|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 12 (June 15), 1999:
pp. 4277-4283
Variation at the von Willebrand Factor (vWF) Gene Locus Is
Associated With Plasma vWF:Ag Levels: Identification of Three Novel
Single Nucleotide Polymorphisms in the vWF Gene Promoter
By
Angela M. Keightley,
Y. Miu Lam,
Jolene N. Brady,
Cherie L. Cameron, and
David Lillicrap
From the Departments of Pathology and Community Health and
Epidemiology, Queen's University, Kingston, Ontario, Canada.
 |
ABSTRACT |
Both genetic and environmental factors contribute to the normal
population variability of plasma von Willebrand Factor (vWF) levels,
however, regulatory mechanisms at the vWF gene locus itself have not
yet been identified. We have investigated the association between
polymorphic variation in the 5'-regulatory region of the vWF gene
and levels of plasma vWF:Ag in a study of 261 group O blood donors.
Three novel single nucleotide polymorphisms (SNPs) were identified in
the vWF promoter: C/T at -1234, A/G at -1185, and G/A at -1051. These
SNPs had identical allele frequencies of 0.36 for the -1234C, -1185A,
and -1051G alleles and 0.64 for the -1234T, -1185G, and -1051A alleles
and were in strong linkage disequilibrium. In fact, these polymorphisms
segregated as two distinct haplotypes: -1234C/-1185A/-1051G (haplotype
1) and -1234T/-1185G/-1051A (haplotype 2) with 12.6% of subjects
homozygous for haplotype 1, 40.6% homozygous for haplotype 2, and
42.5% of subjects heterozygous for both haplotypes. Only 4.3% of
individuals had other genotypes. A significant association between
promoter genotype and level of plasma vWF:Ag was established (analysis
of covariance [ANCOVA], P = .008; Kruskal-Wallis test,
P = .006); individuals with the CC/AA/GG genotype had the
highest mean vWF:Ag levels (0.962 U/mL), intermediate values of vWF:Ag
(0.867 U/mL) were observed for heterozygotes (CT/AG/GA), and those with
the TT/GG/AA genotype had the lowest mean plasma vWF:Ag levels (0.776 U/mL). Interestingly, when the sample was subgrouped according to age,
the significant association between promoter genotype and plasma vWF:Ag
level was accentuated in subjects > 40 years of age (analysis of
variance [ANOVA], P = .003; Kruskal-Wallis test, P
= .001), but was not maintained for subjects  40 years of age
(ANOVA, P > .4; Kruskal-Wallis test, P > .4). In
the former subgroup, mean levels of plasma vWF:Ag for subjects with the
CC/AA/GG, CT/AG/GA, and TT/GG/AA genotypes were 1.075, 0.954, and 0.794 U/mL, respectively. By searching a transcription factor binding site
profile database, these polymorphic sequences were predicted to
interact with several transcription factors expressed in endothelial
cells, including Sp1, GATA-2, c-Ets, and NF B. Furthermore, the
binding sites at the -1234 and -1051 SNPs appeared to indicate allelic
preferences for some of these proteins. Electrophoretic mobility shift
assays (EMSAs) performed with recombinant human NFB p50 showed
preferential binding of the -1234T allele (confirmed by supershift
EMSAs), and EMSAs using bovine aortic endothelial cell (BAEC) nuclear extracts produced specific binding of a nuclear protein to the -1051A
allele, but not the -1051G allele. These findings suggest that
circulating levels of vWF:Ag may be determined, at least in part, by
polymorphic variation in the promoter region of the vWF gene, and that
this association may be mediated by differential binding of nuclear
proteins involved in the regulation of vWF gene expression.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
THE FORMATION OF a primary platelet plug
at sites of vascular injury is an essential component of hemostasis,
which is mediated in the initial stages by the adhesive multimeric
glycoprotein von Willebrand factor (vWF). A deficiency or qualitative
defect of vWF results in the inherited bleeding disorder von Willebrand disease (vWD), thereby illustrating the physiologic importance of this
protein. In contrast to its role in bleeding disorders, the involvement
of vWF in the pathogenesis of thrombotic disease is less apparent.
Nevertheless, there is growing evidence to implicate vWF in at least
the terminal thrombotic complications of processes such as
atherosclerosis. Porcine models of vWD, in which there is a marked vWF
deficiency, have helped to establish that occlusive coronary thrombosis
is a vWF-dependent condition (reviewed in Brinkhous et
al1). Elevated levels of vWF have been associated with
cardiovascular disorders such as ischemic heart disease, cerebrovascular disease, peripheral and pulmonary vascular disease, and
several conventional risk factors for thrombotic disease, such as
diabetes, hypertension, hypercholesterolemia, and smoking have also
been linked with increased levels of plasma vWF (reviewed in Lip and
Blann2). In some instances, the concentration of plasma vWF
has been established as a significant and independent predictor of
cardiovascular risk in individuals with coronary artery
disease.3-5
vWF, synthesized exclusively by endothelial cells and megakaryocytes,
performs two critical hemostatic functions, mediating platelet
adhesion6 and thrombus formation7 at sites of
vascular damage and serving as the carrier for procoagulant factor VIII in circulating blood.8 The gene encoding vWF, approximately 180 kb in length and containing 52 exons, has been localized to chromosome 12.9,10 Characterization of the vWF regulatory sequence has indicated that control of vWF gene expression occurs through the interaction of positive and negative regulatory elements located within this region. A cell-type-specific promoter was identified in bovine aortic endothelial cells (BAECs) from -487 bp
upstream of the transcription start site to +247 bp, which contains a
ubiquitous core promoter between -90 and +22 bp, a strong negative
regulatory element upstream of the core promoter (-313 to -487 bp) and
a cell-specific positive regulatory region located downstream of the
core promoter in the first exon.11 Endothelial
cell-specific gene expression requires the presence of the positive
regulatory region with an intact GATA binding site11; the
inhibitory effect of the negative regulatory element has been
attributed to interactions with an NF1-like protein12 and
Oct-1.13 An additional element in the vWF promoter, a
polymorphic dinucleotide repeat, (GT)n, which starts at
-668 bp has also been identified,14 but the functional
significance of this element remains to be determined.
