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Blood, Vol. 96 No. 3 (August 1), 2000:
pp. 1106-1112
PHAGOCYTES
Molecular analysis of 9 new families with chronic granulomatous
disease caused by mutations in CYBA, the gene encoding
p22phox
Julie Rae,
Deborah Noack,
Paul G. Heyworth,
Beverly A. Ellis,
John T. Curnutte, and
Andrew R. Cross
From the Department of Immunology, Genentech Inc, South San
Francisco; and the Department of Molecular & Experimental Medicine, The
Scripps Research Institute, La Jolla, CA.
 |
Abstract |
Chronic granulomatous disease is a rare inherited disorder caused by
nonexistent or severely decreased phagocyte superoxide production that
results in a severe defect in host defense and consequent
predisposition to microbial infection. The enzyme responsible for
generating the superoxide, NADPH oxidase, involves at least 5 protein
components. The absence of, or a defect in, any 1 of 4 of these
proteins (p22phox, p47phox,
p67phox, or gp91phox) gives
rise to the known types of chronic granulomatous disease. One of the
rarest forms of the disease is due to defects in the CYBA gene
encoding p22phox, which together with
gp91phox forms flavocytochrome
b558, the catalytic core of NADPH
oxidase. To date, only 9 kindreds with p22phox
deficiency have been described in the literature comprising 10 mutant
alleles. Four polymorphisms in the CYBA gene have also been
reported. Here we describe 9 new, unrelated kindreds containing 12 mutations, 9 of which are novel. In addition, we report 3 new polymorphisms. The novel mutations are (a) deletion of exons 2 and 3, (b) a missense mutation in exon 3 (T155 C), (c) a splice site
mutation at the 5' end of intron 3, (d) a missense mutation in
exon 2 (G74T), (e) a nonsense mutation in exon 1 (G26A), (f) a missense mutation in exon 4 (C268T),
(g) a frameshift in exon 3 due to the insertion of C at C162, (h) a
nonsense mutation in exon 2 (G107A), and (i) a missense
mutation in exon 2 (G70A).
(Blood. 2000;96:1106-1112)
© 2000 by The American Society of Hematology.
| |
Introduction |
Chronic granulomatous disease (CGD) is a rare inherited
disorder of the innate immune system caused by genetic defects in the
superoxide-generating NADPH oxidase of phagocytes.1 In the
absence of superoxide (O2 ) production by
these cells, microbial pathogens are not killed efficiently, and the
host is left vulnerable to recurrent, life-threatening infections.
NADPH oxidase activity requires the participation of at least 5 proteins. Two of them, gp91phox and
p22phox, together form a heterodimeric flavin and
heme-containing protein, flavocytochrome b558, the
catalytic core of the enzyme. Flavocytochrome b558
is present in the specific granule and plasma membranes of resting
neutrophils. In contrast, p47phox,
p67phox, and p40phox form a
complex located in the cytosolic compartment of resting neutrophils.
This complex translocates to the membrane and associates with
flavocytochrome b558 during oxidase activation. The
small GTP-binding protein, Rac2 is also required for oxidase activity, and associates with the membrane during the activation process (review
by Clark2). Defects in the genes encoding 4 of the phox proteins (gp91phox,
p22phox, p47phox, and
p67phox) are known to cause CGD. The protein
p40phox has been strongly implicated in NADPH
oxidase regulation, but its role is unclear at present. No disorders
have been recognized that are due to mutations in the
p40phox gene (NCF-4), although
p40phox levels are reduced in
p67phox-deficient CGD.3,4 Until very
recently, no genetic defects in Rac2 have been reported, which may be
because such a defect produces a lethal phenotype, or because there are
sufficient levels of the closely related Rac1 present in the phagocytes
to compensate for any loss of Rac2. (Rac1 can substitute for Rac2 in
cell-free systems of oxidase activation.)5-7 However, a
single missense mutation in Rac2 has recently been reported to cause a
CGD-like condition in 1 individual. The patient was heterozygous for
the mutation but severely affected, suggesting that the amino acid substitution acts in a dominant-negative fashion. 8,9
In myeloid cells, the absence of p22phox protein
because of genetic defects also results in the loss of
gp91phox expression and vice versa, indicating that
each of these proteins requires the other for mutual stability.
