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CLINICAL OBSERVATIONS, INTERVENTIONS, AND THERAPEUTIC TRIALS
Neutrophil-specific granule deficiency: homozygous recessive inheritance of a
frameshift mutation in the gene encoding transcription factor
CCAAT/enhancer binding protein–⑀
Adrian F. Gombart, Masaaki Shiohara, Scott H. Kwok, Kazunaga Agematsu, Atsushi Komiyama, and H. Phillip Koeffler
Neutrophil-specific granule deficiency
(SGD) is a rare congenital disorder. The
neutrophils of individuals with SGD display atypical bi-lobed nuclei, lack expression of all secondary and tertiary granule
proteins, and possess defects in chemotaxis, disaggregation, receptor up-regulation, and bactericidal activity, resulting in
frequent and severe bacterial infections.
Previously, a homozygous mutation in
the CCAAT/enhancer binding protein–⑀
(C/EBP⑀) gene was reported for one case
of SGD. To substantiate the role of C/EBP⑀
in the development of SGD and elucidate
its mechanism of inheritance, the mutational status of the gene was determined
in a second individual. An A-nucleotide
insertion in the coding region of the
C/EBP⑀ gene was detected. This mutation
completely abolished the predicted translation of all C/EBP⑀ isoforms. Microsatellite and nucleotide sequence analyses of
the C/EBP⑀ locus in the parents of the
proband indicated that the disorder may
have resulted from homozygous recessive inheritance of the mutant allele from
an ancestor shared by both parents. The
mutant C/EBP⑀32 protein localized in the
cytoplasm rather than the nucleus and
was unable to activate transcription. Consistent with this, a significant decrease in
the levels of the messenger RNAs
(mRNAs) encoding the secondary granule protein human 18-kd cationic antimi-
crobial protein (hCAP-18)/LL-37 and the
primary granule protein bactericidal/permeability-increasing protein were observed in the patient. The hCAP-18 mRNA
was induced by overexpression of
C/EBP⑀32 in the human myeloid leukemia
cell line, U937, supporting the hypothesis that C/EBP⑀ is a key regulator of
granule gene synthesis. This study
strongly implicates mutation of the
C/EBP⑀ gene as the primary genetic defect involved in the development of neutrophil SGD and defines its mechanism of
inheritance. (Blood. 2001;97:2561-2567)
© 2001 by The American Society of Hematology
Introduction
Neutrophil-specific granule deficiency (SGD) is a rare congenital
disorder, possibly inherited in an autosomal recessive fashion.
Individuals with SGD (5 reported worldwide) possess atypical
bi-lobed nuclei and lack expression of secondary and tertiary
granule messenger RNAs (mRNAs) and protein, including lactoferrin, transcobalamin, gelatinase B, and collagenase.1-8 Additionally,
they display a marked decrease in levels of the primary granule
defensins; however, expression of the primary granule genes
myeloperoxidase (MPO) and lysozyme are unaffected.9,10 The
neutrophils of SGD patients are defective in chemotaxis, dissaggregation, receptor up-regulation, and bactericidal activity.1-8 More
recently, the deficiency in granule gene expression was extended to
eosinophils.11 These cells from SGD patients lacked eosinophilspecific granule contents, including eosinophil cationic protein,
eosinophil-derived neurotoxin, and major basic protein.11 Because
of these numerous deficiencies and functional defects, SGD
individuals are severely immunocompromised and develop frequent bacterial infections, including Pseudomonas aeruginosa and
Staphylococcus aureas.
Since SGD individuals express normal levels of lactoferrin and
transcobalamin in their saliva but not in either their plasma or neutrophils, the molecular basis for SGD was hypothesized to involve the
mutation of a myeloid-specific transcription factor.8,12-14 A candidate
gene encoding such a transcription factor is CCAAT/enhancer binding
protein–⑀ (C/EBP⑀).15,16 It is expressed primarily during granulocytic
differentiation.15-19 Targeted disruption of the gene in mice leads to
defects in terminal differentiation of neutrophils with increased numbers
of morphologically atypical neutrophils.20 The phenotypic and functional defects of neutrophils from C/EBP⑀-deficient mice closely
parallel those of SGD. They include bi-lobed nuclei, a significantly
reduced capacity to produce superoxide when treated with phorbol
12–myristate 13–acetate, and impaired chemotaxis and bactericidal
activity.20-22 The null mice are susceptible to gram-negative bacterial
sepsis, particularly with P aeruginosa, and succumb to systemic
infection at 3 to 5 months of age.20 Consistent with SGD, the
C/EBP⑀-deficient mice lack expression of mRNAs encoding the same
secondary and tertiary granule proteins. In addition, mRNAs for the
cathelicidin B9 and murine cathelin-related antimicrobial peptide
(CRAMP) are severely reduced in the bone marrow.21,22 These cathelinlike peptides possess potent activity against gram-negative bacteria,
From Cedars-Sinai Medical Center, Burns and Allen Research Institute,
Division of Hematology/Oncology, UCLA School of Medicine, Los Angeles, CA,
and Department of Pediatrics, Shinshu University School of Medicine,
Matsumoto, Japan.
