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PHAGOCYTES
From the Central Laboratory of the Netherlands Blood
Transfusion Service (CLB), the Laboratory for Experimental and Clinical
Immunology and the Emma Children's Hospital, both of the Academic
Medical Center, University of Amsterdam, The Netherlands; Department of
Biochemistry and Molecular Biology IV, University Complutense de
Madrid, Spain; Dr von Haunerschen Kinderspital, Ludwig Maximilians
Universität, München, Germany; Sophia Children's Hospital,
University Hospital Rotterdam, Erasmus Medical Center, The Netherlands;
and Department of Haematology, Imperial College School of Medicine,
Hammersmith Hospital, London, United Kingdom.
In this study the blood cells of 4 male patients from 2 unrelated
families with chronic nonspherocytic anemia and recurrent bacterial infections were investigated. The activity of
glucose-6- phosphate dehydrogenase (G6PD) in the red blood cells and
in the granulocytes of these patients was below detection level.
Moreover, their granulocytes displayed a decreased respiratory burst
upon activation. Sequencing of genomic DNA revealed a novel 3-base pair (TCT) deletion in the G6PD gene, predicting the deletion of a
leucine at position 61. The mutant G6PD protein was undetectable by
Western blotting in the red blood cells and granulocytes of these
patients. In phytohemagglutinin-stimulated lymphocytes the G6PD protein
was present, but the amount of G6PD protein was strongly diminished in
the patients' cells. Purified mutant protein from an Escherichia
coli expression system showed decreased heat stability and
decreased specific activity. Furthermore, we found that the messenger
RNA of G6PD180-182delTCT is unstable, which may contribute
to the severe G6PD deficiency observed in these patients. We propose
the name "G6PD Amsterdam" for this new variant.
(Blood. 2002;100:1026-1030) In cells lacking mitochondria, such as red
blood cells (RBCs), the only source of reduced nicotinamide adenine
dinucleotide phosphate (NADPH) is the hexose monophosphate
pathway.1 In the first step of this pathway,
glucose-6-phosphate (G6P) is converted into
6-phosphogluconolactone, catalyzed by glucose-6-phosphate dehydrogenase (G6PD), and accompanied by the reduction of nicotinamide adenine dinucleotide phosphate (NADP) into NADPH. A sufficient amount
of NADPH is essential for the integrity of RBCs, because NADPH reduces
glutathione, which protects these cells against oxidative stress. G6PD
deficiency therefore leads to hemolytic anemia, ranging from mild
hemolytic anemia induced by infections or drugs to chronic
nonspherocytic anemia with attacks of severe anemia induced by
infections or drugs.1
Because the G6PD gene is located on the X chromosome, G6PD
deficiency usually becomes manifest in hemizygous men. The severity of
the G6PD deficiency depends on the effects of the mutation on protein
stability and activity. So far, only missense and small in-frame
deletions are known; nonsense mutations or large deletions have not
been described, consistent with the idea that complete G6PD deficiency
is incompatible with life. G6PD deficiency is usually restricted to
RBCs, because these cells have a relatively long survival time (3 months) after release from the bone marrow but lack protein
synthesis. Thus, G6PD protein instability is first manifested in
RBCs.1
Although rare, severe G6PD deficiency can lead to symptoms of chronic
granulomatous disease (CGD), such as recurrent bacterial and fungal
infections.2-5 This disease is normally caused by a defect
in one of the components of the NADPH oxidase. This enzyme catalyzes the generation of superoxide in phagocytes, which is used by the phagocytes to kill ingested
microorganisms.6 In severe G6PD deficiency,
superoxide cannot be formed in the phagocytes due to a lack of NADPH in
these cells, an impairment that is seen when G6PD activity in the
phagocytes is less than 5% of normal values.
Here, we investigated the genetic defect underlying the severe
G6PD deficiency found in patients of 2 different families with mild
chronic hemolytic anemia and recurrent bacterial or fungal infections.
In both families a novel deletion of 3 nucleotides, the TCT triplet at
position 180-182 in the G6PD gene, was found. This deletion predicts a
G6PD protein that lacks a leucine at position 61. Our results indicate
that the deletion of this leucine leads to the expression of an
unstable and less active G6PD protein. These altered enzyme properties
and the unstable messenger RNA (mRNA) of this variant probably cause
the chronic nonspherocytic anemia and CGD-like symptoms in these
patients. We propose to call this variant enzyme "G6PD Amsterdam."