However, this is apparently not true of all cell types, as
gp91phox and p22phox are stably
expressed in the absence of their partners in COS7 cells.10
The primary structure of p22phox suggests it
contains 4 membrane-spanning domains in the N-terminal two-thirds of
the molecule, and a proline-rich domain in the C-terminal cytoplasmic
tail. Such proline-rich regions can mediate protein-protein association
by binding to SH3 domains that are found in a variety of proteins
involved in signal transduction, including the cytosolic phox
proteins. The proline-rich domain of p22phox binds
the N-terminal SH3 domain of p47phox, and this
interaction is believed to play a dominant role in promoting the
association of the cytosolic complex, containing p40phox, p47phox, and
p67phox, with flavocytochrome
b558 (reviewed in Heyworth et al11 and DeLeo and Quinn12).
The incidence of CGD is estimated to be approximately 1 in 200 000 to
250 000 individuals. The most common form (approximately 65%) is
X-linked and is due to defects in the CYBB gene that codes for
gp91phox. The remaining approximately 35% of cases
are inherited in an autosomal recessive manner. A22 CGD (ie, CGD
resulting from a defect in p22phox) is one of the
rarest forms of the disease, accounting for only 6% of cases. The
protein p22phox is encoded by the CYBA
gene, located on chromosome 16q24.13 The approximately
600-base pair (bp) open reading frame is divided into 6 exons spanning
about 8.5-kilobase (kb). Carriers of the autosomal recessive forms of
CGD can be difficult to detect, as they typically appear normal by the
nitroblue tetrazolium slide test and have rates of
O2production within the normal range.
Identification of mutations in individuals with autosomally inherited
forms of CGD provides the only effective basis for detecting carriers
among family members or performing prenatal diagnoses. In the case of
A22 CGD, patients from only 9 families (18 alleles, 10 different
mutations) have been reported so far in the
literature.13-17 Here we report an additional 9 families
and describe 12 mutations, 9 of which are novel, and 3 new
polymorphisms. These polymorphisms are of potential interest because of
a recent report that the C214 wild-type genotype (His72) is
associated with a higher risk of coronary heart disease than the rarer
T214 (Tyr72) genotype18 although this association could not be confirmed by others.19-21 Another recent
report describes a positive association of a polymorphism in the
3' untranslated region with heart disease.22 These
associations may be due to a putative role for
p22phox in the activity of a recently described
isoform of flavocytochrome b558 that is involved in
mitogenic signaling 23.24
| |
Patients, materials, and methods |
Patients with chronic granulomatous disease and family members
Patient 1 is a 2-year-old Hispanic boy whose mother and father are
second cousins. He has a history of recurrent cervical adenitis,
inguinal lymphadenitis, and perirectal abscess from the age of 3 months. A male sibling died at the age of 4 months from a sudden onset
of a gastrointestinal infection. A male cousin died at birth secondary
to respiratory problems, and another male cousin is thought to have
died of multiple infections associated with swollen joints.
Patient 2 is the son of unrelated parents with no family history of
CGD. He had recurrent infections and liver abscesses by the age of 2, a
kidney infection at age 4, and hepatic abscesses at age 8, at which
time he was diagnosed with CGD. After treatment with prophylactic
antibiotics, and with the exception of a liver abscess at age 12, he
remained well until age 19. After discontinuing prophylactic antibiotic
treatment, he developed 2 hepatic abscesses and a soft tissue abscess
over his lower rib cage at age 25. He died at age 29 from
gram-negative septicemia after a brief hospitalization.
Patient 3 is a 3-month-old girl with no family history of CGD. She was
diagnosed after pneumonia caused by Aspergillus.
Patient 4 is a female with no clinical or family history available.
Only DNA from the patient and her mother could be obtained.
Patient 5, a 2-year-old boy of unrelated parents, was referred after a
history of chronic otitis media, recurrent skin infections, chronic
dacryocystitis, intermittent diarrhea, and a leg abscess. There is no
known family history of CGD.
