Molecular analysis in three cases of X91- variant chronic granulomatous disease

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1995 86: 3575-3582
Molecular analysis in three cases of X91- variant chronic
granulomatous disease
HN Bu-Ghanim, AW Segal, NH Keep and CM Casimir
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Molecular Analysis in Three Cases of X91- Variant Chronic
Granulomatous Disease
By Hanan N. Bu-Ghanim, Anthony W. Segal, Nicholas H. Keep, and Colin M. Casimir
Defects in gp91-phox, the large subunit of cytochrome bm
(b& give rise t o X-linked chronic granulomatous disease
(CGD), a rare inherited condition characterized by an extreme susceptibility t o bacterial and fungal infection. In the
majority of cases, the phagocytes are unable t o generate
any superoxide owing t o complete absence of the flavocytochrome. However, a small minority of these patients do
have somephagocytic oxidase activity. We describehere an
analysis of the molecular basis ofthe disease in three such
variant patients with lesions in the gene coding for gp91phox on the X chromosome. Threedifferent genetic lesions
were found, resulting in thesubstitution of tyrosine for cysteine 244, a deletion of oneof three lysines 313 through 315,
and the deletion of the six C-terminal amino acids, respectively. The functional consequences of these defectson oxi-
dase activity was a reduction t o 12%. 3.6%. and 2.1% of the
normal levels, respectively. Corresponding levels of gp91phox were 20%. 8%. and 16% of normal classifving these
patients as X91-. Microbicidal assays showed that killing of
Staphylococcusaureuswasgrossly
impaired in cells in
which there was 12% normal activity. This implies that if
gene therapy is t o be applied, it must restore oxidase activity
t o a much higher level than that present in the cells of this
patient. The sites of two of the mutations were analyzed on
a model of the C-terminal half of the gp9l-phox, based on
the crystal structure of the homologous protein ferrodoxin
NADP reductase. Possible structural consequences of the
mutations were examined.
0 1995 by The American Society of Hematology.
C
in the right axilla. Over the past 2 years he has spent 6 weeks in
the hospital because of severe infections of the skin and scalp. His
mother and only sister were identified as carriers, and he has one
affected brother who has also had numerous infections, most notably
of the nasal sinuses, some of which required hospitalization.
Patient 2 was born in 1982 to healthy unrelated parents. He developed skin abscesses at 3 weeks of age, and subsequently a colitis
resembling Crohn's disease. He has had repeated skin abcesses and
chronic mouth ulcers, and at the age of 9 years, he developed a liver
abcess.
Patient 3 was born in 1976 to healthy unrelated parents. He developed axillary lymphadenitis after a Bacille Calmette-Gukrin (BCG)
vaccination and developed an axillary abcess at the age of 3 years,
which required surgical drainage. At the age of 4.5 years, he developed a bilateral interstitial pneumonia.
Nitroblue-tetrazolium slide test was performed as described by
Borregaard et al.'* Superoxide generation was measured by the superoxide dismutase inhibitable reduction of cytochrome c.~'
Neutrophils were purified from peripheral blood taken into 5 IU/
mL heparin by dextran sedimentation, followed by separation on
Ficoll-Hypaque (Lymphoprep; Nycomed Pharma AS, Oslo Norway)
and hypotonic 1ysk3*
Western blotting. Neutrophils were suspended in (6% [wUwt]
sucrose; 1.8 mmoVL NaCl; 60 mmoliL KCI; 2.3 mmoliL M g Q ; 6
mmol/L Pipes, pH 7.3) supplemented with 1 p m o K diisopropyl
flourophosphate, 1 pmoVL phenylmethylsulphonyl flouride, 1 pmoV
L chymostatin, and 1 p m o m leupeptin and were disrupted by sonication on ice for 3 to 5 seconds in a sonicator (MSE Instruments;
Sanyo Gallenkamp, Middlesex, UK) at 8 pm amplitude, and centri-
HRONIC granulomatous disease (CGD) is a rare inherited disorder in which phagocytic cells are unable to
generate superoxide, resulting in the defective killing of
some ingested microorganisms and an increased predisposition to bacterial and fungal infections.' Superoxide is produced by a membrane-bound NADPH oxidase system that
is assembled from both membrane and cytosolic components. The membrane-bound component is flavocytochrome
b558(also b-245)rZ.3
a heterodimer composed of a 22-kD ( a )
subunit, p22-phox, and a heavily glycosylated 76- to 92-kD
(p) subunit,4.'gp91-phox. gp91-phox is a member of the
flavin nucleotide reductase (FNR)family of reductases, having binding sites for NADPH and flavin adenine dinucleotide
(FAD) cofactors.6 The location of the heme is uncertain.
