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Blood, Vol. 93 No. 10 (May 15), 1999:
pp. 3432-3441
Two Common Functional Polymorphisms in the Promoter Region of the
Coagulation Factor VII Gene Determining Plasma Factor VII Activity
and Mass Concentration
By
Ferdinand M. van 't Hooft,
Angela Silveira,
Per Tornvall,
Anastasia Iliadou,
Ewa Ehrenborg,
Per Eriksson, and
Anders Hamsten
From the Atherosclerosis Research Unit, King Gustaf V Research
Institute and the Cardiology Unit, Department of Medicine, Karolinska
Hospital, and the Division of Genetic Epidemiology, Institute of
Environmental Medicine, Karolinska Institute, Stockholm, Sweden.
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ABSTRACT |
Recent studies have provided evidence for associations between
common polymorphic markers in the coagulation factor VII (FVII) gene
and plasma FVII levels. Here we describe two common, nonrelated, functional polymorphisms in the promoter region of the FVII gene, a G
to T substitution at position  401 and a novel G to A substitution at
position 402. Both polymorphisms strongly influence the binding properties of nuclear protein(s). The rare 401T allele is associated with a reduced basal rate of transcription of the FVII gene in human
hepatoblastoma cells and with reduced plasma concentrations of total
FVII (VIIag) and fully activated FVII molecules (VIIa). In contrast,
the rare 402A allele confers increased transcriptional activity and
is associated with increased plasma FVII levels. Together, the two
polymorphisms explained 18% and 28% of the variation in VIIag and
VIIa, respectively, in a group of 183 healthy, middle-aged men. It is
concluded that these polymorphisms are important for the regulation of
the plasma levels of FVII and that they are likely to be useful genetic
markers to resolve the issue of whether a causal relationship exists
between FVII levels and risk of coronary heart disease.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
COAGULATION FACTOR VII (FVII) is a
vitamin K- dependent protease that plays an important role in the
extrinsic pathway of blood coagulation. FVII is synthesized principally
in the liver and is secreted as an inactive single-chain glycoprotein.
In the presence of tissue factor, inactive FVII is converted by limited proteolysis to its fully activated two-chain form, VIIa. Activation can
be effected by a number of activated coagulation factors, including Xa,
IXa, XIIa, and thrombin. After activation, VIIa rapidly converts FIX
and FX into their active forms, thus initiating the generation of
thrombin and fibrin clot formation.
The coagulant activity of FVII has been identified as a potential risk
indicator for coronary heart disease (CHD). In the Northwick Park Heart
Study, FVII coagulant activity was an independent predictor of
myocardial infarction in initially healthy middle-aged men,1,2 and particularly of fatal coronary
events.3 However, two recent large-scale prospective
studies, the Prospective Cardiovascular Münster
Study4 and the Edinburgh Artery Study,5 failed to identify the FVII level as a predictor of future CHD. Results from
cross-sectional studies are also contradictory. Some studies have
reported increased plasma FVII levels in groups with manifest CHD or at
risk of CHD,6-10 whereas others did not.11-13
Recent studies have provided evidence for associations between common
polymorphic markers in the FVII gene and plasma FVII levels. Two common
polymorphisms, a G to A substitution in the codon for amino acid 353 (R353Q) causing a substitution of arginine by glutamine in the FVII
protein14 and a decanucleotide insertion/deletion at
position 323 in the promoter region
(323P0/1015), have been extensively studied in
relation to FVII levels in the past few years.10,16-25
Heterozygotes for the rare alleles have 20% to 40% lower FVII
coagulant activity than homozygotes for the wild-type
alleles,17,21,22 and allele frequencies differ among populations at different risk of CHD.25 FVII gene
polymorphisms may explain up to about one third of the FVII level
variation in plasma.21 This indicates that plasma FVII
levels are to a major extent determined by genetic influences. However,
there is considerable controversy over whether these genetically
determined variations in plasma FVII levels influence the risk of
CHD.10,23,25-27 It is also notable that there is no
distinct evidence at present that any of the FVII gene polymorphisms
described to date are directly related to changes in FVII metabolism.
It is therefore likely that some of these polymorphisms are in linkage
disequilibrium with other polymorphisms in the FVII gene, which are of
physiological relevance for FVII metabolism. In view of the proposed
role of FVII as a risk factor for CHD, it is important to define the
nature of these functional polymorphisms.