Clinical histories
Patient II1 (born in 1993 to Hindustan parents) was healthy until the
age of 3.5 years, when he was admitted to the hospital with high fever,
coughing, tachypnea, and tachycardia. A chest x-ray showed pneumonia of
the right basal lobe. Blood cultures revealed Chromobacterium
violaceum, an uncommon human pathogen that can cause serious
infections in patients with neutrophil dysfunction. He also had anemia
(Hb 75 g/L [7.5 g/dL]), which was thought to be due to the
septicemia. After initial treatment with cephalosporin (cefuroxime),
the therapy was changed upon the antibiogram into meropenem, which was
continued for 14 days. He responded well, although the anemia
persisted. One day after antibiotic discontinuation, he relapsed with
sepsis. Again Chromobacterium violaceum was cultured from
the blood. Magnetic resonance imaging showed osteomyelitis of the
thoracic spine at T10. Meropenem was started again in combination with
ciprofloxacin, both intravenously for 28 days, followed by
ciprofloxacin orally for another 2 weeks. He recovered without
developing sequelae. During a follow-up of 4 years without prophylactic
antibiotic treatment, he had no serious infections. His Hb levels
fluctuated around 109 g/L (10.9 g/dL), with 35 Purification and culture of cells
Enzyme determinations G6PD activity in RBCs and granulocytes was determined as described previously.7Neutrophil function tests Purified neutrophils were suspended in a medium that contained 138 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4, 0.6 mM CaCl2, 1.0 mM MgCl2, 5.5 mM glucose, and 0.5% (wt/vol) human albumin (pH 7.4). Oxygen consumption was measured with an oxygen electrode.8Molecular genetic studies Genomic DNA and complementary DNA (cDNA) sequencing were performed as described earlier5 using an ABI prism 377 automated sequencer (Applied Biosystems, Foster City, CA). Primers for polymerase chain reaction (PCR) amplification and DNA sequencing are described in Table 1.
Immunoblotting of G6PD For immunodetection, 105 cells were boiled in sodium dodecyl sulfate sample buffer (125 mM Tris, pH 6.8; 20% [wt/vol] sodium dodecyl sulfate; and 12.5% [vol/vol] -mercaptoethanol) and
were loaded on a 12.5% polyacrylamide gel, according to Laemmli, in a
gel apparatus (Mini-Protean II, BioRad, Hercules, CA). Western blotting was performed (Mini Trans-Blot cell, BioRad) according to the
manufacturer's recommendations. Detection of proteins was performed as
described previously with a polyclonal antibody against recombinant G6PD.
Expression of G6PD in E coli G6PD in Escherichia coli was expressed as described by Roos et al.5 Purification of recombinant G6PD was performed by affinity chromatography as previously reported.9,10 The electrophoretic mobility of the recombinant enzymes was determined by means of electrophoresis in native polyacrylamide in Tris buffer (pH 8.8) at 4°C,11 followed by G6PD activity staining with phenazine methosulfate and methylthiotetrazole. Thermostability assays were performed according to the World Health Organization protocol.12 Briefly, samples with purified recombinant enzyme were incubated for 60 minutes at 51°C; aliquots were drawn every 20 minutes and assayed for G6PD activity.
G6PD activity in the RBCs and granulocytes of the patients from
both families was below the detection limit of our assay (Table 2). Both mothers of the patients
displayed about half the enzyme activity in their RBCs and granulocytes
compared with healthy controls. Because the patients suffered
from recurrent infections, a defect in granulocyte function due to the
impaired G6PD activity was suspected. Indeed, oxygen consumption of the
granulocytes of the patients after addition of opsonized zymosan
particles was drastically diminished (Table 2). Granulocytes of both
mothers consumed about half the amount of oxygen compared with healthy controls. The granulocyte NADPH oxidase defect was confirmed with other
tests (nitroblue tetrazolium [NBT] test, dihydrorhodamine [DHR]
test, and cytochrome c reduction; not shown). In the
NBT test, the mothers showed a mosaic of formazan-positive and
-negative cells (not shown). Cytochrome b558
spectrum, reaction with a monoclonal antibody against
gp91phox, and sequencing of CYBB (the X-linked
gene encoding gp91phox) revealed no abnormalities in
the patients.