The cytosolic factors that are required for the activation of
electron transport and translocate to the membrane upon
tim mu la ti on^.^ include p47-phox,p67-phox,
1 4 3 and p40phox.I6 The system also requires a small guanosine triphosphate binding protein, p21'".'7,'8
Genetic lesions in any of the oxidase components (other
than p40-phon and p21'" for which lesions have not yet been
identified) result in CGD, but defects in the X-linked gene
for the p chain of the flavocytochrome are the most prevalent, leading to about two thirds of cases." The classical Xlinked phenotype is characterized by total absence of
NADPH oxidase activity and both subunits of the flavocytochrome, which are required for mutual stability." In addition, several cases of a less common form of X-linked CCD,
which is referred to as "X-variant" CGD and which differs
from the classical phenotype in having reduced rather than
absent oxidase activity, have been de~cribed.'"~'We have
studied the molecular defect in three unrelated X-variant
CGD cases with different levels of residual oxidase activity
and variable abundance of the two subunits of the flavocytochrome.
MATERIALS AND METHODS
Patients. Patient 1, a male,wasbornin
1980 to two healthy
unrelated parents. At the age of 2.5 years was treated for an abscess
in the occiput. He had continuous ear infections between the ages
of 5 and 9 years but was otherwise fairly well until the age of 10
years, when he developed an abscess and then a chronic infection
Blood, Vol 86, No 9 (November l), 1995:pp 3575-3582
Fromthe Division of Molecular Medicine, University College
London; and the Division of Cell and Molecular Biology, Institute
of Child Health, London, UK.
Submitted February 21, 1995; accepted July 6, 1995.
Supported by the Welcome Trust, Medical Research Council,
NETHRA, and EC BIOMED I program (PI-1321).
Address reprint requests to Anthony W. Segal, MD, PhD, Division
of Molecular Medicine, University College London. 5 University St,
London WClE SJJ, UK.
The publication costs ofthis article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1995 by The American Society of Hematology.
0006-4971/95/8609-07$3.00/0
3575
e
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3576
BU-GHANIM ET AL
Table 1. Primers Used for the PCR Amplification
of gp9l-phOX cDNA
DNA, where 2 pg was used to amplify the region from base 1.573
(exon 12) to 1,746 (exon 13), thus amplifying intron 12 in the process
using primers @7-/36 (Table I ) . The resulting PCR product of = l .2
Oligonucleotide
kb was blunt ended by the addition of S U Klenow fragment to I
81
5' CCT
CTG
CCA CCA TGG GGA
AC
3'
1
p g amplified DNA i n a reaction containing 1 X PCR buffer and 6
P2*
5' GAC l T C A A A GTAAGA CCTCCG GATG 3' 624
mmol/L deoxynucleotide triphosphates. The reaction was incubated
83
5' GCA TCA CTG GAG TTG TCA TCA 3'
539
for 20 minutes at room temperature, The tilled DNA was agarose
CCT
CAT
AGC
TGA
ACA CA 3'
1,222
84*
5' CCA
gel purified and precipitated with ethanol. The purified DNA fragp5
5' ATA
AGC
AGG
AGT
TTC AAG
AT
3'1,127
ment was ligated to Snlu I cut blue-script plasmid (Stratagene, La
86*
5' TTT CCT CAT
GGA
AGA
GAC AAG 3' 1,746
Jolla, CA) using T4 DNA ligase (NBL, Northumberland, UK). The
5' AAG
ACA
ATT
GCA AGT
CAA
CA 3'
1,573
87
products of the ligation reaction were used to transform XLI-Blue
(Stratagene)competentcells
that were plated onampicillinagar
Oligonucleotides 81 through 87 were used for PCR amplification
plates. Positiveclones were picked by screening plate lifts with
(01 and 82;83 and /34;85 and 86;and 87 and 06).They were derived
to this region. Patient 3
radiolabeledDNAprobescorresponding
from the gp91-phoxcDNA ~ e q u e n c e . 3Position
~
within the cDNA seand eight positive clones from each normal control were used for
quence of the most 5' nucleotide of the primer is indicated
sequencing by US Biochemicals' sequenase method.