The rate of FVII gene transcription is indicated to be a critical
factor in the regulation of plasma FVII levels. Three common mutations
in the promoter of the FVII gene have been described: the
323P0/10 polymorphism, a T to C substitution at position 122 (122T/C), and a G to T substitution at position
401 (401G/T).15,28 It has been proposed that
the 323P0/10 polymorphism may influence the rate of
transcription of FVII.28 However, in preliminary studies
using electromobility shift assay (EMSA), we found no evidence that
either the 323P0/10 polymorphism or the 122T/C polymorphism affected the interaction with hepatic nuclear proteins, suggesting that these mutations may be irrelevant for FVII gene transcription. We therefore made a more extensive screening of the
promoter of the FVII gene to uncover additional polymorphisms and
performed functional studies in vitro of all promoter polymorphisms encountered. Here we describe a new, common polymorphism at position 402 that is unrelated to the previously described FVII
polymorphisms. It is also shown that the rare alleles of the
polymorphisms at positions 401 and 402 are associated
with marked changes in the rate of FVII gene transcription in vitro in
human hepatoblastoma (HepG2) cells and independently contribute to
altered plasma levels of FVII in vivo in healthy middle-aged men.
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MATERIALS AND METHODS |
Subjects.
A total of 183 men aged 35 to 50 years (40.1 ± 2.8 years, mean ± SD) were investigated in this project. They were recruited at
random from the general population of the greater Stockholm area using
a registry containing all permanent residents in Stockholm County. Of
those initially invited, 81% agreed to participate in the research
program. All the men were interviewed to exclude individuals with a
history of cardiovascular disease. Additional exclusion criteria were
the presence of concomitant disorders, like severely impaired renal
function, arteritis, collagenosis, and diabetes mellitus, a history of
alcohol abuse or other forms of addiction, and non-Swedish origin of
the subject.
Blood sampling, DNA procedures, and biochemical methods.
Blood sampling and preparation of plasma for FVII analyses were as
described.10 For DNA procedures, nucleated cells from frozen whole blood were prepared according to Sambrook et
al29 and DNA was extracted by a salting-out
method.30 The FVII protein concentration was determined as
FVII antigen (VIIag), using an enzyme immunoassay (Factor VII EIA kit,
Dako A/S, Glostrup, Denmark, a kind gift from Dr Mirella Ezban, Novo
Nordisk A/S, Gentofte, Denmark). Plasma levels of activated FVII (VIIa)
were determined with a clotting assay31,32 using soluble
recombinant truncated tissue factor (a kind gift from Prof James H. Morrissey, Oklahoma Medical Research Foundation, Oklahoma City,
OK). Coagulation times were converted to VIIa
concentration by comparison with a standard curve constructed from
varying concentrations of purified recombinant factor VIIa (a kind gift
from Dr Mirella Ezban, Novo Nordisk A/S). Data were analyzed using the
Windows Research Software supplied with the ACL-300 coagulometer
(Instrumentation Laboratories, Milano, Italy), as
described.17 The within and between run coefficients of
variation, respectively, were 1.8% and 2.9% for VIIag and 3.9% and
9.1% for VIIa.
Gene sequencing.
For the nucleotide sequencing of the promoter of the FVII gene, a 705 base pair (bp) section spanning from position 491 to +210 was
amplified by polymerase chain reaction (PCR) using the forward primer
5'-GGCTCACCTAAGAAACCAGC and the reverse primer 5'-AAGAAATTGAACAGGAGCCG. This PCR-fragment was used as template for further amplifications as part of the Taq DyeDeoxy Terminator Cycle
sequencing system (Perkin Elmer, Applied Biosystems Division, Foster
City, CA). Nested primers, designed on the basis of the published
sequences of the promoter of the FVII gene, were used for the analysis
of overlapping sections of 150 to 250 bp in both directions.
Genotyping.
Genotyping for the 401G/T and the 402G/A polymorphisms
was performed using a PCR fragment amplified by the forward primer 5'-GGCTCACCTAAGAAACCAGC and the reverse primer
5'-GTTGACATTCCCCATGGGAC, followed by digestion with the
restriction enzymes TaiI (for the 401G/T polymorphism)
and BslI (for the 402G/A polymorphism). TaiI
recognizes the nucleotide sequence ACGT and therefore cleaves the
401T allele. In contrast, the restriction enzyme BslI
recognizes the nucleotide sequence CC(N7)GG, and will
therefore only cleave the wild-type allele. Because it was observed
that the 401G/T and the 402G/A polymorphisms are not
related (see Results), it was possible to deduce the genotype of the
402 G/A polymorphism by subtraction of the genotype for the
401G/T polymorphism from the genotype obtained by digestion with
BslI. The conditions for the genotyping were: PCR in a 25 µL
reaction mixture containing 50 to 500 ng of genomic DNA, 1.2 µmol/L
of the primers, 50 mmol/L KCl, 1 mmol/L MgCl2, 10 mmol/L
Tris-HCl pH 9.0, 0.1% Triton X-100, 0.2 mmol/L of each dNTP, and 1 unit Termostable Taq polymerase. The reaction mixtures were incubated
for 3 minutes at 94°C, followed by 35 cycles of denaturation at
94°C for 30 seconds, annealing at 59°C for 1 minute, and
extension at 72°C for 2 minutes. Digestion with the appropriate
restriction enzyme was performed in 25 µL of the PCR product using
2.7 units of TaiI at 65°C for 3 hours or 2.7 units of
BslI at 55°C for 4 hours.