Normal levels of plasma vWF can exhibit a sixfold variability, ranging
from approximately 40% to 240% of the mean population level, which is
designated a value of 1 U/mL, based on an earlier report describing
serial studies of vWF:Ag in normal individuals.15 Several
genetic and environmental factors, such as ABO blood
group,16 age,17 pregnancy,18 and
the acute phase response,19 have been described as
influencing levels of plasma vWF, however, knowledge of mechanisms at
the vWF locus itself, which might influence its expression, remains
elusive. Numerous recent reports have documented an association between
variation within regulatory regions of gene loci involved in hemostasis
and circulating levels of these proteins. Furthermore, these sequence
variations, which include polymorphisms in the
 -fibrinogen,20 plasminogen activator inhibitor-1 (PAI-1),21 and protein C22 gene promoters, and
infrequent mutations of the thrombomodulin23 gene promoter
have also been established as independent risk factors for thrombotic
vascular disease. These studies suggest that regulatory sequence
variations in "hemostatic" genes may be critical elements in the
regulation of gene transcription, protein biosynthesis, and possibly,
determination of risk for thrombotic disease. This report has examined
the potential association of three novel single nucleotide
polymorphisms (SNPs) in the vWF regulatory sequence with the
concentration of plasma vWF in vivo; the binding of nuclear proteins
involved in vWF transcription at each of these polymorphic sites has
also been investigated.
| |
MATERIALS AND METHODS |
Subjects.
Blood samples were collected from 261 blood donors attending the
Canadian Red Cross donor center in Ottawa, Ontario, Canada. The study
protocol had been approved by the local institutional ethics review
board, and all samples were given a unique coded identifier. Blood
donors at this clinic represent a variety of ethnic groups, but are
predominantly Caucasian. Samples were collected exclusively from group
O donors to minimize the variability of plasma vWF:Ag levels that is
recognized to result from differences in ABO blood type.
Measurement of plasma vWF:Ag.
Plasma vWF:Ag was determined by enzyme-linked immunosorbent assay
(ELISA) using a polyclonal goat antihuman vWF antibody (Affinity Biologicals, Hamilton, Ontario, Canada). A "CryoCheck" normal reference plasma (Precision Biologicals, Dartmouth, Nova Scotia, Canada), for which a variety of hemostatic parameters
including vWF:Ag have been quantified using World Health Organization
standards (WHO Lot 91/666), was used to calculate levels of plasma
vWF:Ag for each subject. Each individual assay for vWF:Ag was performed in triplicate.
Genomic amplification and sequencing.
Genomic DNA was extracted from EDTA-anticoagulated whole blood by a
previously described salt extraction method.24 A portion of
the vWF gene, from -1380 to -534 bp upstream of the transcription start
site, was screened for promoter polymorphisms. This 846-bp fragment was
amplified by polymerase chain reaction (PCR) from genomic DNA samples
under the following conditions: 94°C for 1 minute, 56°C for 30 seconds, and 72°C for 30 seconds (30 cycles in total). The
downstream and upstream oligonucleotide primers were
5'-ATAAGAGCTGGAAGTGGAAA-3' and
5'-GGGAGTGATGGTTTGAGTCT-3' (Cortec DNA Service
Laboratories, Kingston, Ontario, Canada), respectively. Several of
these amplified products were inserted into the multiple cloning site
of the pCR2.1 vector (Invitrogen, Carlsbad, CA). SNPs were detected in
these clones by either manual direct dideoxy sequencing using a T7
Sequenase v. 2.0 kit (United States Biochemical, Cleveland, OH) or by
automated sequencing (Mobix, Hamilton, Ontario, Canada).
SNP genotyping by allele-specific oligonucleotide (ASO)
hybridization.