* Antisense primer.
RNu.se protecfion experimenfs. Probeconstructs used forthe
RNase protection experiment were madeby digesting a cDNA clone
fuged at 250g at4°C for 5 minutesin a microcentrifuge. The resulting (extending from the Psf I site at base number 179 to the Kpn I site
at 3,654) in pGEM-4 vector (Promega) with restriction enzymes that
postnuclearsupernatant(PNS)wassubjectedtosodiumdodecyl
cut once in the vector and once in the coding sequence to eliminate
sulfate-polyacrylamide gel electrophoresis on a 12.5% gel (30 to 40
unwanted cDNA sequences at the3' end of the cDNA, thus bringing
p g protein per track). The proteins were then transferred to a reinthe SP6 RNA promoter into proximity with different regions of the
forcednitrocellulosemembrane
(0.45 pm, Schleicher & Schuell,
cDNA,for the synthesis of antisenseRNAprobes.
ASuc I was
Dassel,
Germany)
using
the
semidry
transfer
system
(LKB
prepared by digesting with Suc I, which cuts the cDNA insert of the
00.8 mA/cm' for120
Multiphor;LKB,Uppsala,Sweden)at
gp9 1 -phox at base number 1,903. The clone was then recircularized
minutes. The blots were blocked with (5% dried skimmed milk,I %
and digested with the Stu 1 restriction enzyme, which cuts at base
bovineserumalbumin,
5% fetalbovineserum,1
m o m glycine,
number 1,523. The other probe, A5.w 361, was made in three steps
0.01% Tween, in TRIS-buffered saline), incubated with monoclonal
of digestionandligation: ( I ) the Pst I-Kpn 1 clone was cut with
antibodies (MoAbs) 449 or 48 reacting
with p22-phox and gp91EcoRI, which cuts once in the polylinker and once in the insert at
phox, respectively(agiftfrom
A.J. Verhoeven3'), anddeveloped
1,567 bp. andrecircularized:(2)
this was then cut with Hindlll
with
horseradish
a
peroxidase-conjugated
goat-antimouse
IgG
(polylinker) and Avu 1 (insert S' end 398 bp) and recircularized; ( 3 )
( 1 :3500; Promega, Madison, WI) by enhanced chemiluminescence
the product of step 2 was cut with EcoRI and Bsu 361 (insert 3' end
detection (ECL; Amersham Ltd, Bucks, UK). The flavocytochrome
1,049 bp). and recircularized; and (4) finally. the product of step 3
level in each patient was estimatedby densitometry (Enhanced Laser
by
was cut with Ncn I (insert 675 bp). The precise areas covered
Densitometer; LKBPharmacia, Uppsala, Sweden) of Western blots
thetwoprobesareasfollows:
ABsu 361 probe,nucleotides 67.5
and developed by ECL. A standard curve was prepared by loading
through 1,049; ASuc I probe nucleotides 1,523 through 1,903.
serial dilutionsofthenormalPNS,where
40 pg of normalPNS
Radiolabeled cRNA probes, ASuc I , and ABsn 361 were prepared
corresponded to 100%. Forty micrograms of the patient's PNS was
using 1 p g of the linearized cDNA clone and SP6 RNA Polymerase
loaded on the gel and comparedwith the normal standards by densiunder conditions recommended by the manufacturer (Promega). Five
tometry of both subunits bands. Care was taken to ensure
that the
nanogram!, of each cRNA radiolabeled probe was mixed with 1 5 to
signal produced by the patients cells fell within a linear region of
20 pg of total cellular RNA and 20 pg of yeast tRNA and incubated
the standard curve.
under oil overnight at 42°C. Theresultinghybridsweredigested
Mutationanalysis.