Additional assays were developed to genotype for the 401G/T and
402 G/A polymorphisms using primers containing nucleotide mismatches, which made it possible to use restriction enzymes specific
for the two polymorphisms. The forward primers were
5'-CAAATATTTACATCCACACCCA ATAC (401G/T
polymorphism) and
5'-CAAATATTTACATCCACACCCAAG T C
(402G/A polymorphism), whereas the same reverse primer
5'-GTTAAGGTGTGTGTGGCACC was used for both PCR amplifications
(mismatches in primers are underlined). Because it was found in
preliminary experiments that the forward primers influenced the
cleavage efficiency of the restriction enzymes, a second PCR reaction
was introduced using the forward primer 5'-CAAATATTTACATCCACACCC
and the reverse primer 5'-AGGAGAAAGGTCAGGTGACC. The 401G/T
and 402G/A polymorphisms were subsequently assayed with the
restriction enzymes XmnI and PstI, respectively.
Complete agreement was observed between the different assays for
genotyping the 401G/T and 402G/A polymorphisms in all DNA
samples analyzed for this study.
Genotyping for the 122T/C and 323P0/10 polymorphisms was
performed using the forward primer 5'-TCGCATGATTGCTATGGGAC and the reverse primer 5'-GTTGACATTCCCCATGGGAC. The 122T/C
polymorphism was analyzed using the restriction enzyme Bsp1286
I. For the analysis of the 323P0/10 polymorphism, the PCR
fragment was cleaved by the restriction enzyme EcoRI, followed
by electrophoresis using 3% MetaPhor (FMC Bioproducts, Rockland, ME).
In addition to the four promoter polymorphisms, the MspI
polymorphism in exon 8 of the FVII gene was analyzed as described by
Green et al.14 All restriction enzymes were purchased
either from New England Biolabs (Beverly, MA) or from Boehringer
Mannheim (Mannheim, Germany).
EMSA.
Nuclear extracts were prepared according to Alksnis et
al.33 All buffers were freshly supplemented with leupeptin
(0.7 µg/mL), aprotinin (16.6 µg/mL), phenylmethyl sulfonyl fluoride
(PMSF) (0.2 mmol/L) and 2-mercaptoethanol (0.33 µL/mL). The protein
concentration in the extracts were estimated by the method of Kalb and
Bernlohr.34 Incubation for EMSA was conducted as
described35 and the reaction products were applied to 7%
polyacrylamide gel (80:1
acrylamide/N,N'-methylene-bisacrylamide weight ratio),
whereafter electrophoresis was performed in 22.5 mmol/L Tris/22.5
mmol/L boric acid/0.5 mmol/L EDTA buffer for 2.5 hours at 200 V. Nonradioactive competitor DNAs, either identical, or of the opposite
allelic variant, or of nonspecific origin, were added in 100-fold
excess of the labeled DNA.
DNA constructs.
Two sets of double-stranded oligonucleotides were constructed,
constituting the 30 bp sequence around the polymorphic region of the
401G/T and 402G/A polymorphisms, flanked by BamHI
and BglII ends. The double-stranded oligonucleotides were
ligated head to tail into BamHI-digested HCAT
vector.36 The correct sequence and orientation of the
inserts were tested by DNA sequencing. Promoter-CAT plasmids were
constructed using 607 bp promoter fragments (position 490 to
+117) ligated into a SplI- and SalI-restricted pCAT-Basic vector as described by the supplier (Promega, Madison, WI).
The promoter fragments were obtained by PCR amplification of DNA
samples from subjects homozygous for either the 402A or the
401T alleles, or homozygous for the wild-type condition, using
the forward primer 5'-ACATGCATGCGCTCACCTAAGAAACCAGCC and the
reverse primer 5'-ACGCGTCGACGTTTATGGAGAAAACCTCCCC.
Transient transfection assay.
HepG2 cells were cultured in 90-mm dishes in Dulbecco's modified
Eagle's medium (DMEM) supplemented with 10% fetal calf serum. Confluent cells were transfected using the calcium-phosphate DNA coprecipitation method, essentially as described by Sambrook et al.29 pSV- -galactosidase (Promega) was cotransfected as
an internal control. In all experiments, 5 µg of CAT-construct and 5 µg of -galactosidase plasmid were added to the medium. CAT activity was analyzed using the method described by Sambrook et al29 and quantified using a phosphorimager (Bio-imaging
Analyzer BAS-2500, Fuji Photo Film Co, Tokyo, Japan). -galactosidase
activity was determined as described by the supplier (Promega). CAT
levels were expressed in arbitrary units after standardization for
-galactosidase activity. All constructs were tested in triplicates
in four independent transfection experiments.