vWF -1234C/T, -1185 A/G, and -1051 G/A promoter genotypes were
established for each subject by ASO hybridization; the sequence and
melting temperature (Tm) for each centrally mismatched ASO is listed in Table 1. Using 15 U of T4
polynucleotide kinase (New England BioLabs, Beverly, MA), 20 pmol of
each ASO were end-labelled with 10 µCi [ -32P]
adenosine 5' triphosphate (ATP) and applied to a Sephadex
G-50 (Pharmacia Biotech, Piscataway, NJ) spin column to remove
unincorporated isotope. Amplified genomic DNA (5 µL, conditions as
previously described) was denatured, applied to Hybond-N+ nylon
membrane (Amersham, Arlington Heights, IL) in a Minifold II slot blot
apparatus (Schleicher and Schuell, Keene, NH), and baked at 80°C
for 2 hours. Membranes were prepared in duplicate and prehybridized in
5X SSPE (0.75 mol/L NaCl, 50 mmol/L NaH2PO4, 5 mmol/L EDTA), 5X Denhardt's solution (0.1% Ficoll 400, 0.1%
polyvinylpyrrolidone, 0.1% bovine serum albumin), and 0.5% sodium
dodecyl sulfate (SDS) for 1.5 hours at 37°C. Each of the
end-labelled ASOs was hybridized separately to one of the membranes in
the prehybridization solution at 37°C for 16 hours. Membranes were
subsequently washed to a stringency of 150 mmol/L NaCl and 15 mmol/L
sodium citrate (1X SSC, pH 7.0) at temperatures corresponding to the
Tm of each ASO probe (Table 1) and autoradiographed for 2 hours at -70°C with Kodak X-OMAT AR film (Eastman Kodak, Rochester,
NY). Genomic DNA samples with known SNP genotypes, confirmed by direct
sequence analysis, were included on each membrane as controls for
hybridization and washing conditions.
Electrophoretic mobility shift assays (EMSAs).
The polymorphic promoter sequences at -1234, -1185, and -1051 were
assessed for potential transcription factor binding sites with the
TFSEARCH program (v. 1.3),25 which searches sequence fragments against the MATRIX table of TRANSFAC,26 a
transcription factor binding site profile database.
Nuclear extracts were prepared from BAECs using a previously described
rapid extraction method.27 Double-stranded oligonucleotides (200 ng), designed for each type of polymorphic sequence (Table 1),
were labelled with 15 µCi [ -32P] dATP and 5 U of the
Klenow fragment of DNA polymerase I (Gibco BRL/Life Technologies,
Gaithersburg, MD) in a reaction containing (final concentrations) 10 mmol/L Tris (pH 7.5), 50 mmol/L NaCl, 10 mmol/L MgCl2, 1 mmol/L dithiothreitol (DTT), and 0.5 mmol/L deoxynucleotide triphosphates (dNTPs; excluding dATP). After a 15-minute incubation at room temperature (RT), 5 mmol/L
nonradiolabelled dATP was added and incubated for an additional 10 minutes. Finally, labelled oligos were purified with chloroform:isoamyl
alcohol (24:1) and applied to a Sephadex G-50 spin column to remove
unincorporated isotope. For EMSAs, protein-DNA complexes were formed
essentially as described,28 but with a few modifications.
The EMSA reaction contained 25 mmol/L HEPES (pH 7.6), 34 mmol/L KCl, 5 mmol/L MgCl2, 1 µg of double-stranded poly (dI-dC)
competitor DNA, and 10 µg of bovine serum albumin. To the
radiolabelled oligo/buffer mixture, 5 µg of BAEC nuclear extract or
0.1 µg of recombinant human NFB (p50 subunit) (Promega, Madison,
WI) was added and incubated at RT for 20 to 30 minutes. For supershift
assays, the EMSA protocol was identical, but with a subsequent
incubation (15 minutes at RT) with 0.1 to 0.2 µg of polyclonal
antibodies directed against NFB p50 (Santa Cruz Biotechnology, Santa
Cruz, CA). Competition EMSAs were performed with 10-fold, 50-fold, and
100-fold excess of unlabelled oligonucleotide for -1234C/T and
-1051G/A.
Statistical analysis.
Allele frequencies for each SNP were estimated by gene counting;
 2 analysis was used to evaluate deviation of genotype
distributions from Hardy-Weinberg equilibrium and also to test for
allelic association between these SNPs. The skewness of the plasma
vWF:Ag distribution was normalized by logarithmic (ln) transformation,
however, untransformed mean vWF:Ag levels are reported in all tables
for convenience. Transformed levels of vWF:Ag were compared between
genotype groups using analysis of covariance (ANCOVA), in which age was
incorporated as a covariate into a one-way analysis of variance
(ANOVA). In addition, subjects were divided into two age groups (40
years and >40 years); subgroup analysis was performed using one-way ANOVA. Alternatively, a nonparametric Kruskal-Wallis test was performed
to assess differences in untransformed vWF:Ag levels between genotype
groups. Student's unpaired t-tests were used to compare age
and transformed plasma vWF:Ag levels between male and female subjects.
| |
RESULTS |
Range of plasma vWF:Ag values.
The general characteristics of the subjects in this study are
summarized in Table 2. The mean (standard
deviation [SD]) plasma vWF:Ag level of the entire sample was 0.831 (0.390) U/mL, which is in a range previously reported for group O
individuals.16 The normal range of plasma vWF:Ag levels in
these subjects was considered to be 0.332 to 1.994 U/mL (40% to 240%
of the sample mean).15 Therefore, according to this
criterion, 95% of subjects in this study had normal levels of plasma
vWF:Ag, while subnormal levels of vWF:Ag were identified in 3.8% of
subjects, and 1.2% of individuals had abnormally high vWF:Ag levels.
Mean age and mean level of plasma vWF:Ag did not differ significantly
between male and female subjects (P > .1).
Characterization of vWF promoter SNPs.