Patient 6 is the 25-year-old daughter of first cousins from Southern
India. Two siblings (1 male, 1 female) both died at age 6, probably as
a result of CGD as determined from a review of pathology sections
showing the presence of multiple granuloma in lung and lymph nodes. The
patient had recurrent pulmonary infections and fever from the age of 10 months, and was diagnosed with CGD at age 8. She has continued to have
recurrent lung infections, an episode of malaria, and a bone abscess
from which Pseudomonas aeruginosa was isolated.
Patients 7 and 7a, are sisters, 15 and 19 years of age. The elder
sister has had multifocal osteomyelitis from which Burkholderia gladioli was cultured, and also chronic immune thrombocytopenia.
Patient 8 is a 5-year-old girl with no known family history of CGD or intermarriage.
Patient 9 is a 6-year-old Hispanic girl diagnosed with CGD after an
episode of Serratia osteomyelitis. A chest x-ray examination showed the presence of multiple tiny granuloma. A male sibling died in
childhood. The parents were not known to be related.
Blood samples
Blood samples were obtained from patients and their family members
(where available) by their physicians, following the procedures and
appropriate consent protocols approved by the Human Subjects Committee
of The Scripps Research Institute.
Neutrophil functional assays
Clinical diagnoses of CGD were confirmed by examination of the
capacity of neutrophils to produce O2
using the nitroblue tetrazolium (NBT) slide test, spectrophotometric assay of cytochrome c reduction, and/or flow cytometry (using dihydrorhodamine [DHR] or dichlorofluorescein [DCF]), as previously described.25 The presence of flavocytochrome
b558 was ascertained using reduced-minus-oxidized
difference spectroscopy26; protein immunoblotting27 and/or by flow cytometry using monoclonal
antibody 7D5.28-29
Preparation of DNA, amplification by polymerase chain reaction, and
sequencing
Genomic DNA was isolated from whole blood using Puregene DNA
Isolation Kits (Gentra Systems, Inc, Minneapolis, MN) or from EBV-transformed cell lines. In the latter case,
1 × 107 cells in phosphate-buffered saline (PBS)
were pelleted, all but 200 µL of the supernatant removed
and the remainder processed using the QIAamp Blood kit (Qiagen,
Valencia, CA). In one patient, total RNA was isolated from whole blood
using the RNeasy Blood Mini kit (Qiagen, Valencia, CA) and reverse
transcriptase-polymerase chain reaction (RT-PCR) was performed using
the Omniscript Reverse Transcriptase and Taq DNA Polymerase (Qiagen,
Valencia, CA).
The following buffer was used for the amplification of each exon: 33.5 mmol/L Tris-HCl, (pH 8.8), 8.3 mmol/L
(NH4)2SO4, 3.35 mmol/L MgCl2,
85 µg/mL bovine serum albumin (BSA), 5% DMSO, 0.125 mmol/L
each dNTP, 90 ng each specific primer, 2.5 units AmpliTaq polymerase,
500 ng genomic DNA. The primers used are shown in Table
1.
For thermocycling, the following conditions were used: For exons 1 through 5, an initial denaturation at 94°C for 3 minutes then 30 cycles of 94°C for 5 seconds, 70°C for 1 minute, followed by a
7-minute extension at 72°C. For exon 6, an initial denaturation at
94°C for 3 minutes then 30 cycles of 94°C for 30 seconds,
63°C for 15 seconds, 72°C for 30 seconds, followed by a
7-minute extension at 72°C.
Amplified segments were purified using a QIAquick PCR purification kit
(Qiagen, Valencia, CA) and sequenced in both directions using the ABI
Prism BigDye Terminator Cycle Sequencing Ready Reaction Kit (PE Applied
Biosystems, Foster City, CA). Exons 1 through 5 were sequenced using
reactions mixtures as follows: 2 µL Ready Reaction Premix, 3 µL 5X
Sequencing Buffer, 10 ng primer, and 2 µL PCR product in a 20 µL
total reaction volume. Exon 6 was sequenced using undiluted BigDye
Terminators: 8 µL Ready Reaction Premix, 20 ng primer, and 4 µL PCR
product in a 20 µL total reaction volume. Sequencing reactions were
purified in 96-well MicroAmp Trays (PE Applied Biosystems,
Foster City, CA) by precipitating with 80 µL 75% isopropanol.