Thenumbering of nucleotidesandamino
with 4.2 p.g/mL RNase A and 200 U/mL RNase
T I . The reaction
acids used in this paper was according to the system of Orkin."
productwereseparatedona6%denaturingpolyacrylamidegel,
Total cellular RNA was isolated from Epstein-Barr virus-transdried,andautoradiographed
with Hyperfilm MP(Amersham) at
formed B-Iymphocytes3'~'' as described by Chomczynski and Sac-70°C with intensifying screens for up to 1 week.
chi." Avian myeloblastosisvirusreversetranscriptase(Promega)
Iodinationreactionswereperformedasdescribed
by Klebanoff
was used for the synthesis of the first cDNA strand using l p g total
and Clark.'" The reaction mixture consisted of 4 mmol/L sodium
RNA primed with (dT),z~,x
or sequence specific primer 0 6 in 20 pL
phosphatebuffer pH 7.4;128mmol/L NaCI; 12 mmol/L KCI: 10
I .7-kb
reaction as described by the manufacturer (Promega). The
mmol/L CaClz; 2 mmol/L MgCI,; 80 pmol/L Nal (40 nmol in 0.5
open reading frame of the flavocytochrome b wasamplified in three
mL reaction; 0.8 pCi of "'1); 2 mmol/L glucose; 3% normal serum;
overlapping fragments (1-624, 539-1222, and 1127-1746 bp) using
0.5 mg of Zymosan boiled, washed, and resuspended in phosphatethree pairs of synthetic primerswith one primerof each pair biotinylbuffered saline (PBS), and 2.5 X 10' polymorphonuclear leukocytes
ated at its 5' end (oligonucleotides pl and p2; p3 and p4; and 05
(PMNs) in a final volume of 0.5 mL. Iodination by resting cells was
and 8 6 , Table I). For each pair of primers, 5
p L of the reverse
measured in theabsence of zymosan.The reaction mixture was
transcriptase reaction was amplified by polymerase chain reaction
incubated at 37°C for 30 minutes before starting the reaction by the
I
(PCR)in a 50 pL reaction(40cycles of 30 secondsat93°C
additionofthePMNs.The
reaction was terminatedaftervarying
minute at 60°C. 2 minutes at 74OC)."
times by the addition of 0. I mL of 10 mmol/L sodium thiosulphate
Solid phase sequencing3' was performed on the biotinylated PCR
and I mL of cold 10% trichloroacetic acid (TCA). The precipitate
product adsorbed to streptavidin-coated magnetic beads (Dynal AS,
was pelleted by centrifugation at IS,000g for 5 minutes in a microOslo, Norway).Sequencingwasperformed
as describedin the
centrifuge at 4"C, was washed four times with I .0 mL of 10% TCA,
Sequenase manual (US Biochemicals, Cleveland, OH) and the reactions analyzed on standard 6% polyacrylamide denaturing gels. Tem- and the radioactivity was counted in a gamma counter (Universal
Gamma Counter; LKB).
plate for DNA sequencing on the
3' end of the gp91-phox coding
Phagocytosis and killing. Staphylococcus uureus, isolatedfrom
sequence of patient 3 was preparedby PCR amplification of genomic
~~
~
~~
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VARIANT
Table 2. NADPH Oxidase Activity, Levels of gp91-phox and p22-phox, Site of Mutationin the cDNA for gp9l-phox,
and the Predicted Conseauences on Its Primarv Structure
Oxidase Activity
Superoxide Produced
(nmol/lO' cells/rnin)
(mean SD)
=
Patient
1
16.9
2
3
2
% Normal.
2.3 (n = 5)
-t
p22-phox
96 Normal
Mutation
30
Deletion of one of three
AAG repeats starts from
base 949
G743 A
Duplication of bases
1,672-1.702. StOD codon
20 12
5.1 (n = 2)
3.0 (n = 2)
Mean
gp91-phox
% Normal
3.6
2.1
Deletion 315
of Lys
-
9
24
8
16
Amino Acid Change
CysZu Tyr
Deletion of C-terminus 6
amino acids 565-570
+
SD of four normal subjects is 143 2 30 nmol/107 cells/min.
an abscess i n patient I , was grown overnight i n Luria-Broth (LB)
supplemented with 25 pCilmL 'H-glucose (ICN Radiochemicals.