Statistical methods.
Distributions of continuous variables in groups were expressed as means ± standard deviation. Logarithmic transformation was performed on
all skewed variables to obtain a normal distribution before statistical
computations and significance testing were undertaken. Allele
frequencies were estimated by gene counting. A  2 test
was used to compare the observed numbers of each FVII genotype with
those expected for a population in Hardy-Weinberg equilibrium. The
normalized linkage disequilibrium coefficient (D') for all pairs of
FVII polymorphisms was calculated according to Ott.37 One-way analyses of covariance (with age or age and plasma
triglycerides as covariates) and two-way analyses of variance performed
by the general linear model procedure were performed to test whether genetic variation within the FVII locus was associated with differences in VIIag or VIIa. The Scheffé multiple comparison test was used as a post-hoc test. The percentage of genotype-based variation in FVII
levels was calculated according to Sing and Davignon.38 Differences in binding affinity of nuclear proteins for the polymorphic sites and differences in transcriptional activity between promoter constructs were evaluated by paired Student's t-test.
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RESULTS |
Detection of a new, common polymorphism at position 402 of the
FVII promoter.
A 705 bp section of the proximal promoter of the FVII gene was
sequenced in both directions using DNA samples from 10 subjects with a
broad range of plasma FVII levels. Minor differences were observed
compared with the sequence reported by O'Hara et al39: an
extra C was observed at 149, and an extra G was observed at 459. No differences were found compared with the sequences
published by Pollak et al28 and Erdmann and
Heim.40 The nomenclature introduced by the latter authors
will be used throughout this report. Three previously described
mutations were observed in several subjects: the decanucleotide
sequence insert (5'-CCTATATCCT-3') at position 323
(323P0/10), the T to C substitution at position 122
(122T/C), and the G to T substitution at position 401
(401G/T). In addition, a G to A substitution at position
402 was found in several subjects.
Assays were developed for the detection of all four promoter mutations,
and the nucleotide sequence of the promoter of the FVII gene was
determined in DNA samples from additional subjects who were homozygous
for the rare haplotype 122C/323P10/401T. No
additional polymorphisms were detected when in total nine alleles with
this haplotype were analyzed. The nucleotide sequence was also
determined in DNA samples from subjects who were homozygous for the
rare 402A allele. Again, no additional polymorphisms were
detected when in total 11 alleles with the 402A allele were analyzed.
Allele-specific binding of two nuclear proteins to the 401G/T
and 402G/A polymorphic sites.
The possibility that the decanucleotide insertion at 323 and/or the
nucleotide substitutions at positions 122, 401, and 402 might affect the interaction with nuclear proteins was
analyzed with EMSA using nuclear extracts derived from HepG2 cells. No evidence was found for differences in binding characteristics of a 30 bp DNA fragment containing either the 122T or the 122C site of the FVII promoter (data not shown). Furthermore, no differences were found in the binding characteristics of a 35 bp DNA fragment containing the decanucleotide insertion and a comparable 25 bp DNA
fragment without the insertion (data not shown).
The binding characteristics of the 401G/T and 402G/A
polymorphisms were analyzed using three different 30 bp DNA fragments. The wild-type fragment was a 30 bp fragment containing the common 401G and 402G sites. For the analysis of the
401G/T polymorphism, a 30 bp fragment containing the rare
401T site and the common 402G site was used. This
fragment is referred to as the 401T fragment. The 30 bp fragment
termed 402A contained the rare 402A site and the common
401G site. Considerable quantitative differences were observed
for two protein-DNA complexes when the binding characteristics of the
three DNA fragments were analyzed in detail. As shown in Fig 1A, substantial quantities of
protein-DNA complex 1 (indicated with arrow 1) were observed for the
401T fragment. In contrast, only minor quantities of complex 1 were noted for the wild-type and the 402A fragments. Lower
quantities of protein-DNA complex 2 (indicated with arrow 2) were
observed with the 402A and the 401T fragments than with
the wild-type fragment. Occasionally, we noted that the protein-DNA
complex 2 was split in two bands with slightly different mobility. The
observed differences between the three fragments were quantified in
five independent experiments, using different nuclear extracts from
HepG2 cells. In agreement with the data presented in Fig 1A,
significant quantitative differences were observed between the three
fragments for the two protein-DNA complexes (Fig 1B and 1C). These
quantitative differences were substantiated by the results from EMSA
studies in which the binding characteristics of the three fragments
were analyzed using different concentrations of nuclear extract (data
not shown). Competition experiments showed that all three fragments are
able to compete effectively for the two protein-DNA complexes when
either the 401T fragment (Fig 2),
the 402A fragment, or the wild-type fragment (data not shown)
were used as radiolabeled probe, indicating specific protein-DNA
interactions. Taken together, the EMSA studies provided evidence for
differences in the binding characteristics of the three fragments with
regard to two nuclear proteins.