Direct sequence analysis of genomic clones containing 846 bp of vWF
promoter sequence, from -1380 to -534, showed the presence of three
novel SNPs: C/T at -1234, A/G at -1185, and G/A at -1051. These SNPs
had identical allele frequencies of 0.36 for the -1234C, -1185A, and
-1051G alleles and 0.64 for the -1234T, -1185G, and -1051A alleles. The
distribution of vWF -1234C/T, -1185A/G, and -1051G/A genotypes, shown
in Table 3, was not significantly different from the
distribution expected from Hardy-Weinberg equilibrium (P > .1). Of the 27 possible genotypes, only three main genotypes were
represented (-1234/-1185/-1051): CC/AA/GG (12.6%), CT/AG/GA (42.5%),
and TT/GG/AA (40.6%). Only 4.3% of subjects had genotypes different
from those listed above. Using contingency tables constructed for each
pair of SNPs, it was determined that the vWF -1234C/T, -1185A/G, and
-1051G/A polymorphisms were in strong linkage disequilibrium (P = .001). Therefore, these SNPs were segregating as two distinct haplotypes: -1234C/-1185A/-1051G (haplotype 1) and -1234T/-1185G/-1051A (haplotype 2).
Association of vWF promoter genotype with level of plasma vWF:Ag.
Mean levels of plasma vWF:Ag were significantly different between SNP
genotype groups (Table 4) such that individuals
homozygous for haplotype 1 (ie, CC/AA/GG) had a higher mean vWF:Ag
level than subjects homozygous for haplotype 2 (ie, TT/GG/AA) (0.962 U/mL v 0.776 U/mL), with heterozygotes possessing an
intermediate level (0.867 U/mL) of vWF:Ag (ANCOVA, P = .008;
Kruskal-Wallis test, P = .006). Although age was not
significantly different between genotype groups (ANOVA, P > .5), it was determined to be significantly associated with the level of
plasma vWF:Ag (ANCOVA, P = .011). Before the ANCOVA analysis,
the relationship between age and plasma vWF:Ag within each specific
genotype group was examined and compared. This relationship was not
significantly different between SNP genotype groups, indicating that
the association between age and plasma vWF:Ag was essentially
identical, regardless of SNP genotype, which was the assumption of the
ANCOVA model. Interestingly, the effect of vWF:Ag variation associated
with these SNPs differed dramatically when individuals were grouped according to age (40 years and >40 years) in the subgroup analysis. The correlation between SNP genotype and level of plasma vWF:Ag was
accentuated in subjects >40 years of age (Table 4), with a difference
of 0.28 U/mL between mean plasma vWF:Ag levels in subjects homozygous
for haplotypes 1 and 2 (ANOVA, P = .003; Kruskal-Wallis test,
P = .001). In contrast, the association between promoter genotype and vWF:Ag level was not maintained in subjects 40 years of
age (ANOVA, P > .4; Kruskal-Wallis test, P > .4),
as shown in Table 4. It should be noted that allele frequencies for
these SNPs did not differ between age groups.
Binding of endothelial cell nuclear proteins to vWF promoter SNPs.
To investigate the possibility that these SNPs alter binding of nuclear
proteins, EMSAs were performed using double-stranded oligonucleotides
corresponding to the allele sequences at -1234, -1185, and -1051. Incubation of these allele sequences with nuclear extracts from
unstimulated ("resting") BAECs produced a different pattern of
protein-DNA complex formation for each polymorphic site
(Fig 1A). For the -1234C/T polymorphism,
both alleles bound one major factor present in the BAEC nuclear
extract, however, the gel-retarded complex is less intense in the assay
with the C allele. EMSAs with the -1051G/A SNP showed a nuclear protein bound by the A allele that was not bound by the G allele. Neither of
the alleles at -1185 (A or G) bound proteins in the BAEC nuclear extract. The formation of protein-DNA complexes (or lack thereof) in
EMSAs as described was verified by five independent assays. Additionally, competition EMSAs, performed with 10-fold, 50-fold, and
100-fold excess of unlabelled oligonucleotide for both -1234C/T and
-1051G/A, produced a marked reduction in protein-DNA complex formation
in all reactions (data not shown), which indicated specificity of
binding. A database search (TFSEARCH/TRANSFAC) with the sequences for
these SNPs identified several transcription factors expressed in
endothelial cells, which might bind these polymorphic sites, including
Sp1, GATA-2, c-Ets, and NFB. Furthermore, it appeared as though some
of these binding sites demonstrated allelic preference; for example,
NFB was predicted to bind preferentially to the T allele at -1234, and at -1051, a binding site for GATA-2 was evident for the A allele,
but not the G allele. The preferential binding of NFB (p50) by the
-1234T allele was confirmed by EMSAs with rhNFB (p50) (Fig 1B).
Protein-DNA complex formation was evident only for the -1234T allele,
and this complex was supershifted by antibodies directed against NFB
(p50).
View larger version (56K):
[in this window]
[in a new window]
| Fig 1.
Differential binding of nuclear proteins to -1234C/T,
-1185A/G, and -1051G/A allele sequences. (A) EMSAs performed with
nuclear extracts from unstimulated BAECs. Arrow, -1234C/T-specific
factor; arrowhead, -1051A allele-specific factor; free, free (unbound)
probe. Allele sequences for -1234C/T and -1051G/A exhibited
differential DNA-protein complex formation, while the -1185A/G
sequences did not appear to bind factors present in BAEC nuclear
extracts. (B) EMSAs performed with recombinant human NFB (p50).
