-spe-
cific restriction enzyme analysis. Also single-strand The approach to carrier status establishment in pre-
conformation polymorphism or simple restriction natal diagnosis involves identifying the particular
fragment length polymorphism can be informative in mutation in a given family and then analyzing fetal
defined cases (Fig. 4). DNA directly for the mutant allele(s).

Carrier detection is of great importance for genetic PERSPECTIVES

counseling and prenatal diagnosis of CGD. In the
case of X-linked CGD, this is usually relatively easy, The mainstay of current CGD therapy is antibacteri-
because female carriers (who are mostly healthy) al and antifungal prophylaxis. Antibiotic dosages of
generally exhibit two populations of cells (due to ran- trimethoprim sulfamethoxazole as the drug of choice
dom X-chromosome inactivation): one positive for prevent bacterial and intraconazole fungal infec-
NADPH oxidase activity and the other negative. tions19. In case of infection, therapy includes aggres-
These distinct populations can readily be distin- sive and prolonged application of antibiotics and
guished biochemically using the NBT slide test or prednisone and surgical drainage of abscesses or
flow cytometry. resection (when possible) of granulomas.
Immunomodulatory agents, such as interferon γ, also
In the case of the autosomal recessive CGD forms, play a role in the treatment and prevention of
the strategy is more complicated. Generally, carriers intractable infection. Bone marrow transplantation
of autosomal recessive CGD have uniform popula- and gene therapy may offer the promise of complete
tions of neutrophils that are capable of generating cure in the future.
amounts of O2- within the normal range, and the indi-
viduals concerned have no obvious clinical manifes- Some studies show that CGD in adults may be more
tations. In the relatively few heterozygotes for p22phox common than previously assumed25. In view of the
deficiency in whom cellular flavocytochrome b558 con- possibility of treatment, infection prophylaxis and
centrations have been measured, these too appear genetic counseling for affected families, CGD should
within normal limits. Consequently, the only reliable be excluded in any patient with unexplained infec-
tions or granulomas.

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