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Blood, Vol. 92 No. 5 (September 1), 1998:
pp. 1639-1645
Severe Factor VII Deficiency Due to a Mutation Disrupting an Sp1
Binding Site in the Factor VII Promoter
By
Josephine A. Carew,
Eleanor S. Pollak,
Katherine A. High, and
Kenneth A. Bauer
From the Hematology-Oncology Section, Department of Medicine,
Brockton-West Roxbury Department of Veterans Affairs Medical Center,
and Harvard Medical School, Boston, MA; and the Departments of
Pediatrics and Pathology and Laboratory Medicine, University of
Pennsylvania and The Children's Hospital of Philadelphia, PA.
 |
ABSTRACT |
We have identified a point mutation in the promoter of the factor
VII gene responsible for a severe bleeding disorder in a patient from a
large French-Canadian family with known consanguinity. The proband has
an extremely low plasma level of factor VII antigen and factor VII
coagulant activity (<1 percent of normal) and suffers from hemarthroses and chronic arthropathy. Sequencing of the patient's factor VII 5 flanking region, intron/exon junctions, and coding regions showed a homozygous point mutation, a C to G transversion at
position  94 relative to the translation start site. We show here
that this mutation prevented binding of transcription factor Sp1 and of
other nuclear proteins to this region of the factor VII promoter and
resulted in a 20-fold reduction in reporter gene expression in HepG2
cells. These data underscore the importance of this region of the
factor VII promoter for in vivo expression of the factor VII gene.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
HUMAN COAGULATION factor VII is a 406 amino acid, vitamin-K-dependent glycoprotein that is synthesized in
the liver and circulates in blood as an inactive
zymogen.1,2 Factor VII, in its activated form (factor
VIIa), participates in the initiation of coagulation via the extrinsic
pathway in association with tissue factor, an integral membrane protein
that is exposed to the circulation upon vascular injury or stimulation
of monocytes and endothelial cells.3-5 The factor
VIIa-tissue factor complex initiates coagulation by activating both
factor IX and factor X, leading to the localized generation of
thrombin.6,7
A reduced plasma level of factor VII coagulant activity (VII:C) leads
to factor VII deficiency, a bleeding diathesis of variable severity. It
is inherited as an autosomal recessive disorder, and its incidence is
estimated to be 1 per 500,000 in the general population.8,9
In recent years, at least 30 different mutations in the factor VII
structural gene have been identified in patients with factor VII
deficiency.10-15 Although the majority of the mutations described are missense mutations, several nonsense mutations, small
deletions, and splice site abnormalities have been identified as well.
Recognition of defects in the promoter of the human factor VII gene has
also become possible with the identification and analysis of its
5 regulatory region by several groups.16-18 Unlike
the promoters of many eukaryotic genes, that of the factor VII gene has
neither a CAAT nor a TATA box. However, it does include binding sites
for both ubiquitously distributed and liver-enriched transcription factors upstream of the major transcriptional start site at position 51 (all numbering is with respect to the translational start site at position +1.) Among these, the sites for Sp1, a ubiquitous transcription factor of the zinc-finger family, and for hepatocyte nuclear factor-4 (HNF-4), a tissue-restricted orphan receptor, were
shown to be important for expression of the factor VII
gene.17,18
In contrast to the large number of mutations identified within the
structural factor VII gene, there are few reports of alterations within
regulatory regions that influence its expression. We have recently
identified and characterized a point mutation within the 5
regulatory region of the factor VII gene of a patient with severe
factor VII deficiency.19 This mutation is a T to G
substitution at position 61 that markedly reduces promoter
activity by preventing HNF-4 binding and transactivation. In addition,
there is a polymorphism in the 5 regulatory region, a
decanucleotide insertion at position 323, which is associated
with modest reductions in plasma levels of factor VII antigen (VII:Ag)
and VII:C.20,21 Although the decanucleotide insert is often
found in allelic association with the Arg353Gln substitution, a
polymorphism within exon 8, which itself reduces plasma levels of
VII:Ag and VII:C,22 in vitro analysis of the insert
alone indicated that it causes an approximately 33%
reduction in factor VII promoter activity in human hepatoma cells.18
In this report, we describe a new mutation in the factor VII promoter
that is responsible for factor VII deficiency in a large French-Canadian kindred with known consanguinity. The mutation, a C to
G transversion at position 94, is within the core binding site
of transcription factor Sp1. It disrupted binding of both Sp1 and other
nuclear proteins to the factor VII promoter, thereby severely
compromising the expression of the factor VII gene in reporter gene
assays.