Oncology and a member of the Jonsson Cancer Center.
Submitted July 26, 2000; accepted January 3, 2001.
Supported by National Institutes of Health grant CA26038-20, the Ko-So
Foundation, the Horn Foundation, the Parker Hughes Fund, and the C & H
Koeffler Fund. A.F.G. is a recipient of a Lymphoma Research Foundation of
America fellowship; H.P.K. is a recipient of the Mark Goodson Chair in
BLOOD, 1 MAY 2001 䡠 VOLUME 97, NUMBER 9
Reprints: Adrian F. Gombart, Cedars-Sinai Medical Center, Division
of Hematology/Oncology, UCLA School of Medicine, Davis Bldg 5065,
8700 Beverly Blvd, Los Angeles, CA 90048; e-mail: gombarta@csmc.
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.
© 2001 by The American Society of Hematology
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2562
BLOOD, 1 MAY 2001 䡠 VOLUME 97, NUMBER 9
GOMBART et al
including P aeruginosa.23,24 As with SGD, C/EBP⑀-deficient mice
exhibit abnormalities in their eosinophils and lack expression of
mRNAs encoding eosinophil peroxidase and major basic protein (our
unpublished observations, March 1999).20
Because of the striking similarities between SGD patients and the
C/EBP⑀-deficient mice, the C/EBP⑀ locus was examined for mutations
in 1 of the 5 known individuals.25 A homozygous 5–base pair (bp)
deletion in the second exon was described.25 In humans, 4 C/EBP⑀
isoforms, referred to as p32, p30, p27, and p14, are generated.16,19 The
p32, p27, and p14 forms are encoded by 4 mRNA isoforms generated by
transcription from 2 different promoters, P␣ and P␤, combined with
differential splicing (see Figure 1B).19 The p30 isoform is synthesized
by translation from a downstream start codon at amino acid position
32.16 The predicted 5-bp frameshift resulted in a truncation of the
transcriptionally active p32 and p30 isoforms with the loss of the
dimerization and DNA-binding domain regions.25 The p27 and p14
isoforms, which are unable to activate transcription, were unaffected
(unpublished results, March 1999).19 The truncated C/EBP⑀32 was
unable to activate transcription efficiently.25 These results implicated
alteration of the C/EBP⑀ gene in the development of one case of SGD.
Comparison of the phenotypes of individuals suffering from
SGD indicate that it is likely to be a heterogenous disease and
suggests the possibility of different underlying genetic defects.4
The purpose of this study is to test the hypothesis that alteration of
the C/EBP⑀ gene is the primary defect involved in the development
of SGD. We identified a mutation in the C/EBP⑀ locus of a second
individual with SGD and elucidated the mechanism of inheritance
by analysis of the locus in the parents of the individual.
Patient, materials, and methods
SGD proband and genomic DNA isolation
The proband is a 27-year old female who has suffered from recurrent
pyogenic infections, such as otitis media, skin abscesses, and pneumonia,
since infancy. As previously reported,3,8 the neutrophils of this individual
exhibit unique bi-lobed nuclei and decreased cytoplasmic granularity.
These cells display, by histochemical staining, normal peroxidase-positive
products and no alkaline phosphatase activity, an absence of specific
granules upon electron microscopy, impaired chemotaxis, defective bactericidal activity against S aureus and Escherichia coli, and markedly
decreased lactoferrin and transcobalamin content. Peripheral blood was
obtained from the proband and her parents after informed consent. The
peripheral blood mononuclear cells (PBMCs) were purified by Ficoll
gradient centrifugation, and genomic DNA was prepared. For the normal
control, genomic DNA was prepared from the bone marrow of healthy
volunteers after informed consent. The parents of the proband are healthy
with no history of recurrent infections. The number and morphology of the
neutrophils of both parents were normal.