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| Fig 1.
Differential binding of nuclear proteins to the 401
and 402 polymorphic sites. (A) EMSA of nuclear extract derived from
HepG2 cells bound to a 30 base pair DNA fragment containing either the
402A site (lanes 1 and 2), the wild-type (WT) site (lane 3), or the
401T site (lane 4) of the FVII promoter. Arrows 1 and 2 denote the
allele specific factors. Lane 1, without extract; lanes 2 to 4, 0.20 mg/mL HepG2 extract. (B and C) The relative intensity of the
protein-DNA complexes 1 and 2 obtained with the three DNA fragments
were quantified in five independent experiments. Bars indicate mean
values and standard deviations. The statistical significance of
differences was determined by Student's paired t-test.
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| Fig 2.
Binding of nuclear proteins is specific. EMSA of nuclear
extract derived from HepG2 cells bound to the 401T fragment in the
presence of unlabeled DNA as competitor. Arrows 1 and 2 refer to the
allele-specific factors. Lane 1, without extract; lane 2, 0.20 mg/mL of
HepG2 extract in the absence of competitor; lanes 3 to 6, 0.20 mg/mL of
HepG2 extract in the presence of 200-fold excess of unlabeled DNA as
competitor. Competitors used were: 402A site (lane 3), WT site (lane
4), 401T site (lane 5), and nonrelated 30 bp fragment (lane 6).
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The differences in the binding characteristics of the three fragments
were further explored by quantitative competition experiments. In
Fig 3, the abilities of unlabeled
401T and 402A fragments to compete with a labeled
401T fragment are compared. As expected, the unlabeled
401T fragment was a more efficient competitor for the
protein-DNA complex 1, as compared with the unlabeled 402A fragment, whereas no significant differences were observed between unlabeled 401T and 402A fragments regarding competition
for protein-DNA complex 2 (Fig 3A). These differences were quantified in three independent experiments (Fig 3B and 3C). Similar experiments were performed comparing the abilities of unlabeled 402A and wild-type fragments to compete with the labeled wild-type fragment. These studies showed that the unlabeled 402A fragment is less effective than the unlabeled wild-type fragment in competing for protein-DNA complex 1 (data not shown). In all, the results from the
EMSA studies indicate that both the 401G/T mutation and the 402G/A mutation influence the specific binding properties of two
nuclear proteins.
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| Fig 3.
Differences in binding affinity of the nuclear proteins
for the polymorphic sites. (A) EMSA of nuclear extract derived from
HepG2 cells bound to the 401T fragment in the presence of increasing
concentrations of unlabeled 402A fragment (lanes 2 to 5) or 401T
fragment (lanes 7 to 10) as competitor. Arrows 1 and 2 refer to the
allele-specific factors. Lanes 1 and 6, 0.20 mg/mL of HepG2 extract in
the absence of competitor; lanes 2 to 5 and 7 to 10, 0.20 mg/mL of
HepG2 extract in the presence of threefold (lanes 2 and 7), ninefold
(lanes 3 and 8), 27-fold (lanes 4 and 9) and 81-fold (lanes 5 and 10)
excess DNA as competitor. The intensity of the protein-DNA complexes 1 and 2 were quantified in three independent experiments. The relative
intensities of the protein-DNA complexes 1 and 2 in the presence of
increasing concentrations of unlabeled DNA fragments are shown in B and
C, respectively. Line plots indicate mean values and standard
deviations. The statistical significance of differences was determined
by Student's paired t-test. *P < .05; **P < .01.
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The 401G/T and 402G/A polymorphisms modulate the
transcription of the FVII gene.
Transfection studies in HepG2 cells were conducted to evaluate whether
the 401G/T and the 402G/A polymorphisms influence the
rate of transcription of the FVII gene. CAT activities were compared
between constructs harboring either a single or two tandemly arranged
30 bp fragments of the FVII promoter. These constructs contained either
the 401T, the 402A or the wild-type sites. As shown in
Fig 4, higher CAT activities were observed
with the 402A than with the wild-type constructs. In contrast,
significantly lower CAT activities were observed with the 401T
than with the wild-type constructs (Fig 5).
It is notable that the effects of the tandemly arranged constructs were
greater than those of the single-fragment constructs.
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| Fig 4.