The complementary DNA (cDNA) numbering system we have used here follows
the standard convention that +1 is the A of the initiator ATG codon.
This differs from the numbering of some sequences deposited in GenBank
(accession numbers M21186 and J03774). Twenty-eight should be
subtracted from the GenBank sequence number to make the initiator +1.
We have followed the standard system of designating the CGD phenotype
thus: A22+ represents normal levels of expression of
nonfunctional p22phox protein;
A22 represents diminished expression of
p22phox, and A22° represents an absence of
p22phox expression.
| |
Results |
Absence of NADPH oxidase activity and flavocytochrome
b558 in patients with CGD
The biochemical findings on the patients are reported in Table
2. In all cases, no significant
O2 production was detected by NBT
staining, cytochrome c reduction, or by flow cytometry using DHR or
DCF, or a combination of these methods. Patient 6 showed a small amount
of O2 generation by DCF assay, but
gp91phox or p22phox were
undetectable by immunoblot, suggesting a complete, or near complete
absence of flavocytochrome b558. Similarly, none of
the other patients showed any evidence for
gp91phox or p22phox
polypeptides by immunoblot. Where possible, the parents of the patients
were analyzed biochemically; values for
O2, and flavocytochrome
b558 content were in the normal to low-normal range.
Having demonstrated the absence of flavocytochrome
b558 in these patients, genetic analysis was
undertaken. Because the most common cause of CGD is X-linked and
involves defects in the CYBB gene (coding for
gp91phox), the male patients in this study
(patients 1, 2, and 5) were initially analyzed for defects in the
gp91phox gene by single strand conformational
polymorphism.30 In each case, the results were normal.
Conversely, it was considered most likely that female patients
deficient in flavocytochrome b558 had a primary
defect in CYBA. Except in individuals with a highly skewed
X-chromosome inactivation, or with a XO karyotype, a defect in
CYBB is unlikely to be the cause of CGD in females.
Consequently, all 6 exons of the CYBA gene (coding for
p22phox) were sequenced in each patient. Primers
were chosen such that were greater than or equal to 16 intronic
nucleotides at the 5' ends, and at least 12 intronic nucleotides
at the 3' ends of each exon were sequenced, to increase the
chances of detecting mutations that result in splicing errors (Table
1).
Patient 1.
Attempts to amplify exons 2 and 3 individually from genomic DNA of
patient 1 were unsuccessful. Exons 1, 4, 5, and 6 amplified normally,
testifying to the integrity of the DNA and raising the possibility of a
deletion within the gene. PCR amplification of the patient's genomic
DNA from exon 1 to exon 4 (the shortest PCR practicable) produced a
single fragment of approximately 3.0-kb, in contrast to the normal
4.0-kb fragment (Figure 1A).
These results indicate that the patient is homozygous for a genomic
deletion of exons 2 and 3. Products of both sizes were amplified from
his mother (Figure 1A) and father (not shown), demonstrating that they
are both carriers of this deletion. Analysis of the cDNA of the patient
revealed 2 products, a major messenger RNA (mRNA) species of 443 bp,
corresponding to the skipping of exons 2 and 3, and a minor species of
359 bp, corresponding to the skipping of exons 2, 3, and 4 (Figure 1B).
The patient and his mother are also homozygous for a previously
unreported polymorphism, the father is heterozygous. This novel
polymorphism was the substitution of A for G at nucleotide 403 in exon
6. This causes the nonconservative substitution of lysine for glutamic
acid at amino acid 135. Seventy-five normal individuals were sequenced
without finding another allele of this type (Table 4),
but in view of the mother's good health and positive DHR test, we conclude this represents a rare polymorphism.
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| Fig 1.
Molecular mass analysis of
PCR products derived from amplification of genomic DNA and of mRNA
species derived by reverse transcriptase PCR.
(A) PCR amplification of exons 1 to 4 of genomic DNA from
patient 1, his mother, and a normal control was performed as described
in "Patients, materials, and methods." The normal product is
approximately 4.0-kb, and the product derived from amplification of
exons 1 and 4 with exons 2 and 3 deleted is approximately 3.0-kb. (B)
RT-PCR of mRNA prepared from whole blood was performed as described in
"Patients, materials, and methods." Left lane, markers; right
lanes, 5, 10 and 13 µL of PCR product. The upper band is derived from
a mRNA with exons 2 and 3 skipped, the lower band is derived from a
mRNA species with exons 2, 3, and 4 skipped.
|
|
Patient 2.