RNA isolation and analysis
Total RNA from all nucleated cells present in the peripheral blood was
prepared from the proband and her father after informed consent. The RNA
was extracted by lysis of cells in TRIzol reagent as described by the
manufacturer (Gibco/BRL, Rockville, MD). For reverse-transcription polymerase chain reaction (RT-PCR) analysis, RNA was treated with DNaseI
and reversed transcribed by means of random oligonucleotides as described
previously.26 Each reverse transcription reaction contained approximately
1 to 2 ␮g total RNA. Northern blot analysis was performed essentially as
described,27 with the use of 10 ␮g total RNA per lane. Probes (described
below) were labeled by means of the StripEZ system (Ambion, Austin, TX),
and blots hybridized in ULTRAhyb solution (Ambion) as instructed by the
manufacturer. Autoradiographs were exposed to X-OMAT AR film (Kodak,
Rochester, NY).
Table 1. Primers used for amplification of C/EBP⑀ by polymerase chain reaction
Primer name
Nucleotide sequence
Nucleotide position*
Prom-S
5⬘-agtcggagggaggaggttgc-3⬘
15-34
Prom-AS
5⬘-tggcttcacggcaaagagatc-3⬘
691-671
R66
5⬘-atgtgtgagcatgaggcctccatt-3⬘
602-625
440
5⬘-cggcagtggccaaaggggcct-3⬘
1790-1770
EX2-S
5⬘-cagcctctgcgcgttctcaag-3⬘
995-1015
EX2-AS
5⬘-gtccgcagagttaggccgtgc-3⬘
2163-2143
NFM-1
5⬘-ccacaccagcctctccagc-3⬘
1070-1052
404
5⬘-cagacaggaaggcgctggg-3⬘
795-813
NFM-2
5⬘-gggagggcgccttcaggag-3⬘
1826-1808
*Numbering based on published sequence available from EMBL/GenBank/
DDBJ under accession No. U80982.
PCR of genomic DNA and complementary DNA
The primers used to amplify the C/EBP⑀ genomic locus are described in Table 1.
The primers were used in the following combinations to amplify overlapping
regions of the proband’s genomic DNA: (1) Prom-S ⫹ Prom-AS; (2) R66 ⫹ 440;
(3) EX2-S ⫹ EX2-AS; (4) R66 ⫹ NFM-1; and (5) 404 ⫹ NFM-1. PFU
(Stratagene, La Jolla, CA) or Advantage Taq (Clontech Laboratories, Palo Alto,
CA) polymerases were used to amplify the fragments as described by the
manufacturers. The PCR conditions were as follows: 94°C, 3 minutes; then 35
cycles 94°C, 30 seconds; 60°C (for primer sets 1 and 2) or 64°C (for primer sets 3
through 5), 30 seconds; and 72°C, 2 minutes. To amplify the complementary
DNA (cDNA) of C/EBP⑀, primer pair 404 ⫹ NFM-2 was used with a 64°C
annealing temperature. The products were cloned into either pST-Blue (Novagen,
Madison, WI) or pcR2.1 (PE Applied Biosystems, Carlsbad, CA) as
described by the manufacturer. Products were sequenced with an ABI Prism
Dye Terminator Cycle Sequencing Ready Reaction kit (Invitrogen, Foster
City, CA) by means of primer binding sites available in the plasmids (T7,
SP6, and M13R) and the primers described in Table 1 and were analyzed by
an ABI 377 sequencing machine.
The primers for MPO, lactoferrin, glyceraldehyde-3-phosphate dehydrogenase, and 18S ribosomal RNA (rRNA) and Southern blot analysis of PCR
products were described previously.17,27,28 The primers for amplification of
bactericidal/permeability-increasing (BPI) protein were BPI-S, 5⬘cagaagggcctggactac-3⬘ (nucleotide [nt] 154-171); BPI-AS, 5⬘-tgctgcagctggagcag-3⬘ (nt 516-531); and, for hybridization, BPI-Int, 5⬘-ctgcagaaggagctgaagaggatc (nt 196-219). The primers for amplification of human 18-kd
cationic antimicrobial protein (hCAP18) were CAP18-F1, 5⬘-agctacaaggaagctgtgcttcg-3⬘ (nt 115-137); CAP18-R1, 5⬘-tcactgtccccatacaccgc-3⬘ (nt
333-352); and, for hybridization, CAP18-PR, 5⬘-caggattgtgacttcaagaaggacg-3⬘ (nt 299-322). The primers for amplification of human neutrophil
peptide 3 (HNP3) were HNP3-S, 5⬘-gccatgaggaccctcg-3⬘ (nt 48-63); and
HNP3-AS, 5⬘-gcagcagaatgcccagag-3⬘ (nt 332-315). These primers also
amplified HNP-1 because the genes are nearly identical.29 The hCAP18
PCR product was subcloned into pcR2.1 (Invitrogen) and sequenced to
verify its identity. The insert was excised with EcoRI and used as a probe in
Northern blot analysis.