Difference in transcriptional activity of the 402A
allele. The CAT activities of constructs harboring one or two tandemly
arranged 30 base pair fragments with either the wild-type (WT) site or
the 402A site were compared in transfection studies using HepG2
cells. The constructs were tested in triplicate in four independent
experiments. The CAT activities of the 402A-1x and 402A-2x
constructs were expressed relative to the activities of the WT-1x and
WT-2x constructs, respectively, the latter jointly indicated as WT.
Bars indicate mean values and standard deviations. The statistical
significance of differences was determined by Student's paired
t-test.
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| Fig 5.
Difference in transcriptional activity of the 401T
allele. The CAT activities of constructs harboring one or two tandemly
arranged 30 base pair fragments with either the wild-type (WT) site or
the 401T site were compared in transfection studies using HepG2
cells. The constructs were tested in triplicate in four independent
experiments. The CAT activities of the 401T-1x and 401T-2x
constructs were expressed relative to the activities of the WT-1x and
WT-2x constructs, respectively, the latter jointly indicated as WT.
Bars indicate mean values and standard deviations. The statistical
significance of differences was determined by Student's paired
t-test.
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Additional transfection experiments were performed using 609 bp FVII
promoter constructs. In agreement with other reports,36 it
was found that the activity of the FVII promoter is remarkably weak.
The expression of the FVII promoter in HepG2 cells was only two- to
threefold higher than the control plasmid without promoter, whereas in
the same experiments the SV40 promoter gave 25- to 50-fold higher CAT
values. This phenomenon considerably hampered the detection of
differences in CAT activity between the long FVII promoter constructs.
In four experiments a significant reduction (-34% ± 17%,
P < .05) in CAT activity was observed for the 401T construct compared with the wild-type construct. A small increase (+13% ± 21%) in CAT activity was noted for the 402A
construct, but this increase did not reach the conventional level of
statistical significance.
Allele frequencies and degree of linkage disequilibrium.
Genotyping was performed in 183 healthy, population-based men aged 35 to 50 years. All FVII polymorphisms were found to be in Hardy-Weinberg
equilibrium. Allele frequencies were as follows: 122T/C
(0.91/0.09), 323P0/10 (0.91/0.09), 401G/T (0.91/0.09), 402G/A (0.71/0.29), and R353Q (0.92/0.08). The 122T/C,
323P0/10, and 401G/T polymorphisms exhibited complete
allelic association. The normalized linkage disequilibrium coefficient
(D') was 0.4123 (P < .0009) for the 401G/T and
402G/A polymorphisms, 0.9985 (P < .0001) for the
401G/T and R353Q polymorphisms, and 0.4076 (P<0.0026) for the 402G/A and R353Q polymorphisms.
Associations between the 401G/T and 402G/A
polymorphisms and the plasma concentrations of activated FVII and FVII
mass.
The relationships between the 401G/T and 402G/A
polymorphisms and FVII plasma levels were analyzed in detail in the 183 apparently healthy, population-based men. As shown in Table
1, the rare 401T allele was
associated with significantly lower plasma concentrations of FVII
protein (VIIag) and fully activated FVII molecules (VIIa),
respectively, than the common 401G allele. In contrast, the rare
402A allele was associated with significantly higher FVII levels
than the common 402G allele. Furthermore, subjects who were
homozygous for a rare allele had a greater change in FVII levels than
subjects who were heterozygous for a rare allele. The influences of the
401G/T and 402G/A polymorphisms on the VIIag
concentration were about equally strong, accounting for 11.0% and
10.1% of the variation in VIIag, respectively (Table 1). In contrast,
the impact of the 401G/T polymorphism on the VIIa concentration
was stronger (% attributable phenotypic variation 19.1%) than the
corresponding effect of the 402G/A polymorphism (% attributable
phenotypic variation 4.6%). By comparison, the R353Q polymorphism in
exon 8 accounted for 11.1% of the variation in VIIag (P < .0001) and 23.7% of the variation in VIIa (P < .0001).
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Table 1.
Associations Between the 401G/T and 402G/A
Polymorphisms in the Promoter Region of the Factor VII Gene Locus
and Plasma Concentrations of Factor VII Protein and Activated Factor
VII in Healthy Men
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To further determine the impact of genetic variation on FVII levels in
plasma, genotypes for each pair of polymorphisms were defined and
ranked in order of FVII mean values (Table
2). In addition to the two functional
401G/T and 402G/A polymorphisms in the FVII promoter, the
R353Q polymorphism in exon 8 was included in this analysis. The highest
VIIag and VIIa concentrations were encountered in subjects who were
homozygous for the 401G, 402A, and 353R alleles.