Costa Mesa, CA). These were washed and opsonized as described
previously.'' The reaction containing S X IO7 PMNs and I X IOx
bacteria i n 5 mmol/L glucose PBS was incubated i n a rapidly stirred
chamber at37°C. Aliquots ( S O pL) were taken at intervals into I
m L ice-cold Hanks' buffer (GIBCO-BRL, Gaithersburg. M D ) containing I mmol/L N-ethylmaleimide and 1 0 0 pglmL lysostaphin
(Sigma Chemical Co. Poole. UK), incubated at 37°C for 15 minutes,
and centrifuged at 7.000~at 4°C for 4 minutes. Radioactivity remaining in the supernatant was estimated by scintillation counting
(LKB). A second aliquot (50 pL). was taken at the same time into
I O m L o f ice-cold H,O, incubated for I hour at4°C and dilutions
were plated for colony counts onto blood agar plates.
Patient 2 had very little oxidase activity (3.6%)and a similarly reduced flavocytochrorne b (8% to 9%). In patient 3,
there was a discrepancy between the very low oxidase activity (2.1%) and proportionally greater amounts of flavocytochrome (16% to 24%).
Killing of S aureus by cells from patient 1 was markedly
defective and comparable with the levels observed in cells
from a patient with classical CCD (Fig 2) despite normal
phagocytosis (not shown).
1
RESULTS
All three patients showed weakly staining cells on the
nitroblue tetrazolium (NBT) slide test with a mosaic pattern
of weak and strong staining of cells from their mother, confirming the X-linked pattern of inheritance.
Table 2 and Fig 1 show the levels of the NADPH oxidase
activity and the two subunits of the flavocytochrome b on
Western blots. Patient I retained significant levels of oxidase
activity (12%) and flavocytochrorne protein (20% to 30%).
a
b
~
.-
c-
d
e
f
gp9 l -phox
I
2o
0
l
5
'
"
-
l
~
IO
'
"
l
'
'
'
15
Tlme (min.)
Fig 1. Western blot of neutrophil postnuclear supernatant with
MoAbs 48 and 449. (a) normal control; (b) patient 1; (c) affected
brother of patient 1; (dl patient 2; (e) patient 3; and (f) a classical Xlinked patient. Forty micrograms postnuclear
of
supernatant protein
were loaded in each track. The location of p22-phox and gp9l-phox
are indicated.
Fig 2. Killing ofS aureus by neutrophils fromtwo normal subjects
(0.0);
the heterozygotecarrier mother of patient
1 (A);two measur1 at two different occasions (0,.I; and a patient
ments from patient
with classic X-CGD l*). The mean t SE of 3 t o 5 measurments are
shown where errorbars are included. Other points indicate theaverage of duplicate measurements.
From www.bloodjournal.org by guest on October 15, 2014. For personal use only.
BU-GHANIM ET AL
3578
A-
.
.
Patient 1
A C GT
-e
L
3'
Lys
31!
5'
B
Control
A CGT
Patient 2
A C G T
Fig 3. Sequence analysis of cDNA of segments of thegene coding for gp9l-phoxfrom
patients. A, B, and C correspond t o sequence obtained
from patients1.2. and 3, respectively. The sequence ladders on the leftin each figure were obtained from
a normal control.The deleted bases
in (A) are indicated by threesquares, and they correspond t o L ~ S ~(B)
, ~Cys,,
;
is mutatedt o Tyr; In (C), the duplicated region is indicated by
a bracket in thenormal, and is shownt o be repeated in patient 3. Patient 3 ladder shows theantisense strand reversed and inverted t o clarify
the interpretation of the amino
acid sequence.
Iodination by PMNs in patient 1 was found to be reduced
to approximately the same level as superoxide production.
In the patient 5.4 2 0.8 (n = 3 ) nmol of iodine were incorporated/107 cellsh, amounting to 1 1 % of the normal levels of
49.0 2 4.4 ( n = 3) nrn0l/l0~hcomparable with the 12% of
oxidase activity.
Northern blot analysis of mRNA showed the presence of
a normal amount of a normal sized transcript in a l l the patients (data not shown).