Arrow, oligo/rhNFB p50 complex formation; arrowhead, supershift with
anti-NFB (p50) antibody (Ab). Markedly enhanced binding of rNFB
p50 occurred to the -1234T allelic probe; this complex was
supershifted by antibodies directed against NFB p50. A NFB
consensus oligonucleotide was included in this EMSA as a positive
control.
|
|
| |
DISCUSSION |
In this study, three novel SNPs of the vWF promoter are described,
-1234C/T, -1185A/G, and -1051G/A; the strong allelic association of
these polymorphisms was expected, given their close physical proximity
within the promoter. The objective of this study was to evaluate the
effect of genotypic variation at the vWF locus on levels of plasma
vWF:Ag in vivo. A clear correlation between vWF promoter SNP genotype
and plasma vWF:Ag levels was observed in this sample of 261 group O
blood donors; because of the unbiased manner in which subjects were
ascertained for this study, it is likely that this sample is
representative of a larger population of normal individuals. The mean
levels of plasma vWF:Ag differed significantly according to SNP
genotype, with higher levels of vWF:Ag found in subjects with the
CC/AA/GG genotype, intermediate levels in CT/AG/GA subjects, and the
lowest levels in subjects with the TT/GG/AA genotype. ABO blood type
and age, factors known to influence plasma levels of vWF, did not
account for the differences described above. To our knowledge, this is
the first report of an association between genotypic variation at the
vWF locus and levels of plasma vWF:Ag. Although this association has
not yet been assessed in nongroup O subjects, it is likely that the
findings of this study will be applicable to other blood groups. One of these polymorphisms, -1051G/A, and an additional SNP, C/G at -1792, were recently reported in a large population of patients with ischemic
heart disease (IHD) and healthy, age-matched controls not to be
correlated with circulating levels of vWF.29 However, in
that study, which did not control for ABO blood group, any potential
association may have been obscured by the variability of plasma vWF
levels known to result from this factor. Interestingly, in this
preliminary report, both the -1792C/G and -1051G/A polymorphisms were
identified as potential predictors of risk for IHD. This finding
confirms the observations of previous studies of hemostatic variables,
such as -fibrinogen,20 PAI-1,21 protein
C,22 and thrombomodulin,23 which have suggested
that some individuals may be at greater risk for manifesting thrombotic
disease because of inherited differences in hemostatic protein
expression. The relative contributions of genetic and environmental
factors to vWF expression have not been clearly defined, however, the
significant finding in this study that promoter genotype is related to
plasma levels of vWF:Ag would imply a significant role for genetic
determinants of vWF:Ag concentration, particularly when combined with
the well-documented relationship between ABO blood type and circulating
levels of vWF. However, the magnitude of this genotypic effect is
likely modulated by several environmental factors, as interactions
between genotype and environmental variables have been reported for the determination of plasma levels of other hemostatic proteins such as
fibrinogen (reviewed in Humphries et al30), and
PAI-1.31 In fact, a genotype-environment interaction might
account for the significant correlation between promoter genotype and
plasma vWF:Ag levels in subjects >40 years of age and the lack of
such an association in subjects 40 years of age. Although reports of
similar findings are rare, it is not unreasonable to propose that some
types of genotypic variation may only be functionally relevant within a
specific environmental context, for example, that of the aging
circulation. Although the mechanisms mediating expression of vWF in
endothelial cells are largely unknown, the biomechanical forces
generated by blood flow, such as fluid shear stress, may well play a
role in this regulation, and blood flow characteristics are well known
to change with advancing age. Interestingly, a contrasting result was
recently reported in the study of the relationship between a SNP in the
-fibrinogen promoter (-455G/A) and plasma fibrinogen levels within
different age groups. When grouped by decade of age, the association of
-455G/A genotype with plasma fibrinogen levels, although significant in
the 45 to 55-year old age group, was diminished in the older age groups (55 to 65 years and >65 years).32
Several different mechanisms could account for the association between
vWF promoter SNP genotype and plasma vWF:Ag level. The three
polymorphisms determining the promoter genotype might affect the
binding affinity of nuclear proteins involved in regulating transcription of the vWF gene. Differential binding of nuclear proteins
has been demonstrated at the site of the 4G/5G PAI-1 polymorphism; both
alleles bound a transcriptional activator, whereas the 5G allele also
bound a repressor protein, which was associated with reduced basal
levels of PAI-1 transcription.21 Alternatively, these SNPs
may be in linkage disequilibrium with functionally important sites
elsewhere, either in the coding region of the gene, which might affect
posttranscriptional processing of vWF, or in regulatory sequences in
close proximity to the vWF gene. Preliminary evidence that the genotype
of two of these three SNPs alters the binding affinity of nuclear
proteins involved in transcriptional regulation has been presented in
this study. Although these sequences are greater than 1 kb upstream of
the transcriptional start site, the in vivo study of vWF regulatory sequences has suggested that elements in addition to the region of the
promoter from -487 to +250 bp are required for widespread cell-type-specific expression.33 Thus, sequences further
upstream of -487 may very well play an important role in
transcriptional regulation of this locus in the intact organism.