Covariance analysis controlling for the influence of age on VIIag and
VIIa (Pearson correlation coefficients: r = 0.06, ns, and r = 0.23, P < 0.01, respectively) showed that the mean FVII level
differences between combined genotypes were highly significant (Table
2). Of note, the genetic contribution to the variation in VIIa
concentration was much higher than the genetic contribution to the
variation in VIIag. Whereas genetic variation associated with genotypes
for each pair of polymorphisms accounted for 12% to 18% of the total
variation in VIIag, the corresponding figures for VIIa were 26% to
28%. The joint effect on VIIag of the 401G/T and R353Q
polymorphisms, which are in strong linkage disequilibrium, was as
expected significantly smaller (11.6%) than the joint effects of the
other two pairs of weakly linked FVII polymorphisms (17.8% for both
the 401G/T and 402G/A polymorphisms and the 402G/A
and R353Q polymorphisms). In contrast, the three pairs of FVII
polymorphisms had a similar impact on the plasma concentration of VIIa.
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Table 2.
Mean Values of Plasma Concentrations of Factor VII
Protein and Activated Factor VII in Groups of Healthy Men With
Different Combinations of Factor VII Genotypes
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Because there is evidence that the plasma FVII levels are influenced by
factors like age, body mass index (BMI), and plasma lipid
concentrations (reviewed in41), we tested whether these factors differed according to 401G/T and 402G/A
genotypes. However, no relationships were observed between the two
polymorphisms and age, BMI, and plasma concentrations of cholesterol
and triglycerides in the major lipoprotein classes VLDL, LDL, and HDL
(data not shown).
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DISCUSSION |
In this study we conducted an extensive screening of the promoter
region of the FVII gene to detect new polymorphisms that might
influence the plasma levels of FVII and the ensuing risk of CHD. One
novel common polymorphism was found, a G to A substitution at position
402. This polymorphism was evaluated with respect to effects on
transcriptional regulation in vitro, along with previously detected
polymorphisms in the promoter region of the FVII gene locus. The
401G/T and 402G/A polymorphisms proved to be functional
in vitro in HepG2 cells and associated with altered plasma
concentrations of FVII in vivo in population-based healthy human
volunteers. Together the 401G/T and 402G/A polymorphisms accounted for 18% and 28% of the variation in the plasma
concentrations of total FVII and fully activated factor FVII molecules, respectively.
Three basic observations were made in relation to both the
401G/T and 402G/A polymorphisms. Firstly, results from
the EMSA studies using nuclear extracts from HepG2 cells indicated that the two mutations influence the specific binding properties of either
both (401T) or one (402A) of two nuclear protein
complexes. Secondly, transfection studies provided evidence that the
rare 401T allele is associated with a decreased basal
transcription rate, whereas the rare 402A allele appeared to
confer increased transcriptional activity compared with constructs
harboring the wild-type allele. Thirdly, significant associations were
found between the two polymorphisms and both the FVII mass
concentration and the plasma level of the fully activated FVII
molecule. The hypothesis that ensues from these observations is that
they are interrelated and part of a sequence of events starting at the level of the FVII promoter and ultimately leading to modulation of the
FVII levels in plasma. Thus, it is envisaged that the 401G/T and
402G/A polymorphisms affect the binding of two hepatic nuclear proteins to the promoter of the FVII gene, with accompanying changes of
FVII expression in hepatocytes and FVII secretion by the liver. This,
in turn, results in decreased (401T allele) or increased (402A allele) plasma levels of FVII.
The molecular mechanisms remain to be determined in detail. The present
data could be interpreted to suggest the hypothesis that the nuclear
protein contained in complex 1 in the EMSA studies functions as a
transcriptional repressor by interfering with the binding/activation of
the protein contained in complex 2 that would act as a transcriptional
activator. The repressor protein, which binds strongly to the rare
401T allele but only to a limited extent to the rare 402A
allele, thus seems to determine the differences in the activity of the
FVII promoter related to the 401G/T and 402G/A
polymorphisms. However, further experiments using for example
methylation interference assays are needed to show that the proteins
contained in complexes 1 and 2 compete for binding at the same or
overlapping sites.
The physiological significance of the 401G/T and 402G/A
polymorphisms was analyzed in transfection studies in HepG2 cells. Initially, we evaluated the effects of these polymorphisms using long
promoter constructs. In agreement with Pollak et al,28 a
reduction in CAT activity was observed for the construct containing the
122C/323P10/401T alleles when compared with a
construct harboring the wild-type alleles. As was also observed by
these authors, the activity of the FVII promoter was found to be
remarkably weak. This phenomenon seriously limits the application of
long promoter constructs for the analysis of the basal rate of
transcription of the FVII gene. We therefore evaluated the effects of
the 401G/T and 402G/A mutations using single or tandemly
arranged 30 bp fragments of the FVII promoter. The use of these
constructs made it possible to evaluate more accurately the impact of
the two mutations on the basal rate of transcription of the FVII gene. These studies clearly showed that the 401T allele is associated with a reduced rate of transcription, whereas the 402A allele is
related to an increased rate of transcription of the FVII gene.