Microsatellite sequence polymorphism and single-strand
conformation polymorphism analyses
The polymorphic CA-repeat microsatellite sequence is located 35-bp 3⬘
of the polyadenylation site of the C/EBP⑀ gene (Figure 1B). For analysis,
the microsatellite was amplified by PCR (as described above) with the
use of the primers KO691 (5⬘-ggcaaagagggcaggacccagc-3⬘) and KO692
(5⬘-ggtgcagacctagccacatgc-3⬘) and an annealing temperature of 55°C.30 The
reactions were denatured by heating at 95°C for 5 minutes and electrophoresed through a denaturing 8M-urea, 5% polyacrylamide gel. PCR–singlestrand conformation polymorphism (PCR-SSCP) analysis with primers 404
and NFM-1 was performed as described.31 The products were denatured
and electrophoresed through a nondenaturing polyacrylamide Mutation Detection Enhancement gel (Biowhittaker Molecular Applications, Rockland, ME)
containing 10% glycerol. [33P]–deoxyadenosine triphosphate was added to the
reactions in both procedures to facilitate visualization of the products by
autoradiography (NEN Lifesciences Products, Boston, MA).
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BLOOD, 1 MAY 2001 䡠 VOLUME 97, NUMBER 9
C/EBP⑀ MUTATION IN HUMAN NEUTROPHIL SGD
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Construction and characterization of mutant C/EBP⑀
The A-nucleotide insertion was introduced into the wild-type C/EBP⑀32
cDNA by means of a 2-step PCR approach as described previously.32
Briefly, in one reaction, 100 ng template (pCMV-C/EBP⑀32) was amplified
with the vector-specific primer 32N (5⬘-tcgccggaattcatgtcccacgggacctactacgagtgtgagccccgg-3⬘) and the gene-specific primer SGDMUT-AS
5⬘-ggcctttgagaacgcgcagaggctggccgg-3⬘). In the second reaction, the vectorspecific primer NdelC 5⬘-agcctggtcgacgtgcccacaatccaccagcca-3⬘) was mixed
with the gene-specific primer SGDMUT-S (5⬘-gttctcaaaggccccctttggccactgccgc-3⬘). The products were amplified in a 100-␮L reaction by means of
Advantage Taq as described by the manufacturer. The resulting products
(1 ␮L each) were mixed and amplified by means of the the 2 vector-specific
primers. The full-length product was gel purified, digested with EcoRI and
SalI, and ligated with 100 ng pCMV-SPORT (Gibco/BRL) cut with EcoRI
and SalI. The resulting transformants were sequenced to verify the presence
of the mutation and verify the integrity of the remainder of the insert.
COS-1 and NIH3T3 cells were maintained in Dulbecco’s modified Eagle’s
medium supplemented with either 10% fetal bovine serum or bovine calf serum,
respectively. For cellular localization, COS-1 cells, plated at 70% confluency on a
60-mm dish, were transfected with 3 ␮g pCMV-SPORT, pCMV-C/EBP⑀32, or
pCMV-SGD⑀ by means of 15 mL GenePorter (Gene Therapy Systems, San
Diego, CA), as described by the manufacturer.At 24 hours post-transfection, cells
were harvested, and nuclear and cytoplasmic fractions prepared as described
previously.16 Whole cell lysates were prepared by lysis in RIPA buffer, and
Western blot analysis was performed as described previously.26 The blots were
incubated overnight with 0.1 ␮g anti-C/EBP⑀ antiserum (rabbit) or 0.5 ␮g
anti–␤-actin mouse monoclonal antibody (Santa Cruz Biotechology, CA) diluted
in phosphate-buffered saline containing 5% powdered milk.16 The primary
antibodies were detected with either donkey anti–rabbit horseradish peroxidase
(HRP) (1:5000) or anti–mouse-HRP (1:500). The complexes were developed
with SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL),
as described by the manufacturer and detected by autoradiography.
For transcriptional activation assays, NIH3T3 cells were plated in a
12-well dish at 70% confluency. For each triplicate, the plasmids were
prepared as a master mix of 1.0 ␮g pGCSF-R and 0.1 ␮g pSV40 Renilla
Luciferase (Promega, Madison, WI) plus expression vector plus empty
vector for a final total of 3 ␮g DNA. The combinations and amounts of
expression vectors are indicated in the legend of Figure 3. The plasmids in
0.5 mL Opti-MEM (Gibco/BRL) were mixed with 15 ␮L of GenePorter in
0.5 mL Opti-MEM, incubated 45 minutes, and 0.33 mL aliquoted to each
well of the 12-well plate (1 ␮g DNA per well). The pGCSFR-Luc reporter
plasmid was kindly provided by Dan Tenen (Harvard Medical School,
Boston, MA). At 24 hours post-transfection, cells were lysed in passive
lysis buffer, and luciferase activity was measured by means of a dual
luciferase assay (Promega).