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MATERIALS AND METHODS |
Clinical history.
The patient is a member of a large kindred from a small town in Quebec
province. Members of his extended family have been described previously
in the medical literature. Two sisters with factor VII deficiency
identified in 1955,23 shortly after the original
description of the disorder,24 were found in 1971 to be
related to 11 additional individuals (eight women and three men) with
factor VII deficiency.25 These 13 patients exhibited plasma
levels of VII:C ranging from 1.5% to 6% of normal with variable
severities of bleeding. The female patients experienced menorrhagia;
several patients had postsurgical hemorrhage, bruising, and epistaxis;
and one patient suffered a cerebral hemorrhage. The parents of affected
individuals were themselves free of clinical symptoms but had plasma
levels of VII:Ag and VII:C that were approximately 50% of normal,
suggesting that the trait was inherited in an autosomal recessive
manner. All 13 patients were descendants of a couple who emigrated to
Canada from France at the end of the 17th century. Although the
incidence of severe factor VII deficiency is quite rare, it was
calculated to be approximately 1 in 335 individuals in the descendants
of this French couple.25
Informed consent was obtained from the patient and his family. This
study was approved by the Human Studies Committee of the Brockton-West
Roxbury Department of Veterans Affairs Medical Center.
Factor VII assays.
Plasma levels of VII:C and VII:Ag were measured on citrated plasma with
a one-stage clotting assay using Automated Simplastin (Organon Teknika
Corp, Durham, NC) and an enzyme-linked immunoabsorbent assay (American
Bioproducts Co, Parsippany, NJ), respectively. The levels of VII:Ag and
VII:C were expressed as a percentage of the level in a normal pool
constructed from equal volumes of plasma from 30 healthy individuals.
DNA isolation and polymerase chain reaction (PCR).
Genomic DNA was purified from leukocytes isolated from whole blood of
the patient and his parents by standard methods.26 The
immediate 5 flanking region of the factor VII promoter (spanning nucleotides 404 to +98) was amplified by PCR and subcloned into the vector pT7blue (Novagen, Madison, WI) for sequencing. The inserts
were sequenced on a 373A DNA Sequencer (Applied Biosystems, Foster
City, CA) by the dideoxy chain termination method.27 A
clone carrying the patient's mutation provided the template for the
subsequent PCR reaction spanning nucleotides 185 to +1 (with
base +1 designated as the translational start site), which was
subcloned into the promoterless pOGH reporter plasmid containing the
human growth hormone structural gene (Nichols Diagnostics Institute,
San Juan Capistrano, CA); the presence of the mutation was confirmed by
sequencing. A plasmid containing the wild-type sequence of the
185 to +1 fragment of the factor VII promoter was prepared
similarly and used as a positive control. The exons of the factor VII
gene were amplified by PCR from the patient's genomic DNA as described
previously.12 The PCR reactions of exons 1a, 5, 7, and 8 were then subcloned and sequenced, whereas those of exons 2, 3, 4, and
6 were analyzed by direct sequencing. All PCR amplifications were done
with Taq DNA polymerase in a Perkin-Elmer-Cetus DNA Thermal Cycler
(Norwalk, CT). Primers for PCR and sequencing were obtained from
Integrated DNA Technologies (Coralville, IA).
Detection of polymorphic alleles in genomic studies.
Restriction analyses were performed as previously
described22 to determine the genotype of the patient and
his parents with respect to two polymorphisms that affect plasma levels
of factor VII, the decanucleotide insert in the 5 regulatory
region and the Arg353Gln substitution in exon 8. Briefly, to detect the
presence of the decanucleotide insert, a 431-bp fragment spanning
position 323 (the insertion site for the decanucleotide) was
amplified by PCR from the genomic DNA of the patient and his parents.
Approximately 200 ng of PCR product was digested with 20 U of
EcoR I (New England Biolabs, Beverly, MA) and then subjected to
electrophoresis on an 8% (wt/vol) polyacrylamide gel. The fragments
generated were 328 and 103 bp in the absence of the insert, or 328 and
113 bp in its presence. To detect the Arg353Gln substitution, a 239-bp fragment of exon 8 surrounding the codon for amino acid 353 was analyzed similarly. In this instance, the presence of the mutation eliminates the only Msp I restriction site within the fragment. In the absence of the mutation, Msp I digestion produced
fragments of 186 and 53 bp.
Cell culture and transient transfection analysis.