This patient was homozygous for a T-to-C transition at nucleotide 155 in exon 3. This missense mutation results in the substitution of
proline for leucine at position 52. No other patient or normal subject
has been found who carry this mutation indicating that it is unlikely
to be a benign polymorphism. No other defects were found in the
patient's CYBA gene. As expected, his mother was heterozygous
for this substitution. DNA from the father was not available.
Patient 3.
This patient was homozygous for the deletion of C244 in exon 4. This
frameshift results in a stop codon at amino acid position 190. The
parents, who are not known to be related, were both found to be
carriers of this single base pair deletion. This defect has been
described previously in an unrelated patient who was heterozygous for
this mutation.13
Patient 4.
Genomic sequencing of DNA from patient 4 revealed a homozygous
splice-site mutation in intron 3, converting the 5'
gtgagttgag. This splicing error results in the loss of exon 3 as detected by RT-PCR. The patient's mother carries this mutation, the
father was not available for study.
Patient 5.
This patient was identified as a compound heterozygote for 2 mutations
in his CYBA gene. The first allele contained a nonsense mutation, a transition of G26A in exon 1, leading to a stop
codon and truncation of the p22phox polypeptide
at amino acid 9 (Figure 2A). This mutation
was inherited from his father and was also found in his sister. The
second allele had a GT transition at nucleotide 74 in exon 2 (Figure 2B). This results in the substitution of glycine 25 with the
larger and more hydrophobic valine. Although this is generally regarded
as a relatively conservative substitution, it apparently leads to an
unstable product, as no protein could be detected in the patient's neutrophils. As expected, the mother is a heterozygous carrier of this
mutation and a normal allele. The patient's sister does not carry the
mutated maternal allele and does not have CGD.
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| Fig 2.
Sequence analysis of exon 1 and 2 in patient 5.
Panel A: Sequence analysis of exon 1 of the CYBA gene of
patient 5 and his family members. The patient, his father, and sister
are all heterozygous for A at nucleotide 26, producing a nonsense codon
at amino acid 9. The normal control shows only G at position 26. Panel
B: Sequence analysis of exon 2 of the CYBA gene of patient 5 and his family members. The patient is homozygous for T at nucleotide
74, causing the replacement of glycine 25 with valine. The patient's
mother is heterozygous as seen by the presence of both G and T at
position 74. The patient's father and sister do not carry this allele
as seen by the appearance of only the G at this position.
|
|
Patient 6.
Through genomic sequencing of DNA from patient 6, we discovered a
homozygous CT transition at nucleotide 268. This causes the
nonconservative replacement of arginine 90 with tryptophan and the loss
of p22phox. The patient's parents are first
cousins, and both are carriers of this mutation.
Patients 7 and 7a.
Two sisters, patients 7 and 7a, were identified as compound
heterozygotes. The maternal allele contained the insertion of C at
nucleotide 162 in exon 3 causing a frameshift and a stop codon at amino
acid 73. The paternal allele contains a missense mutation
C268T; predicting the nonconservative amino acid change arginine 90 tryptophan. This is the same mutation found in
patient 6, but the families are not known to be related.
Patient 8.
This patient is a compound heterozygote, with the allele inherited from
her mother containing a nonsense mutation at nucleotide 107, GA in exon 2, predicting tryptophan 36 being changed to a
stop codon. The patient's second mutation, a transition of
GA at nucleotide 70, also in exon 2, causes a missense defect
predicting the nonconservative substitution of an arginine residue for
glycine at position 24. The father was not available for study.
Patient 9.
This patient is homozygous for the substitution of nucleotide 354 CA, causing the replacement of serine 118 with arginine. As
expected, both parents were carriers of this mutation. This defect has
been described before in another homozygous patient also of Hispanic
heritage.13
Polymorphisms in the CYBA gene
During the course of these studies, we identified a number of
polymorphisms (listed in Table 4) in addition to those described above.