Results
Frameshift mutation in coding region of C/EBP⑀ locus
in SGD individual
Sequencing of PCR products from the genomic DNA of the proband
revealed a single A-nucleotide insertion at nt 1113 (EMBL/GenBank/
DDBJ accession No. U80982) located at the 3⬘-end of exon 2 (Figure
1A-B). This was not detected in the sequence from a normal individual
(Figure 1A). Sequence analysis of this region amplified from the
patient’s cDNA revealed the presence of the A-nucleotide insertion
indicating the mutation was present in the mRNA (data not shown). The
mutation predicts a frameshift and premature termination of the encoded
C/EBP⑀ isoforms p32, p30, p27, and p14 (Figure 1B). The premature
termination would result in the loss of the basic region and leucine
zipper domains that are critical for DNA binding and dimerization,
respectively.
Sequencing of the cloned PCR products from the genomic DNA
of the proband indicated the presence of only the mutant allele.
Figure 1. The C/EBP⑀ gene contains a frameshift mutation in a patient with
SGD. (A) Representative sequence chromatographs from a normal and an SGD
patient from nucleotides 1005 through 1024 based on the previously deposited
sequence.16 The nucleotide sequence is denoted across the top of the chromatograph. The arrows indicate the boundary between exon 2 and intron 2. The codon
encoding amino acid residue lysine 170 (K170) is overlined in the normal sequence.
The sequence was determined from 3 separate PCR reactions on the genomic DNA. A
total of 12 clones from 3 separate PCR reactions were sequenced from both directions to
verify the mutation. (B) Schematic drawing of the C/EBP⑀ genomic locus indicates the 3
exons, translational start codons (ATG) for each isoform, the basic region–leucine zipper
(bZIP) domain, and the 2 alternative promoters, P␣ and P␤. The downward arrowhead
indicates the location of the A-nucleotide insertion in both panels. This would shift the open
reading frame at the K170 codon and result in the lack of the bZIP domain in isoforms p32,
p30, p27, and p14. The upward arrowhead indicates the position of CA-repeat microsatellite that is 35 bp 3⬘ of the polyadenylation site.
This suggested that the mutation was homozygous. To test this, we
used primer pair 404 and NFM-1 to amplify the region of the
mutation from the genomic DNA of the father and mother.
Sequencing of the cloned products revealed the presence in both
parents of wild-type and mutant sequence (data not shown). The
mutation in each parent was an A-nucleotide insertion as described
above for the proband. To confirm the genotype of the proband and
the parents, we performed SSCP analysis using the above primer
pair. Two normal subjects (NHBM-1 and NHBM-2) were homozygous for the wild-type (E) allele (Figure 2A). The proband was
homozygous for the mutant (e) allele, and the parents were
heterozygous for both alleles as indicated by the presence of 2
bands in the parents versus 1 in the normal controls and proband
(Figure 2A). These results support those observed from sequencing
of cloned PCR products.
Mechanism of inheritance of mutant allele
The presence of an identical A-nucleotide insertion in one allele
from each parent strongly suggested that the parents inherited the
same allele from a common distant relative. To test this, we
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GOMBART et al
BLOOD, 1 MAY 2001 䡠 VOLUME 97, NUMBER 9
ment.33 We predicted that the mutant C/EBP⑀ would accumulate in
the cytoplasm and lack the ability to activate transcription from a
promoter containing its binding site. To test this, COS-1 cells were
transfected with either empty vector (⫺), wild-type (W), or mutant
(S) C/EBP⑀32 expression vectors. Whole cell lysates and cytoplasmic (C) and nuclear (N) fractions were analyzed by Western blot
(Figure 3A). The empty-vector transfectants did not express the
C/EBP⑀ proteins whereas the expected wild-type and mutant forms
were expressed at similar levels (Figure 3A). The mutant form did
not appear to be unstable; however, it accumulated in the cytoplasmic fraction and not the nuclear fraction, where the wild-type
protein localizes (Figure 3A, lanes 4-9).
To test the ability of the mutant C/EBP⑀ to activate the
transcription of a promoter containing a C/EBP site, the mutant and
wild-type forms were co-transfected with a G-CSFR-promoter–
luciferase reporter previously shown to be activated by C/EBP⑀32.19
This reporter was activated in a dose-responsive fashion by the
wild-type, but not the mutant, form of C/EBP⑀ (Figure 3B).