Differential binding of at least two proteins (NFB and GATA-2)
potentially involved in the regulation of vWF transcription was
predicted by searching the sequences surrounding these polymorphic
sites against a transcription factor profile database. EMSAs with
rhNFB p50 and BAEC nuclear extracts showed significant differential protein binding between the C and T alleles at -1234 and the G and A
alleles at -1051, respectively. A supershift EMSA performed with the
-1234T allele sequence confirmed the preferential binding of NFB p50
to this allele. Intriguingly, the differential protein binding at both
-1234 and -1051 indicates enhanced binding with the allelic sequences
that constitute part of haplotype 2 (TGA), the haplotype associated
with lower plasma levels of vWF:Ag. If the influence of these SNP
genotypes is mediated through the direct interaction of proteins with
these sites, the implication of these findings is that at least one of
the proteins binding to these sites possesses the properties of a
transcriptional repressor. Transient transfection studies, using
constructs containing either haplotype 1 or haplotype 2 SNP allele
sequences situated upstream of a luciferase reporter gene, are
currently in progress to address this issue. Interestingly, even though
NFB has been, to date, widely regarded as a positive regulator of
gene expression, it was recently reported that developmental silencing
at the human  -globin gene locus was mediated by a NFB site in the
3'-flanking region.34 Regardless, the differential
binding of endothelial cell nuclear proteins to SNP allele sequences
suggests that the polymorphisms in the vWF promoter may have a direct
functional role in the control of gene expression.
While a significant correlation between SNP genotype in the vWF
promoter and circulating levels of vWF:Ag was observed for this group
of subjects, it is quite possible that these SNPs may act in concert
with other polymorphic sequence elements in the vWF promoter, or within
the coding region of the gene, to influence levels of plasma vWF.
However, if the recent assessment of sequence diversity in the human
lipoprotein lipase gene, which reported 79 single nucleotide
substitutions within a 9.7 kb region,35 is any indication
of the extent of sequence variation within other genes, then the
identification of functionally relevant sites, or combination of sites,
could be challenging. Nevertheless, given the apparent clinical
significance of elevated levels of plasma vWF, further studies should
investigate whether the genotype-specific increase in plasma vWF:Ag
levels contributes directly to the increased risk of thrombotic disease
in these individuals and if the promoter genotype could predict for
therapeutic response.
| |
ACKNOWLEDGMENT |
The authors acknowledge Dr Tony Giulivi, of the Canadian Red Cross, for
provision of the blood samples.
| |
FOOTNOTES |
Submitted August 19, 1998; accepted February 10, 1999.
Supported by Grant No. NA-3661 from the Heart and Stroke Foundation of
Ontario, Canada. A.M.K. is the recipient of a Medical Research Council
of Canada Studentship and D.L. is a Career Investigator of the Heart
and Stroke Foundation of Ontario.
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 David Lillicrap, MD, Department of
Pathology, Queen's University, Kingston, Ontario, Canada K7L 3N6;
email: lillicrap{at}cliff.path.queensu.ca.
| |
REFERENCES |
1.
Brinkhous KM, Reddick RL, Read MS, Nichols TC, Bellinger DA, Griggs TR:
von Willebrand factor and animal models: Contributions to gene therapy, thrombotic thrombocytopenic purpura, and coronary artery thrombosis.
Mayo Clin Proc
66:733, 1991[Medline]
[Order article via Infotrieve]
2.
Lip GYH, Blann AD:
von Willebrand factor and its relevance to cardiovascular disorders.
Br Heart J
74:580, 1995[Abstract/Free Full Text]
3.
Jansson J-H, Nilsson TK, Johnson O:
von Willebrand factor in plasma: A novel risk factor for recurrent myocardial infarction and death.
Br Heart J
66:351, 1991[Abstract/Free Full Text]
4.
Thompson SG, Keinast J, Pyke SDM, Haverkate F, van de Loo JCW:
Hemostatic factors and the risk of myocardial infarction or sudden death in patients with angina pectoris.
N Engl J Med
332:635, 1995[Abstract/Free Full Text]
5.
Montalescot G, Philippe F, Ankri A, Vicaut E, Bearez E, Poulard JE, Carrie D, Flammang D, Dutoit A, Carayon A, Jardel C, Chevrot M, Bastard JP, Bigonzi F, Thomas D:
Early increase of von Willebrand factor predicts adverse outcome in unstable coronary artery disease: Beneficial effects of enoxaparin.
Circulation
98:294, 1998[Abstract/Free Full Text]
6.
Tschopp TB, Weiss HJ, Baumgartner HR:
Decreased adhesion of platelets to subendothelium in von Willebrand's disease.
J Lab Clin Med
83:296, 1974[Medline]
[Order article via Infotrieve]
7.
Turitto VT, Weiss HJ, Baumgartner HR:
Platelet interaction with rabbit subendothelium in von Willebrand's disease: Altered thrombus formation distinct from defective platelet adhesion.
J Clin Invest
74:1730, 1984
8.
Weiss HJ, Sussman II, Hoyer LW:
Stabilization of factor VIII in plasma by the von Willebrand factor: Studies on posttransfusion and dissociated factor VIII and in patients with von Willebrand's disease.
J Clin Invest
60:390, 1977
9.
Mancuso DJ, Tuley EA, Westfield LA, Worrall NK, Shelton-Inloes BB, Sorace JM, Alevy YG, Sadler JE:
Structure of the gene for human von Willebrand factor.
J Biol Chem
264:19514, 1989[Abstract/Free Full Text]
10.
Ginsburg D, Handin RI, Bonthron DT, Donlon TA, Bruns GAP, Latt SA, Orkin SH:
Human von Willebrand factor (VWF): Isolation of complementary DNA (cDNA) clones and chromosomal localization.
Science
228:1401, 1985[Abstract/Free Full Text]
11.