The human hepatoma cell line, HepG2 (ATCC HB 8065), was grown in
minimal essential medium supplemented with 10% fetal bovine serum, 1 mmol/L sodium pyruvate, 10 mmol/L HEPES pH 7.4, 2 mmol/L glutamine, 100 U/mL penicillin G, and 100 mg/mL streptomycin, in a 5% CO2
humidified atmosphere at 37°C. Twenty hours before the start of
transfection, 1 × 106 HepG2 cells were seeded on
replicate 60-mm culture dishes. Cells were transfected with mixtures of
reporter plasmid DNA and an internal plasmid control to monitor
transfection efficiency, complexed with the LipofectAmine reagent
(GIBCO-BRL, Gaithersburg, MD) according to the manufacturer's
recommendations. Each transfection mixture contained 3 µg of the
reporter plasmid and 1.5 µg of pSV- -galactosidase control plasmid
(Promega Corp, Madison, WI). After 16 hours of transfection, full
growth media was given to the cells; 48 hours later, the culture media
and cell lysates were assayed for expression of human growth hormone
(hGH) and -galactosidase, respectively. The media were assayed with
the hGH assay kit (Nichols Diagnostics Institute) according to the
manufacturer's directions. Concentrations of hGH were calculated from
a standard curve fitted with a four parameter logistic model. The cell
lysates were analyzed with a colorimetric -galactosidase assay kit
(Promega Corp). Levels of -galactosidase in the samples were
calculated by comparison with a standard curve fitted by linear
regression using the least-squares method. For each dish, the level of
hGH expressed was normalized using the corresponding level of
-galactosidase to correct for differences in transfection
efficiencies. In each experiment, the pOGH plasmid was also transfected
into cells on control dishes; when normalized for expression of
-galactosidase, the values for hGH produced from the promoterless
plasmid were subtracted from the experimental values reported.
Electrophoretic mobility shift assays.
HeLa cells were cultured as described above, and nuclear extracts were
prepared according to the method of Dignam et al.28 The
method of Bradford et al29 was used to determine protein concentrations in the nuclear extracts. Complementary oligonucleotides extending from position 108 to 84 of the factor VII
5 flanking region were annealed and end-labeled with
[ -32P]-adenosine triphosphate (ATP) using T4
polynucleotide kinase, then purified using Sephadex G-50 spin columns
(Boehringer Mannheim, Indianapolis, IN).19 For gel mobility
shift assays, the procedure of Chodosh et al30 was used
with minor modifications. Ten micrograms of HeLa nuclear extract, or 1 footprint unit of human recombinant transcription factor Sp1 (Promega
Corp), was incubated on ice for 10 minutes with 0.5 µg bovine serum
albumin (BSA), 2.5 µg poly (dI.dC), and 1 µg salmon
sperm DNA (Sigma, St Louis, MO) in 20 µl of binding buffer (25 mmol/L
HEPES pH 7.6, 14 mmol/L KCl, 10% glycerol, 0.1 mmol/L EDTA, 0.75 mmol/L dithiothreitol, 5 mmol/L MgCl2). After addition of
0.1 ng of radiolabeled double-stranded oligonucleotide (approximately
100,000 cpm/ng) to the mixture, the reaction was incubated on ice for
an additional 20 minutes. In competition studies, the unlabeled
competitor oligonucleotide was added during the initial incubation on
ice. Following incubation, the reaction mixtures were electrophoresed
on a 5% (wt/vol) polyacrylamide gel and autoradiographed. The
oligonucleotide sequences used in the reactions were as follows:
wild-type, 5 108 GTGTCCTCCCCTCCCCCATCCCTCT 3 84 and mutant 5 108
GTGTCCTCCCCTCCGCCATCCCTCT 3 84; Sp1 consensus
sequence (Promega Corp), 5ATTCGATCGGGGCGGGGCGAGC 3.
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RESULTS |
Patient.
The patient is a 24-year-old Canadian man with a bleeding diathesis,
having plasma levels of both VII:Ag and VII:C that are less than 1% of
normal. He has a history of easy bruising and was diagnosed with factor
VII deficiency at age 11, when he developed hemarthrosis of the left
knee. Between the ages of 12 and 15 years, he required infusions of
fresh frozen plasma at 2- to 3-month intervals for spontaneous bleeds
within his elbow and ankle joints. He was then placed on chronic
prophylaxis with factor VII concentrate (Immuno AG, Vienna, Austria)
three times per week with amelioration of the bleeding diathesis. The
patient's parents do not have a bleeding disorder, although both
exhibit reduced plasma levels of VII:Ag and VII:C
(Table 1). His two sisters exhibit normal hemostasis and have normal factor VII levels (data not shown).