Additionally, an increasing dose of the mutant form did not
Figure 2. Autosomal recessive inheritance of the mutant allele from a distant
relative common to both parents of the proband. (A) SSCP analysis demonstrates that genomic DNA from NHBM is homozygous for the wild-type allele, EE; the
patient is homozygous for the mutant allele containing the A-nucleotide insertion, ee;
and both parents are heterozygous, Ee. (B) Microsatellite analysis reveals that the
mutant, e, but not the wild-type, E, alleles carried by the parents possesses the same
“fingerprint,” indicating they inherited the mutant allele from the same distant relative.
performed microsatellite analysis for a marker that is part of the
C/EBP⑀ gene locus. It is located 35 bp downstream of the
polyadenylation site of the gene.30 The father and mother possessed 2 patterns (E, wild type; and e, mutant) indicative of their
heterozygous status (Figure 2B). Interestingly, they shared one
pattern (e) while the other was unique to each parent (E1 and E2).
The proband was homozygous for the “e” pattern. The inheritance
of an identical microsatellite and nucleotide insertion indicates that
the same mutant allele was inherited from each parent (Figure 2B).
This most likely occurred because the parents inherited the same
mutant allele from a common distant relative. The data support a
homozygous recessive inheritance of the mutant allele.
The mutant C/EBP⑀ accumulates in the cytoplasm and is
unable to activate transcription
The predicted protein resulting from the mutation would lack the
bZIP domain, which is essential for dimerization and DNA binding.
In addition, the basic region contains the nuclear localization
sequence responsible for targeting the C/EBPs to this compart-
Figure 3. Aberrant cellular localization and transcriptional activation by the
mutant C/EBP⑀. (A) Western blot analysis of whole cell lysates (lanes 1-3),
cytoplasmic (C; lanes 4, 6, and 8), and nuclear (N; lanes 5, 7, and 9) fractions for
C/EBP⑀ and ␤-actin expression. The absence of the cytoplasmic protein ␤-actin in the
nuclear fraction serves as a control for the experiment. The cells were transfected
with expression vectors that were either empty (⫺) or encoded wild-type (W) or
mutant (S) C/EBP⑀32 (32 and 24 kd, respectively). The arrows at the left of the panels
indicate the positions of the proteins. (B) NIH3T3 cells were co-transfected with
pG-CSFR-luciferase (firefly) and either empty (⫺), wild-type (WT), or mutant (SGD)
C/EBP⑀32. Luciferase activity was measured and normalized to renilla luciferase used
as a control for transfection efficiency. The experiment was performed twice in
triplicate with results presented in relative light units (RLUs).
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BLOOD, 1 MAY 2001 䡠 VOLUME 97, NUMBER 9
C/EBP⑀ MUTATION IN HUMAN NEUTROPHIL SGD
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demonstrate an ability to interfere with the function of the wild type
in a dominant negative fashion (Figure 3B). Together, these data
indicate the A-nucleotide insertion would significantly impair the
normal function of the C/EBP⑀ protein.
Additional defects in primary and secondary granule
gene expression
The C/EBP⑀-deficient mice have a significant decrease in expression of the cathelin-like genes CRAMP and B9. Because these
peptides possess potent bactericidal activity against gram-negative
bacteria and are components of the secondary granules, we
predicted that the human homologue to murine CRAMP, hCAP18,
would be significantly reduced. We examined the expression of
hCAP18 mRNA in the proband and the father using RT-PCR
analysis and found that its expression was 7-fold less in the
proband (Figure 4A). This reduction corresponded with the expected absence of the mRNA encoding the secondary granule
protein lactoferrin, which was absent in the proband (Figure 4A). In
contrast, the expression of the primary granule gene MPO was
unaffected (Figure 4A).
The primary granule protein BPI possesses very potent antimicrobial activity against gram-negative bacteria.34 Since the expression of the neutrophil primary granule defensins is significantly
reduced in SGD patients, we hypothesized that BPI gene expression may be similarly affected. RT-PCR analysis revealed an
absence of BPI mRNA expression in the proband (Figure 4A). This
corresponded with a significant decrease in the mRNA levels of the
primary granule neutrophil defensins HNP-1 and HNP-3 (Figure
4A, HNP1/3) and was consistent with the absence of defensins
described previously for this patient.10 These results suggest that
significantly reduced levels of BPI protein are present in the
proband’s primary granules.