The mutations found in thethree patients are shown in
Fig 3 . In patient I (Fig 3A), three nucleotides wcre deleted
from an AAG trinucleotide repeat in bases 949 through 957
in the cDNA resulting in the loss of one of a group of three
lysines at positions 313 through 3 IS. In patient 2 (Fig 3B).
From www.bloodjournal.org by guest on October 15, 2014. For personal use only.
3579
VARIANT CHRONIC GRANULOMATOUS DISEASE
a single base G A substitution at nucleotide 743 predicts
the nonconservative replacement of cysteine 244 by a tyrosine. In patient 3 (Fig 3C). a tandem duplication of the 31
bases between 1.672 and 1.702 generates a premature stop
at codon 565. deleting six amino acids from the C-terminus.
The position of the two mutations thatlie in theregion
modeled by Taylor et al" are shown in Fig 4. A and B.
The mutations detected in the PCR-based analysis were
confirmed by RNase protection (Fig S). using mRNA from
B-cell lines established fromthe patients peripheral blood
mononuclear cells. Probe AR.w 361 extends from nucleotide
position 675 through 1.049. A cut at position 949 in patient
I should generate two fragments of 274 and 100 bases.
+
B Sac1
1 2 3
7
'
-
i
2311
A
T-
149
NADPH
C terminus
FAD
1oc
Fig 5. RNase protection experiment. (A) Lane 1, the ABsu 361
probe (bases 675 through 1.049); lanes 2 and 3 represent patients 1
and 2, respectively; lane 4, normal RNA. (B) Lane 1 is ASac I probe
alone (bases 1,523 through 1,903); lane 2, patient 3; lane 3, normal
RNA. The partially protected fragments of the patients
are indicated
with thesmall arrows, whereas the large arrow indicates the normal
fully protected product.
Lys 3 15 deleted
Fig 4. (A) The modeled positionof the lastsix amino acids, which
are missing from patient3, is shown relative
to theNADPH and FAD
cofactors. (B) Ribbon diagram of the whole modeled region of the
flavocytochrome (residues 277 to theC-terminus) in the same orientation asA. The residues deleted in patient 1 (Lys 315) and 3 (last
six amino acids of the C terminus) are shown in black on the wildtype model. Diagrams were drawn withMOLSCRIPT?'
whereas a mutation at position 743 in patient 2 generates two
fragments of 306 and 68 bases. The ASm I probe consists of
the antisense cRNA of the gp91-phox nucleotide sequence
1,523through1,903. In patient 3, a cut at position1,672
generates two fragments of 231 and 149 bases. In all cases,
fragments of the size predicted from PCR analysis were
detected, except for the 68-bp fragment, which was probably
too diffuse to visualize on a 6% gel.
DISCUSSION
The structure of the flavocytochrorne b of the NADPH is
of considerable interest. Its function requires access of the
substrate, NADPH, from the cytosol, and it must also allow
From www.bloodjournal.org by guest on October 15, 2014. For personal use only.
3580
BU-GHANIM ET AL
for hydrophobic, membrane-spanning helices to permit the
translocation of the product, superoxide, to the exterior.
From a structural point of view, gp91-phox can be divided
roughly into two halves; the N-terminus contains a number
of hydrophobic helices,42whereas the C terminus is much
more hydrophilic and contains the FAD and NADPHbinding
sites.' The functional interaction of the two subunits remains
to be established. Both are required for correct processing
in the Golgi, targeting to the vacuole, and ~tability.~'
Analysis of mutations that produce CGD provides important information as to the structural organization of the
flavocytochrome. This has been facilitated by the construction of a model of the C-terminal half of gp91-phox based
uponthe crystal structure of ferrodoxin NADP reductase.