Identification of mutations in the factor VII gene.
Analysis of the entire coding sequence and the intron/exon boundaries
of the patient's factor VII gene revealed only the presence of two
previously described polymorphisms but no alterations sufficient to
account for his bleeding disorder. In exon 5, a C to T transition was
observed at position 7880, which produces a neutral dimorphism in the
codon for His115.31 The only other change in the coding sequence was a G to A substitution at position 10976, which produces the Arg353Gln polymorphism in exon 8. Subsequently, a 404-bp fragment of the 5 flanking region of the patient's factor VII gene was examined and several changes were found. These were the decanucleotide insert at position 323, a C to T substitution at position
122, and a C to G transversion at position 94. As
mentioned, the decanucleotide insert frequently occurs in conjunction
with the Arg353Gln polymorphism; these polymorphisms either alone or
together induce modest reductions in the plasma levels of factor
VII.18,20-22,32 The substitution at position 122 has
been observed in an asymptomatic individual also carrying the
decanucleotide insert18 and therefore is not expected to
contribute to the patient's phenotype.
The inheritance of these alterations in the factor VII gene was
confirmed by sequence analysis of the parents (Table 1). Both parents
were found to be heterozygous for the transversion at position
94. The mother was heterozygous for the decanucleotide insert,
the 122 transition, and the Arg353Gln polymorphism. The father,
like the patient, was homozygous for all three changes, which may
account for the observation that his levels of VII:Ag and VII:C were
slightly lower than the mother's.
Characterization of the 94 C to G mutation.
To analyze the influence of the 94 C to G mutation on promoter
function, a fragment of the 5 flanking region of the factor VII
gene extending from position 185 to position +1 (the
translational start site) was prepared from the patient's DNA and
inserted into the promoterless pOGH reporter plasmid as described in
Materials and Methods. A reporter plasmid containing the wild-type
sequence was similarly constructed. These plasmids were then used to
direct the expression of the hGH structural gene in transient
transfection experiments in HepG2 cells, a human liver hepatoma cell
line expressing factor VII and other coagulation
proteins.33 Figure 1 shows that
the plasmid containing the mutant promoter fragment exhibited only 5.8 ± 2.2% (1 SD) of the activity observed with the plasmid containing
the wild-type promoter fragment (n = 14).
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| Fig 1.
Functional analysis of the wild-type and mutant (94 C
to G) factor VII promoters. One hundred eighty-six base pair fragments
of wild-type or mutant (MT94) human factor VII promoter sequence were
inserted into the hGH reporter vector and used to transiently transfect
HepG2 cells. The data were corrected as described in Materials and
Methods, and the level of hGH expression from reporter plasmid
containing the wild-type sequence was considered 100%. Relative to
this, the level of hGH expression from the MT94 sequence was 5.8 ± 2.2% (1 SD). The data shown were obtained from three experiments in
which a total of 14 dishes were transfected with each expression
vector.
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The 94 mutation occurs within the central hexanucleotide core of
an Sp1 binding site (CCTCCC to CCTCCG) in an area
shown by in vitro mutagenesis experiments to be important for factor VII expression.17,18 Therefore, we examined by
electrophoretic mobility shift assays the behavior of an
oligonucleotide containing the 94 C to G mutation with nuclear
extracts from HeLa cells (which contain abundant amounts of Sp1) and
compared it to that obtained with an oligonucleotide having the
wild-type sequence (Fig 2). The labeled
wild-type oligonucleotide formed four complexes with HeLa nuclear
proteins designated Sp1, B, C, and D (lane 1). The presence of the
ubiquitous transcription factor Sp1 in the slowest-mobility complex has
been shown previously by the formation of a complex of identical
mobility between recombinant human Sp1 and the wild-type factor VII
sequence and by supershift experiments with anti-Sp1
antibody.18 Binding between HeLa nuclear proteins and the
Sp1, B, and D complexes was specific as it could be competed away by
incubation with increasing amounts of unlabeled wild-type oligonucleotide (lanes 2 through 4) but not by equivalent amounts of
unlabeled mutant oligonucleotide (lanes 5 through 7). With the labeled
mutant oligonucleotide, in contrast, only complex C was formed (lane
8). This complex is likely to be nonspecific because it cannot be
competed away by either the mutant or the wild-type oligonucleotide
(lanes 9 through 14).