The lack of expression of secondary and some primary
granule proteins in SGD and C/EBP⑀-deficient mice suggests
that the genes are potential targets of C/EBP⑀. Evidence
supporting this hypothesis comes from a myeloid cell line U937
that is stably transformed with a zinc-inducible expression
vector for C/EBP⑀32.27 Previously, we demonstrated that the
secondary granule genes encoding lactoferrin and neutrophil
collagenase were induced with overexpression of C/EBP⑀.27
Similarly, strong induction of hCAP18 mRNA expression
occurred within 24 hours of zinc treatment (Figure 4B). Taken
together, these results reveal additional defects in neutrophil
primary and secondary granule gene expression and suggest that
C/EBP⑀ directly activates their expression.
Discussion
In this report, we described a 1-bp insertion in the coding region of
the C/EBP⑀ gene that results in a frameshift and a truncated protein.
This protein is predicted to be nonfunctional, because it would lack
the bZIP region that is necessary for subunit dimerization and
binding to DNA. This prediction was supported by our functional
analysis demonstrating its inabilty to properly localize or activate
the G-CSFR promoter. Together with the previous description of a
5-bp deletion in the coding sequence of the C/EBP⑀ gene in another
individual,25 our study strongly supports the hypothesis that
mutation of C/EBP⑀ is the primary genetic defect responsible
for SGD.
Two other transcription factors known to be important in
Figure 4. Expression of mRNA encoding the secondary granule protein
hCAP18 and primary granule protein BPI is severely reduced in the PBMCs of
the SGD patient. (A) RT-PCR analysis of total RNA prepared from the PBMCs of the
patient and father. The cDNAs were analyzed for expression of the primary granule
genes MPO, HNP-1 and HNP-3 (HNP1/3), and BPI; the secondary granule genes
lactoferrin (LF) and hCAP18; and the control 18S rRNA. The products were Southern
blotted and hybridized with either internal oligonucleotide or cDNA probes. (B)
Induced expression of C/EBP⑀32 in U937 activates hCAP18 expression. U937 cells
stably transformed with a zinc-inducible empty vector (pMT) or one containing an
insert for C/EBP⑀32 (pMT-⑀32) were treated either without (⫺) or with (⫹) 100 ␮M
ZnSO4 for 24 hours. RNA was harvested and analyzed by Northern blot hybridization
with a probe for hCAP18. The blot was stripped and subsequently hybridized
for ␤-actin.
myeloid cell differentiation are C/EBP␣ and PU.1. Mice deficient
in C/EBP␣ display an early block in maturation of granulocytes at
the myeloblast stage and do not express secondary granules or their
proteins.35 In addition, these mice have severe defects in hepatic
structure and function, including impaired glycogen storage, and
the mice die soon after birth from hypoglycemia.36,37 Mice deficient
in PU.1 exhibit multiple hematopoietic abnormalities of both
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
2566
BLOOD, 1 MAY 2001 䡠 VOLUME 97, NUMBER 9
GOMBART et al
Table 2. Comparison of SGD and C/EBP⑀ⴚ/ⴚ phenotypes
Phenotype
SGD
C/EBP⑀⫺/⫺
Neutrophils
Granule proteins*
Primary
HNP⫺, BPI⫺
Present†
Secondary
LF⫺, NC⫺, TC⫺, CAP18⫺
LF⫺, NC⫺, NGAL⫺,
Tertiary
NG⫺
NG⫺
Nuclear morphology
Bi-lobed
Bi-lobed
Migration
Abnormal
Abnormal
Bactericidal activity
Impaired
Impaired
CRAMP⫺, B9⫺
Eosinophils
Granule proteins
Bacterial infection
MBP⫺, EDN⫺, ECP⫺, EPO⫹
MBP⫺, EPO⫺
Staphylococcu aureus,
Pseudomonas aeruginosa
Psuedomonas aeruginosa,
Klebsiella
SGD indicates neutrophil-specific granule deficiency (SGD); C/EBP⑀, CCAAT/
enhancer binding protein-⑀; HNP, human neutrophil peptide; BPI, bactericidal/
permeability-increasing; LF, lactoferrin; NC, neutrophil collagenase; TC, transcobalamin; CAP18, 18-kd cationic antimicrobial protein; NGAL, neutrophil gelatinaseassociated lipocalin; CRAMP, cathelin-related antimicrobial peptide; NG, neutrophil
gelatinase; MBP, major basic protein; EDN, eosinophil-derived neurotoxin; ECP,
eosinophil cationic protein; EPO, eosinophil peroxidase. The (⫺) denotes severely
reduced levels or complete absence of messenger RNA or protein. The (⫹) indicates
presence was detected.
*The citations for these data are referred to in the text of this manuscript.