Most of the mutations result in complete instability and loss
of both subunits. Occasional mutations have been described
in which there is high level of expression of the proteins
Gly) in
with total loss of function. A mutation (Asp,,
gp91-phox in the region of a helix predicted to overly the
nucleotide binding groove is characterized by failure of the
translocation of the cytosolic phox proteins,44 giving credence to the theory that these proteins might activate electron
transport by attaching to and moving this helix aside, thereby
providing NADPH access to the FAD moiety.42A mutation
in the Proline-rich segment of the C-terminus of p22-phox
(Prolsh Gln) leads to CGD," this mutation prevents the
binding of the p47-phox protein SH3 domain in ~ i t r o . ~ ~
Mutations resulting in variant CGD with a partial loss of
oxidase activity are of interest because they either result
from structural disorganization leading to incomplete loss of
protein, to partial functional disorganization, or to a mixture
of the two. The model of the C-terminal hydrophilic half of
gp91-phox provides a template upon which to start examining the impact of some of these mutations upon structure
and function. To date, the precise molecular defect has been
identified in seven patients with variant CCD4*of which five
involved amino acid substitutions in gp91-phox (at amino
acid positions 56, 156, 244, 309, and 389).4K.49
The substitution at position 56 located in the N-terminus resulted in a
75% to 80% loss of oxidase activity, and a loss of 40% of
the flavocytochrome.The remaining four resulted in a greater
loss (70% to90%) of the flavocytochrome and a concomitant
(80%to 97%) diminution of NADPH oxidase a~tivity.~'
These mutations appear to have their effect through destabilization of the proteins rather than altering the electron transport. Other variant patients have been described in which
the function of the molecule is disturbed to a much greater
extent than the stability. For example, deletion of amino
acids 488 through 497'" resulted in a normal level of the
cytochrome, but only 6% of oxidase activity. In all three of
our patients there was a loss of most of the flavocytochrome
proteins. In addition, in patient 3, and probably also in 1,
there is a disproportionate loss of oxidase activity suggesting
an additional functional impairment of the residual protein.
Although patient I had 12% of normal oxidase activity,
this wasnot associated with a muchless severe clinical
presentation, and both he and his affected brother were repeatedly hospitalized with frequent and severe infections. In
contrast, the patient described by Schapiro et also had only
+
+
6% oxidase activity, but did not present until the age of 69
years. The basis of the increased susceptibility to infection
in our patient was confirmed as being caused by an inability
of his neutrophils to kill S aureus, which was comparable
with the levels of killing observed in classical X-CGD. This
is important because it indicates that if the gene therapy
approach to treatment isto be effective, it mustresult in
the reconstitution of oxidase activitywell above the 10%
observed in this patient. The results in this patient also indicate that iodination levels in neutrophils are limited by the
levels of substrate.
As referredto above, substitution of a cysteine 244 in
patient 2 has been described previously. Unlike the substitution seen here, where the cysteine is replaced by a tyrosine,
in the previously reported case, a G + C transversion results
in the replacement of cysteine 244 by serine."Although
both cases showed similar, low levels of PMA-stimulated
superoxide production at about 3% of normal, they appear
to have different amounts of proteins with about 8% in our
patient as compared with 39% in the Cys Ser mutation."
Alternatively, because the cytochrome was estimated by
Western blotting in our study and spectroscopically in the
other, this difference could be methodologic. If indeed the
Cys + Tyr is more disruptive than the Cys + Ser, it could
be as a result of the large tyrosine aromatic side chain, which
is likely tocause greater disruption of the surrounding amino
.acids
~ ~ than the smaller aliphatic side chain of serine.
In patient 3, the cDNA analysis indicated the presence of
a duplication of 3 1 bases (1,672 through 1,7021, in the coding
sequence of exon 13, which encodes the C-terminal region
of the protein. Although this patient has a sizeable quantity
of flavocytochrome (16% to 24% of normal), the activity of
the molecule seems more severely affected with superoxide
production at only 2.1 % of normal. The structural model of
Taylor et ai4* may provide an explanation forthis discordance. Itwas suggested from the modelthatthelast
amino acid (570) that is deleted from patient 3 is one of two
amino acids critical to the binding of FAD. The juxtaposition
of the C-terminus and the FAD and NADPH
cofactors is
shown in Fig 4A. Therefore, a probable consequence of the
deletion in patient 3 could be to increase the K,, for one or
both cofactors. Alternatively, the loss of the last six amino
acids could serve to prohibit conformational changes in
gp91-phox that are necessary for the activation of electron
transport.
+
ACKNOWLEDGMENT
We thank Rodolpho Garcia for some ofthe superoxide production
measurements, MargaretChettyfortechnical
assistance, and Dr
Mary-Christine Bohler and professors Alain Fischer and Claude Gricelli for the cells from patient 3.
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