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| Fig 2.
Gel mobility shift assays show loss of nuclear protein
binding with a mutant factor VII promoter sequence (MT94) from a
patient with severe factor VII deficiency. A radiolabeled wild-type
(WT) oligonucleotide including the Sp1 binding site (108 to 84 bp
before the translation start site) in the human factor VII promoter
region showed binding to proteins (arrows) from HeLa nuclear extracts
(lane 1). Specificity of binding is shown by competition with unlabeled
WT oligonucleotide sequence at 10×, 100×, and 500× concentrations
(lanes 2, 3, and 4) but not with an oligonucleotide having the mutant
sequence (lanes 5, 6, and 7). Lanes 8 through 14 show the lack of
binding of nuclear proteins by complexes labeled Sp1, B, and D to
radiolabeled probe containing the MT94 sequence.
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Further confirmation that the patient's mutation disrupted the binding
of Sp1 to the factor VII sequence is provided in
Fig 3. As shown in Fig 3A, a labeled
consensus Sp1 oligonucleotide bound a protein from HeLa nuclear
extracts in the absence (lane 1) but not the presence (lane 2) of
unlabeled Sp1 competitor oligonucleotide. The wild-type factor VII
oligonucleotide (lane 3) competed effectively for binding to this
protein, whereas at the same concentration the mutant factor VII
oligonucleotide (lane 4) did not. Additionally, as shown in Fig 3B,
purified recombinant human Sp1 bound directly not only to its consensus
Sp1 oligonucleotide (lane 1) but also to the wild-type factor VII
sequence (lane 2). However, the purified Sp1 did not bind detectably to
the mutant factor VII oligonucleotide (lane 3). These data suggest that
the 94 mutation prevents transcription of the factor VII gene
through disruption of binding by Sp1 and other DNA binding proteins to
this region of the promoter.
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| Fig 3.
Interactions with an Sp1 consensus sequence and human Sp1
protein. (A) Unlabeled wild-type (WT) but not mutant (MT94) human
factor VII sequence competes for Sp1 binding with radiolabeled Sp1
consensus sequence. Lane 1 shows binding of radiolabeled Sp1 consensus
probe by HeLa nuclear extract in the absence of unlabeled competitor
sequences. Lanes 2 through 4 show Sp1 consensus probe binding with HeLa
nuclear extract in the presence of 1,000× concentrations of unlabeled
competitor Sp1 consensus sequence (lane 2), WT factor VII sequence
(108 to 84; lane 3), and MT factor VII-deficient patient
sequence (108 to 84; lane 4). (B) WT but not MT94 factor
VII-deficient patient sequence binds to the transcription factor Sp1.
Binding of radiolabeled oligonucleotides with recombinant human Sp1 is
shown with radiolabeled Sp1 consensus sequence (lane 1) and WT factor
VII sequence (lane 2) but not with MT factor VII-deficient patient
sequence (lane 3).
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DISCUSSION |
We have identified a naturally occurring point mutation, a C to G
transversion at position 94 in the 5 flanking region of the human factor VII gene, which is responsible for factor VII deficiency in a severely affected homozygous patient. The patient also
has two polymorphisms, the decanucleotide insert at position 323
and Arg353Gln, each of which diminishes factor VII expression to a
modest extent. However, these polymorphisms cannot account for his
phenotype, although it is possible they contribute to a more severe
bleeding diathesis than might have been observed in their absence. The
patient is from a large French-Canadian family in which numerous
members have factor VII deficiency. In view of the known consanguinity
within the family,23,25 it is likely that homozygosity for
the C to G transversion at position 94 is responsible for the
factor VII deficiency observed in the other individuals of this
kindred. Indeed, we have investigated a second severely affected
individual from the same region, presumably a distant relative of the
proband, and confirmed that he was homozygous for the mutation at
position 94 (data not shown).