To identify the mutation responsible for the G6PD defect, DNA
sequencing was performed on genomic DNA of the patients and their
mothers (Figure 1). This revealed a
deletion of 3 nucleotides (TCT) at position 180-182 in the coding
sequence of the G6PD gene, predicting deletion of leucine at position
61 in the protein. This mutation was found in all patients, while both
mothers were heterozygous for the deletion. No other mutations were
found in the G6PD gene of the patients, including 236 base pairs (bp)
in the promoter region, 545 bp of the 5' untranslated region (UTR), and
638 bp of the 3'UTR.
To determine whether this new deletion in the G6PD gene allows
expression of protein, Western blotting was performed on the RBCs and granulocytes of the patients and their mothers. G6PD protein
was not detected in the RBCs or the granulocytes of any of the patients
(Figure 2A). Although diminished compared
with healthy controls, G6PD was detected in the RBCs and
granulocytes of the patients' mothers, mother I and mother II. Because
complete G6PD deficiency is probably incompatible with life, it was
investigated whether the deletion would lead to expression of an
unstable protein. Therefore, rapidly dividing, PHA-stimulated
lymphocytes of patient II1 and his mother were analyzed for G6PD
expression. A low level of G6PD expression was found in PHA-stimulated
lymphocytes of patient II1 and a decreased level of G6PD protein in
those from his mother (Figure 2B).
To test the properties of the mutant protein, it was expressed in an
E coli expression system and purified. Compared with the
wild-type enzyme, G6PD Amsterdam showed reduced specific activity as
well as reduced thermal stability (Table
3 and Figure
3). G6PD Amsterdam had normal
Km values for both NADP and G6P. The utilization
of 2-deoxy-G6P and galactose-6-phosphate was approximately doubled. The
mutant enzyme used a normal percentage of deamino-NADP, and the
Ki for NADPH was normal. Electrophoretic mobility of G6PD Amsterdam was increased 10% compared with the wild-type
enzyme. However, the altered enzymatic properties of the recombinant
G6PD Amsterdam were unlikely to account for the observed severe G6PD deficiency in the patients' cells. Therefore, the stability of the
mRNA of G6PD Amsterdam was assayed. This was done by comparing the
amount of mRNA encoding wild-type G6PD with the amount of mutant G6PD
mRNA by cDNA sequencing (Figure 1). The mRNA was isolated from white
blood cells of mother I and her 3 sons, patient I1, patient II2, and
patient II3, and was subsequently sequenced. Although mother I is a
carrier for the nucleotide 180-182 deletion, as confirmed by genomic
sequencing, this deletion was not detected in the cDNA of her white
blood cells. In contrast, the nucleotide 180-182 deletion was detected
in the cDNA of her 3 sons. This proves that the mRNA encoding G6PD
Amsterdam is less abundantly expressed than the mRNA coding for the
wild-type protein in the mother of these 3 patients. This is strong
evidence that the mutant mRNA is less stable than the wild-type G6PD
mRNA, a property that is very likely to diminish the expression of G6PD
Amsterdam protein.
In this study we describe the finding of a novel deletion of 3 nucleotides in the G6PD gene in 4 patients with mild chronic nonspherocytic anemia and CGD-like symptoms from 2 different families, predicting the deletion of leucine at position 61. This deletion leads to severe G6PD deficiency, confirmed by the absence of residual G6PD activity and of G6PD protein expression in the RBCs and granulocytes of the patients. However, a low level of G6PD protein expression was detected in rapidly dividing PHA-stimulated lymphocytes of one of the patients, consistent with the idea that the deletion of leucine on position 61 in the G6PD protein leads to the expression of an unstable protein. This theory was partially confirmed by expression of the mutant protein
in an E coli expression system. The recombinant enzyme proved to be less stable and less active than the wild-type enzyme, as
shown by increased heat lability and reduced specific activity (Table 3
and Figure 3). However, the enzymatic properties of G6PD Amsterdam were
not severely altered but comparable to another G6PD variant, G6PD
A
The expression of G6PD Amsterdam and the activity in purified blood
cells was much lower than those observed in G6PD A Remarkably, in the family with the 3 G6PD-deficient brothers, only 1 presented with recurrent microbial infections, whereas the other 2 had no known disposition to infections. This might be due to a different exposure to risk factors or to differences in disease-modifying polymorphisms in other genes,16 but that remains to be investigated.
Submitted October 19, 2001; accepted March 21, 2002.
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.