†Defensins (HNPs) are not normally expressed in the neutrophils of mice. A
murine BPI gene has not been described.
myeloid and lymphoid lineages and die either in late embryogenesis or within 2 days of birth.38,39 Those mice that are born soon
succumb to an overwhelming systemic bacterial infection.39 With
intensive antibiotic treatment, the mice can survive approximately
2 weeks. The neutrophils from these mice are not detected until
several days after birth, lack a respiratory burst, are less efficient
than normal neutrophils at phagocytosis and bacterial cell killing,
and do not express mRNAs for secondary granule proteins, but do
contain primary granule proteins.39 Although some of the phenotypic features these mice display are similar to those of SGD, a
human with a germline mutation in either the C/EBP␣ or PU.1 gene
would probably not survive. The only plausible scenario would
involve somatic mutations occurring in the myeloid progenitor cell,
thereby resulting in defective granulocyte differentiation.
Our study shows that a germline mutation was inherited by the
proband. Characterizing the genotype of the parents revealed that
the proband inherited the same A-nucleotide insertion from each
parent in a homozygous recessive manner. Inheritance of an
identical microsatellite marker that is part of the C/EBP⑀ locus
together with the nucleotide insertion indicated that the mutant
allele was most likely inherited from a distant relative shared by
both parents. The first report of a C/EBP⑀ gene mutation was in an
SGD individual whose parents were first cousins once removed.25
In light of our results, we predict that the first patient inherited the
mutation in a similar manner.
The mutation of C/EBP⑀ in this SGD individual resulted in
additional uncharacterized defects in gene expression. The levels of
hCAP18 and BPI mRNAs were severely reduced in the SGD
patient, but not in the father. The lack of hCAP18 expression was
expected since it is a secondary granule protein, but the absence of
BPI, a primary granule protein, was intriguing. Previously, the only
primary granule proteins known to be absent in SGD patients were
defensins. Both hCAP18 and BPI possess potent activity against
gram-negative bacteria. Neutrophils of newborn humans contain 3to 4-fold less BPI than adult neutrophils and exhibit significantly
lower antibacterial activity against gram-negative bacteria.40 This
deficiency of BPI may contribute to the increased incidence of
gram-negative sepsis among newborns.40 Similarly, in SGD patients, we hypothesize that the lack of the granule proteins hCAP18
and BPI may explain their increased susceptibility to gramnegative bacterial infections.
The striking similarity between the phenotypes of the C/EBP⑀-null
mouse and the SGD patients was critical in suggesting that mutation of
C/EBP⑀ in humans was the molecular defect responsible for SGD
(Table 2). Interestingly, we have not observed aberrant primary granule
gene expression in the mouse. This is partly due to a lack of defensin
gene expression in murine neutrophils, and a murine BPI gene has not
been described.41 Another interesting observation is the lack of eosinophil peroxidase (EPO) mRNA expression in the bone marrow of the
C/EBP-deficient mouse, but the presence of EPO protein in the
eosinophils of individuals with SGD.11 Despite these differences,
the C/EBP⑀-null mouse should provide a convenient model system to
elucidate the role of C/EBP⑀ in late maturation of granulocytes and the
development of specific therapeutic approaches, such as supplemental
therapy with antimicrobial peptides to treat SGD. The markedly
decreased level of mRNA expression for the primary granule proteins
BPI and defensins in SGD patients also suggests a role for C/EBP⑀ in
earlier phases of the myeloid differentiation program.9,10 Determining
the role of C/EBP⑀ in regulating the expression of granule genes and
promoting earlier and later stages of differentiation may be important for
understanding other neutropenic disease states, such as acute myeloid
leukemia, that show abnormalities in secondary granule gene expression
and differentiation. Interestingly, our previous studies suggest that
C/EBP⑀ is a critical downstream target gene that is responsible for
retinoic acid–induced granulocytic differentiation of acute promyelocytic leukemia cells.27 In addition, it would be interesting to determine
the role of C/EBP⑀ in other models of specific granule deficiciency,
including neutrophils from neonates and patients following thermal injury.42
Acknowledgments
We thank Dr Seiji Kawano for critically reading the manuscript;
Drs Dorothy Park, Alexey Chumakov, and Seisho Takeuchi for
helpful discussions; Dr Dan Tenen for providing the pG-CSFRluciferase construct; and the patient and her parents for participating in this study.
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2001 97: 2561-2567
doi:10.1182/blood.V97.9.2561
Neutrophil-specific granule deficiency: homozygous recessive inheritance of a
frameshift mutation in the gene encoding transcription factor CCAAT/enhancer
binding protein −ε
Adrian F. Gombart, Masaaki Shiohara, Scott H. Kwok, Kazunaga Agematsu, Atsushi Komiyama and H. Phillip
Koeffler
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