In reporter gene assays performed with HepG2 cells, a fragment of the
mutant promoter extending from position 185 to +1 exhibited very
low activity compared with the corresponding wild-type promoter fragment. In gel mobility shift assays, introduction of the mutant sequence into an oligonucleotide extending from position 108 to
84 disrupted its binding to several HeLa cell nuclear proteins including Sp1. Thus, the reduced activity observed in reporter gene
assays may be due to disruption of binding not only to Sp1 but also to
other transcription factors, as yet unidentified. Numerous examples of
transcription factors that bind either in association with Sp1 or at
overlapping binding sites have been reported, including
Ets,34 GATA-1,35 SREB-1,36 and
H4TF1.37 Other factors such as Sp3 have been reported to
compete with Sp1,38 whereas the specific ratio of Sp1 to
other transcription factors may also be important for determination of
promoter activity.39
Binding of oligonucleotides with the factor VII promoter sequence to
recombinant Sp1 protein was adversely affected by the presence of the
mutation. Additionally, an excess (1,000×) of wild-type promoter
sequence competed away binding of a radiolabeled Sp1 consensus
oligonucleotide to Sp1 protein from HeLa cell extracts. However, excess
mutant factor VII sequence did not, suggesting that the mutant
oligonucleotide did not bind Sp1 at all under these in vitro
conditions. It should be noted that direct binding of purified Sp1 to
even the wild-type factor VII sequence was weak compared with its
binding to the consensus Sp1 oligonucleotide. Within the 6-bp core Sp1
binding region (CCGCCC), the only difference is the central
residue, a G in the consensus but a T in the factor VII sequence. A
previous study investigating the effect of base changes within the core
binding region on the interaction with purified Sp1 found that altering
the central residue from G to T reduces Sp1 binding approximately
threefold.40 Additional reductions in binding may be due to
the effect of factor VII sequence flanking the core binding site.
Greenberg et al 17 have also noted that the factor VII Sp1
site has lower affinity for purified Sp1 than does an Sp1 binding site
identified in the human metallothionein promoter. Weak affinity of even
the wild-type factor VII sequence for Sp1 may explain in part the low
plasma concentration of factor VII that, at 500 ng/mL, is the lowest of
the vitamin-K-dependent coagulation proteins.
Prior studies have shown that the region surrounding position 94
is essential for normal expression of the factor VII gene in hepatoma
cells.17,18 Greenberg et al17 performed
mutational analysis and showed that alteration of the nucleotides at
positions 98 and 100 (CCCCTCCCCC to
CACATCCCCC) or of residues 92 to 96
inclusive (CCCCTCCCCC to CCCCTAAAAA) decreased
activity in reporter gene assays to approximately 12% and 7% that of
the wild-type sequence, respectively. Pollak et al18 made
point mutations at position 100 (CCCCTCCCCC to
CACCTCCCCC), position 94 (CCCCTCCCCC to
CCCCTCCACC), and at both positions. The 100 alteration
diminished specific binding of an oligonucleotide spanning the region
to both HeLa nuclear proteins and purified recombinant Sp1 and reduced the level of expression in reporter gene assays to 35% of that observed with the wild-type sequence. The 94 C to A mutation and
the combined mutations abolished nuclear protein binding and reduced
reporter gene expression to 2% of wild-type. Interestingly, our
patient had nucleotide 94 mutated from C to G, confirming the
importance of wild-type sequence in this region to factor VII
expression.
Naturally occurring mutations involving Sp1 binding sites have recently
been described in the promoters of several other genes with variable
effects on expression. For example, the promoter of 5-lipoxygenase, a
crucial enzyme in the synthesis of leukotrienes, contains five tandem
repeats of a strong Sp1/Egr-1 consensus sequence. Deletions of one or
two repeats, or addition of a sixth, significantly decreases activity
in reporter gene assays.41 Point mutations within the
proximal Sp1 binding site of the human low-density lipoprotein receptor
promoter decrease both Sp1 binding and receptor expression, inducing
moderate42 or severe43 forms of heterozygous familial hypercholesterolemia, depending on the particular base substitution. Point mutations were also associated with increases in
both Sp1 binding and protein expression in the -hemoglobin gene,
resulting in persistence of fetal hemoglobin.44 Naturally occurring mutations such as these, and the one described in this report, validate in vitro analyses of gene regulation and provide insight into mechanisms of human disease.
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FOOTNOTES |
Submitted February 17, 1998;
accepted April 24, 1998.
Supported by the Medical Research Service of the Department of Veterans
Affairs (K.A.B.), the National Institutes of Health Grant No. RO1
HL48322 (K.A.H.), and a fellowship from the Southeastern Pennsylvania
Affiliate of the American Heart Association (E.S.P.).
Address reprint requests to Kenneth A. Bauer, MD, Department of
Veterans Affairs Medical Center, 1400 VFW Parkway, West Roxbury, MA
02132.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
We thank Dr Georges Rivard (Hôpital Sainte-Justine, Montreal,
Canada) for referring the patient and his family for investigation.
| |
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Abstract
Introduction
Abstract