These polymorphisms include 2 nonconservative amino acid substitutions:
lysine 60 threonine caused by an AC transversion at
nucleotide 179 (found 1 allele of 59 studied); and glutamic acid
135 lysine caused by a GA transition at nucleotide 403.
| |
Discussion |
Before this study, only 10 different mutations in the CYBA
gene had been identified as causing A22 CGD. These mutations were found
in patients from 9 kindreds encompassing 18 alleles (Cross et
al31 and references therein). In addition, 4 polymorphisms in the gene had been documented. Here we report on 10 new patients from
9 families in whom we identified 12 mutations, 9 of which are novel. We
also identified 3 previously unreported polymorphisms. Consistent with
previous studies of mutations causing CGD, our results show a wide
variety of defects, insertions, deletions, and substitutions leading to
missense, nonsense, frameshift, and splice-site mutations, with no
preponderance of common affected alleles or "hot-spots." This
heterogeneity has been seen in studies of the X-linked
gp91phox gene
(CYBB),14,30,32 the autosomal
p67phox gene (NCF-2),33-34
and the earlier studies of CYBA.31 The exception to
this pattern is the overwhelming preponderance of a GT deletion ( GT)
at the beginning of exon 2 of the NCF-1 gene, causing
p47phox-deficiency, the second most common form
of CGD. This unusual finding is explained by the presence of at least
1, and probably more, highly homologous pseudogenes that contain the
GT sequence.35-36 Relatively frequent recombination
events between the wild-type gene and the pseudogene(s) account for the
prevalent nature of the GT genotype, and give rise to the single
most common disease-causing allele in CGD.
Two of the mutations identified in this study have been described
previously. The deletion of C244, which we found to be homozygous in
the genomic DNA of patient 3, was first described in a compound heterozygote reported by Dinauer and coworkers.13 In that
case, the other mutant allele carried a missense mutation
G269A, (arginine 90glutamine). These patients are
not known to be related. Patient 9 was found to be homozygous for
C354A, this mutation was found previously in another patient
also heterozygous for this change.13 Although these
patients are not thought to be related, both are of Hispanic origin and
this may reflect the presence of a rare mutant allele in the Hispanic population.
Surprisingly, in the 17 unrelated kindreds with A22 CGD, 4 have changes
causing missense mutations at arginine 90. The first of these patients
was heterozygous for a G269A transition, resulting in the
replacement of arginine 90 with glutamine.13 This same mutation was found in a homozygous state in a patient whose parents were first cousins.16 We have found a second mutation that
causes a change at this amino acid residue in 2 unrelated families.
Patients 7 and 7a were heterozygous, and patient 6 was homozygous for a CT transition at the adjacent nucleotide 268. This mutation
results in the replacement of the arginine with tryptophan rather than glutamine. It is possible that this is a mutational hot spot, because
these 4 events appear to have occurred independently. Both missense
mutations result in the A22° phenotype.
Of the 9 different missense mutations known, including the 4 that are
described here, only 1 results in the expression of stable protein (the
A22+ phenotype). In that instance, the patient was
homozygous for the substitution of proline 156 with
glutamine.15 This particular substitution was very
informative functionally, as biochemical analysis showed that it
resulted in the failure of p47phox to
translocate to the membrane. Proline 156 is within a short proline-rich
region in the cytoplasmic tail of p22phox (amino
acids 151-160) and the profound effect of its alteration to glutamine
highlights the importance of this region of the
p22phox molecule in interactions with an SH3
domain in p47phox.37
It is noteworthy that all the missense mutations reported so far that
result in the A22° phenotype cause amino acid substitutions within
the putative membrane-spanning domains of
p22phox, whereas the mutation causing the
A22+ phenotype and the 4 known polymorphic amino acid
residues fall outside these regions (Figure
3). We speculate that amino-acid substitutions in membrane-spanning domains are poorly tolerated and
lead to unstable protein, particularly if they involve changes in
charge or hydrophobicity. Outside the membrane-spanning domain, even
nonconservative changes seem to be better tolerated. For example, the
histidine 72 tyrosine and glutamic acid
135 lysine polymorphisms both lead to normal levels of
functional p22phox.
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| Fig 3.