Jahroudi N, Lynch DC:
Endothelial-cell-specific regulation of von Willebrand factor gene expression.
Mol Cell Biol
14:999, 1994[Abstract/Free Full Text]
12.
Jahroudi N, Ardekani AM, Greenberger JS:
An NF1-like protein functions as a repressor of the von Willebrand factor promoter.
J Biol Chem
271:21413, 1996[Abstract/Free Full Text]
13.
Schwachtgen J-L, Remacle JE, Janel N, Brys R, Huylebroeck D, Meyer D, Kerbiriou-Nabias D:
Oct-1 is involved in the transcriptional repression of the von Willebrand factor gene promoter.
Blood
92:1247, 1998[Abstract/Free Full Text]
14.
Zhang ZP, Deng LP, Blombäck M, Anvret M:
Dinucleotide repeat polymorphism in the promoter region of the human von Willebrand factor gene (VWF gene).
Hum Mol Genet
1:780, 1992[Free Full Text]
15.
Abildgaard CF, Suzuki Z, Harrison J, Jefcoat K, Zimmerman TS:
Serial studies in von Willebrand's disease: Variability versus "variants".
Blood
56:712, 1980[Abstract/Free Full Text]
16.
Gill JC, Endres-Brooks J, Bauer PJ, Marks WJ Jr, Montgomery RR:
The effect of ABO blood group on the diagnosis of von Willebrand disease.
Blood
69:1691, 1987[Abstract/Free Full Text]
17.
Aillaud MF, Pignol F, Alessi MC, Harle JR, Escande M, Mongin M, Juhan-Vague I:
Increase in plasma concentration of plasminogen activator inhibitor, fibrinogen, von Willebrand factor, factor VIII:C and in erythrocyte sedimentation rate with age.
Thromb Haemost
55:330, 1986[Medline]
[Order article via Infotrieve]
18.
Stirling Y, Woolf L, North WRS, Seghatchian MJ, Meade TW:
Hemostasis in normal pregnancy.
Thromb Haemost
52:176, 1984[Medline]
[Order article via Infotrieve]
19.
Pottinger BE, Read RC, Paleolog EM, Higgins PG, Pearson JD:
von Willebrand factor is an acute phase reactant in man.
Thromb Res
53:387, 1989[Medline]
[Order article via Infotrieve]
20.
Behague I, Poirier O, Nicaud V, Evans A, Arveiler D, Luc G, Cambou J-P, Scarabin P-Y, Bara L, Green F, Cambien F:
-fibrinogen gene polymorphisms are associated with plasma fibrinogen and coronary artery disease in patients with myocardial infarction (The ECTIM Study).
Circulation
93:440, 1996[Abstract/Free Full Text]
21.
Eriksson P, Kallin B, van't Hooft FM, Båvenholm P, Hamsten A:
Allele-specific increase in basal transcription of the plasminogen-activator inhibitor-1 gene is associated with myocardial infarction.
Proc Natl Acad Sci USA
92:1851, 1995[Abstract/Free Full Text]
22.
Spek CA, Koster T, Rosendaal FR, Bertina RM, Reitsma PH:
Genotypic variation in the promoter region of the protein C gene is associated with plasma protein C levels and thrombotic risk.
Arterioscler Thromb Vasc Biol
15:214, 1995[Abstract/Free Full Text]
23.
Ireland H, Kunz G, Kyriakoulis K, Stubbs PJ, Lane DA:
Thrombomodulin gene mutations associated with myocardial infarction.
Circulation
96:15, 1997[Abstract/Free Full Text]
24.
Lahiri DK, Nurnberger JI Jr:
A rapid non-enzymatic method for the preparation of HMW DNA from blood for RFLP studies.
Nucleic Acids Res
19:5444, 1991[Free Full Text]
25. Akiyama Y: TFSEARCH: Searching transcription factor binding
sites, http://www.rwcp.or.jp/papia/
26.
Heinemeyer T, Wingender E, Reuter I, Hermjakob H, Kel AE, Kel OV, Ignatieva EV, Ananko EA, Podkolodnaya OA, Kolpakov FA, Podkolodny NL, Kolchanov NA:
Databases on transcriptional regulation: TRANSFAC, TRRD, and COMPEL.
Nucleic Acids Res
26:364, 1998
27.
Osborn L, Kunkel S, Nabel GJ:
Tumor necrosis factor and interleukin 1 stimulate the human immunodeficiency virus enhancer by activation of the nuclear factor B.
Proc Natl Acad Sci USA
86:2336, 1989[Abstract/Free Full Text]
28.
Lichtsteiner S, Wuarin J, Schibler U:
The interplay of DNA-binding proteins on the promoter of the mouse albumin gene.
Cell
51:963, 1987[Medline]
[Order article via Infotrieve]
29. Heywood DMH, Ossei-Gerning N, Grant PJ: Two novel polymorphisms
of the von Willebrand factor gene promoter and association with
ischaemic heart disease. Thromb Haemost 375, 1997 (abstr,
suppl)
30.
Humphries SE, Thomas A, Montgomery HE, Green F, Winder A, Miller G:
Gene-environment interaction in the determination of plasma levels of fibrinogen.
Fibrinol Proteol
11:3, 1997
31.