In agreement with the report of Dell'Acqua et al,42 the
401G/T polymorphism showed complete allelic association with the 323P0/10 and 122T/C polymorphisms. Because neither of
these latter polymorphisms were found to exhibit allele-specific
interactions with hepatic nuclear proteins, it could be argued that the
401G/T site accounts for the association of the 323P10
allele with significantly lower plasma FVII concentrations, which has
been shown in several populations.21,22,24,25 It should be
emphasized in this context that failure to detect allele-specific
interactions with nuclear proteins on EMSA does not completely rule out
the potential role of a promoter polymorphism. Indeed, Pollak et
al28 proposed that the 323P0/10 polymorphism is of
functional significance. However, in agreement with our results, no
protein-binding sites were found by these authors in the vicinity of
the 323 site, and the 401G/T site appears to have been
contained in the constructs used in the transfection studies.
The impact of the 401G/T and 402G/A polymorphisms on the
plasma FVII levels was evaluated in apparently healthy, middle-aged Swedish men. When the two functional promoter polymorphisms were analyzed in conjunction, they contributed to 18% of the variation in
the total FVII protein concentration (VIIag) in plasma and to 28% of
the variation in the plasma concentration of fully activated FVII
molecules (VIIa). Furthermore, the impact of the 401G/T site on
VIIa was much stronger than the corresponding effect of the
402G/A site, whereas the two promoter polymorphisms had similar effects on VIIag. It seems conceivable that the 401G/T and
402G/A polymorphisms would only affect the synthesis of FVII.
The R353Q site in exon 8 reduces the secretion of FVII43
and is in strong linkage disequilibrium with the 401G/T site in
most populations studied so far. The question then arises of whether
the R353Q site contributes to the high proportion of the variation in
VIIa associated with the 401G/T polymorphism or whether the
401G/T locus is linked to other mutations in coding regions of
the FVII gene. It is notable in this context that the charge change
ensuing from the substitution of a positively charged arginine with a neutral glutamine at amino acid position 353 may affect the interaction of FVII with lipid surfaces with ensuing effects on the activity state
of the FVII molecule.44 It is noteworthy that the in vivo data are in good agreement with the results from the in vitro transfection studies. Indeed, in both transfection experiments and
clinical studies it was found that the 401T allele was
associated with a major reduction in transcriptional activity and
plasma FVII mass concentration, whereas the enhancing effect of the
402A allele on these parameters was relatively small. However,
the overall effects of the 401G/T and 402G/A
polymorphisms at the population level were comparable, due to the
three-fold higher frequency of the 402A allele as compared with
the 401T allele.
There is some epidemiological evidence that FVII coagulant activity is
a predictor of future CHD.1-4 However, data is
contradictory both with respect to plasma FVII level relations to
CHD1-13 and regarding the potential relationship between
genetic markers associated with FVII coagulant activity and risk of
CHD.10,23,25-27 It is noteworthy in this context that none
of the genetic markers used to date to evaluate the relations of FVII
with CHD are known to be related to changes in FVII metabolism. The
results presented in this report indicate that the 401G/T and
402G/A polymorphisms are physiologically relevant for FVII
metabolism and suggest that they may be useful genetic markers for
resolving the issue of whether a causal relationship exists between
FVII levels and CHD. Needless to say, the predictive power of
individual polymorphisms may vary between populations depending on
differences in the overall risk factor burden, environmental
influences, and gene-environment interactions. It is interesting to
note that the frequencies of the protective Q353 and 401T
alleles were considerably lower amongst the healthy 50-year old Swedish
men participating in the present study (0.08 and 0.09, respectively)
compared with healthy Italian populations (0.22 to 0.25 and 0.13, respectively)21,26,43 in which the incidence of myocardial
infarction is much lower.
| |
ACKNOWLEDGMENT |
The authors thank Tobias Burt for help with the genotyping.
| |
FOOTNOTES |
Submitted August 31, 1998; accepted January 12, 1999.
Supported by grants from the Swedish Medical Research Council (8691),
the Swedish Heart-Lung Foundation, the Marianne and Marcus Wallenberg
Foundation, the European Commission (BMH4-CT96-0272), the Petrus and
Augusta Hedlund Foundation, the King Gustaf V 80th Birthday Foundation,
the Professor Nanna Svartz Foundation, and the Foundation for Old Servants.
The publication costs of this
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.
Address reprint requests to Ferdinand M. van 't Hooft,
MD, King Gustaf V Research Institute, Karolinska Hospital,
S-171 76 Stockholm, Sweden; e-mail: gerd{at}instmed.ks.se.
| |
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Haemostatic function and ischaemic heart disease: Principal results of the Northwick Park Heart Study.
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2:533-537, 1986 |
TOP
ABSTRACT
INTRODUCTION