Pictorial representation of
p22phox.
The shaded areas represent the putative transmembrane regions. The
N-terminus and C-terminus are both cytoplasmic. The stippled area is
the proline-rich sequence (amino acids 151-160) that mediates a
protein/protein interaction with p47phox. Note
that all missense mutations that result in the complete loss of protein
(the A22° phenotype) are located within the transmembrane regions,
whereas the mutation causing the A22+ phenotype, as well as
the polymorphic amino acids, are located outside the membrane.
|
|
During the course of this study, we found examples of all 4 of the
polymorphisms previously reported in the literature (Table 4);13,16 in addition, we also found 3 new polymorphisms, 2 of which produced amino acid substitutions. The first, lysine 60 arginine, is a conservative substitution predicted to be
located in a cytoplasmic loop between the central 2 membrane-spanning domains of p22phox. The second, glutamine
135 lysine, is a less conservative change, located close to
the cytoplasmic side of the membrane. Polymorphisms within
p22phox may have significance for certain
disease susceptibilities. Inoue and colleagues18 have
recently reported a significant association between the C214 genotype
(histidine 72) and coronary artery disease, compared with the T214
genotype (tyrosine 72), although these findings have not been confirmed
by others.19-21 It has been postulated that
p22phox forms part of an isoform of NADPH
oxidase that may play a signaling role in nonphagocytic
cells38 and very recently such an enzyme has been
described.24 Polymorphic forms of
p22phox might have differential effects in such systems.
The difficulty of detecting heterozygotes carrying exonic deletions is
illustrated in the family of patient 1. Detection cannot be achieved by
direct sequencing alone, because the primers will always amplify the
normal sequence (for example, the parents of patient 1 appear normal by
sequencing). Instead, analysis of the size of fragments generated by
PCR amplification of genomic DNA or cDNA must be used to identify
carriers as shown in Figure 1.
Carrier detection is of great importance for genetic counseling and
prenatal diagnosis. In the case of X-linked CGD, this is usually
relatively easy, because female carriers (who are mostly healthy)
generally exhibit 2 populations of cells (due to X-chromosome inactivation), 1 positive for NADPH oxidase activity and 1 negative. These distinct populations can readily be distinguished biochemically using the NBT slide test or DHR flow cytometry. The situation with the
autosomal recessive forms of CGD is quite different, however.
Generally, carriers of autosomal recessive CGD have uniform populations
of neutrophils that are capable of generating amounts of
O2 within the normal range and the
individuals concerned have no obvious clinical manifestations. In the
relatively few heterozygotes for
p22phox-deficiency in whom cellular
flavocytochrome b558 concentrations have been
measured, these too appear within normal limits. Consequently, the only
reliable way of testing for such carriers is by molecular genetic analysis.
| |
Acknowledgment |
We are grateful to Valerie Moreau for her skilled secretarial assistance.
| |
Footnotes |
Submitted February 17, 2000; accepted March 31, 2000.
Supported by National Institutes of Health Grant Nos. RO1 AI 24838 (A.R.C), CA68276 (P.G.H.), and RR00833 (to the GCRC at TSRI).
Reprints: Andrew R. Cross, Department of Molecular & Experimental Medicine, MEM-241, The Scripps Research Institute, 10550 N
Torrey Pines Rd, La Jolla, CA 92037; e-mail: scross{at}scripps.edu.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
References |
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Chronic granulomatous disease. In:
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Primary Immunodeficiency Diseases, A Molecular and Genetic Approach. New York, NY: Oxford University Press; 1997.
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Clark RA.
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1999;179:S309-S317.
3.
Tsunawaki S, Mizunari H, Nagata M, Tatsuzawa O, Kuratsuji T.
A novel cytosolic component, p40phox, of respiratory burst oxidase associates with p67phox and is absent in patients with chronic granulomatous disease who lack p67phox.
Biochem Biophys Res Commun.
1994;199:1378-1387[Medline]
[Order article via Infotrieve].
4.
Wientjes FB, Hsuan JJ, Totty NF, Segal AW.
P40phox, a third cytosolic component of the activation complex of the NADPH oxidase to contain src homology 3 domains.
Biochem J.
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Abstract
Introduction