Burzotta F, Di Castelnuovo A, Amore C, D'Orazio A, Di Bitondo R, Donati MB, Iacoviello L:
4G/5G promoter PAI-1 gene polymorphism is associated with plasmatic PAI-1 activity in Italians: A model of gene-environment interaction.
Thromb Haemost
79:354, 1998[Medline]
[Order article via Infotrieve]
32.
Thomas AE, Green FR, Humphries SE:
Association of genetic variation at the -fibrinogen gene locus and plasma fibrinogen levels: Interaction between allele frequency of the G/A 455 polymorphism, age and smoking.
Clin Genet
50:184, 1996[Medline]
[Order article via Infotrieve]
33.
Aird WC, Jahroudi N, Weiler-Guettler H, Rayburn HB, Rosenberg RD:
Human von Willebrand factor gene sequences target expression to a subpopulation of endothelial cells in transgenic mice.
Proc Natl Acad Sci USA
92:4567, 1995[Abstract/Free Full Text]
34.
Wang ZB, Liebhaber SA:
Developmental silencing of human -globin gene transcription is mediated by a 3' flanking NFB site.
Blood
92:697a, 1998 (abstr, suppl 1)
35.
Nickerson DA, Taylor SL, Weiss KM, Clark AG, Hutchinson RG, Stengård J, Salomaa V, Vartiainen E, Boerwinkle E, Sing CF:
DNA sequence diversity in a 9.7-kb region of the human lipoprotein lipase gene.
Nat Genet
19:233, 1998[Medline]
[Order article via Infotrieve]
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
B.-Q. Zhao, A. K. Chauhan, M. Canault, I. S. Patten, J. J. Yang, M. Dockal, F. Scheiflinger, and D. D. Wagner
von Willebrand factor-cleaving protease ADAMTS13 reduces ischemic brain injury in experimental stroke
Blood,
October 8, 2009;
114(15):
3329 - 3334.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. A. Davies, P. W. Collins, L. S. Hathaway, and D. J. Bowen
Effect of von Willebrand factor Y/C1584 on in vivo protein level and function and interaction with ABO blood group
Blood,
April 1, 2007;
109(7):
2840 - 2846.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. L. Lemmerhirt, J. A. Shavit, G. G. Levy, S. M. Cole, J. C. Long, and D. Ginsburg
Enhanced VWF biosynthesis and elevated plasma VWF due to a natural variant in the murine Vwf gene
Blood,
November 1, 2006;
108(9):
3061 - 3067.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. P. Riddel Jr. and B. E. Aouizerat
Genetics of von Willebrand disease type 1.
Biol Res Nurs,
October 1, 2006;
8(2):
147 - 156.
[Abstract]
[PDF]
|
|
|
|
|
|
|
C. Hough, C. D. Cuthbert, C. Notley, C. Brown, C. Hegadorn, E. Berber, and D. Lillicrap
Cell type-specific regulation of von Willebrand factor expression by the E4BP4 transcriptional repressor
Blood,
February 15, 2005;
105(4):
1531 - 1539.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. J. Kunicki, A. B. Federici, D. R. Salomon, J. A. Koziol, S. R. Head, T. S. Mondala, J. D. Chismar, L. Baronciani, M. T. Canciani, and I. R. Peake
An association of candidate gene haplotypes and bleeding severity in von Willebrand disease (VWD) type 1 pedigrees
Blood,
October 15, 2004;
104(8):
2359 - 2367.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J.D. Mills, M.W. Mansfield, and P.J. Grant
Elevated fibrinogen in the healthy male relatives of patients with severe, premature coronary artery disease
Eur. Heart J.,
August 2, 2002;
23(16):
1276 - 1281.
[Abstract]
[PDF]
|
|
|
|
|
|
|
J. O'Donnell, F. E. Boulton, R. A. Manning, and M. A. Laffan
Amount of H Antigen Expressed on Circulating von Willebrand Factor Is Modified by ABO Blood Group Genotype and Is a Major Determinant of Plasma von Willebrand Factor Antigen Levels
Arterioscler Thromb Vasc Biol,
February 1, 2002;
22(2):
335 - 341.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. S. Williams and P. F. Bray
Genetics of Arterial Prothrombotic Risk States
Experimental Biology and Medicine,
May 1, 2001;
226(5):
409 - 419.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
P. W. Kamphuisen, J. C. J. Eikenboom, and R. M. Bertina
Elevated Factor VIII Levels and the Risk of Thrombosis
Arterioscler Thromb Vasc Biol,
May 1, 2001;
21(5):
731 - 738.
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. C. Souto, L. Almasy, E. Muniz-Diaz, J. M. Soria, M. Borrell, L. Bayen, J. Mateo, P. Madoz, W. Stone, J. Blangero, et al.
Functional Effects of the ABO Locus Polymorphism on Plasma Levels of von Willebrand Factor, Factor VIII, and Activated Partial Thromboplastin Time
Arterioscler Thromb Vasc Biol,
August 1, 2000;
20(8):
2024 - 2028.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Allen, A. M. Abuzenadah, J. Hinks, J. L. Blagg, T. Gursel, J. Ingerslev, A. C. Goodeve, I. R. Peake, and M. E. Daly
A novel von Willebrand disease-causing mutation (Arg273Trp) in the von Willebrand factor propeptide that results in defective multimerization and secretion
Blood,
July 15, 2000;
96(2):
560 - 568.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
TOP
ABSTRACT
INTRODUCTION
Age and Level of Plasma
VWF:Ag
1234C/T,