Reprints: D. Roos, Dept of Experimental Immunohematology, Central Laboratory of the Netherlands Blood Transfusion Service, Plesmanlaan 125, 1066 CX Amsterdam, The Netherlands; e-mail: d_roos{at}clb.nl.
1.
Beutler E.
G6PD deficiency.
Blood.
1994;84:3613-3636 2. Cooper MR, DeChatelet LR, McCall CE, et al. Complete deficiency of leukocyte glucose-6-phosphate dehydrogenase with defective bactericidal activity. J Clin Invest. 1972;51:769-778[Medline] [Order article via Infotrieve]. 3. Gray GR, Stamatoyannopoulos G, Naiman SC, et al. Neutrophil dysfunction, chronic granulomatous disease, and non-spherocytic haemolytic anaemia caused by complete deficiency of glucose-6-phosphate dehydrogenase. Lancet. 1973;2:530-534[CrossRef][Medline] [Order article via Infotrieve].
4.
Vives Corrons JL, Feliu E, Pujades MA, et al.
Severe-glucose-6-phosphate dehydrogenase (G6PD) deficiency associated with chronic hemolytic anemia, granulocyte dysfunction, and increased susceptibility to infections: description of a new molecular variant (G6PD Barcelona).
Blood.
1982;59:428-434
5.
Roos D, van Zwieten R, Wijnen JT, et al.
Molecular basis and enzymatic properties of glucose 6-phosphate dehydrogenase volendam, leading to chronic nonspherocytic anemia, granulocyte dysfunction, and increased susceptibility to infections.
Blood.
1999;94:2955-2962
6.
Roos D, de Boer M, Kuribayashi F, et al.
Mutations in the X-linked and autosomal recessive forms of chronic granulomatous disease.
Blood.
1996;87:1663-1681
7.
Loos H, Roos D, Weening R, Houwerzijl J.
Familial deficiency of glutathione reductase in human blood cells.
Blood.
1976;48:53-62 8. Weening RS, Roos D, Loos JA. Oxygen consumption of phagocytizing cells in human leukocyte and granulocyte preparations: a comparative study. J Lab Clin Med. 1974;83:570-577[Medline] [Order article via Infotrieve].
9.
Town M, Bautista JM, Mason PJ, Luzzatto L.
Both mutations in G6PD A- are necessary to produce the G6PD deficient phenotype.
Hum Mol Genet.
1992;1:171-174 10. Bautista JM, Mason PJ, Luzzatto L. Purification and properties of human glucose-6-phosphate dehydrogenase made in E. coli. Biochim Biophys Acta. 1992;1119:74-80[CrossRef][Medline] [Order article via Infotrieve]. 11. Kirkman HN, Hendrikson EM. Sex-linked electrophoretic difference in glucose-6-phosphate dehydrogenase. Am J Hum Genet. 1963;15:241[Medline] [Order article via Infotrieve]. 12. Betke K, Beutler E, Brewer GJ, et al. Standardization of procedures for the study of glucose-6-phosphate dehydrogenase. Report of a WHO Scientific Group. WHO Tech Rep; 1967. Ser no. 366. 13. Eisenhaber F, Imperiale F, Argos P, Frommel C. Prediction of secondary structural content of proteins from their amino acid composition alone. I. New analytic vector decomposition methods. Proteins. 1996;25:157-168[CrossRef][Medline] [Order article via Infotrieve]. 14. Eisenhaber F, Frommel C, Argos P. Prediction of secondary structural content of proteins from their amino acid composition alone. II. The paradox with secondary structural class. Proteins. 1996;25:169-179[CrossRef][Medline] [Order article via Infotrieve]. 15. Beutler E. The genetics of glucose-6-phosphate dehydrogenase deficiency. Semin Hematol. 1990;27:137-164[Medline] [Order article via Infotrieve]. 16. Foster CB, Lehrnbecher T, Mol F, et al. Host defense molecule polymorphisms influence the risk for immune-mediated complications in chronic granulomatous disease. J Clin Invest. 1998;102:2146-2155[Medline] [Order article via Infotrieve].
© 2002 by The American Society of Hematology.
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Top
Abstract
Introduction
). One of his brothers (I2) has
known G6PD deficiency and presented with prolonged neonatal jaundice
and episodes of acute hemolysis, but he has no known disposition to infections. In a period without clinical problems, he had a normal Hb
level but 56
.
about 2% of the protein yield found with
wild-type G6PD. For comparison, the yield of G6PD A