© 1999 American Heart Association, Inc.
Original Contributions |
Mutations in Promoter Region of Thrombomodulin and Venous Thromboembolic Disease
From the Laboratoire d'Hémostase, Hôpital Broussais—AP-HP and Unité INSERM 428, UFR des Sciences Pharmaceutiques et Biologiques, Université René Descartes, Paris, France.
Correspondence to Dr Martine Alhenc-Gelas, Laboratoire d'Hémostase, Hôpital Broussais, 96 rue Didot, F-75674 Paris Cedex 14, France. E-mail martine.alhenc-gelas{at}brs.ap-hop-paris.fr
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Abstract |
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Abstract—The present study was designed to analyze the thrombomodulin proximal promoter region spanning nucleotides -293 to -12 to search for polymorphisms that could modify thrombomodulin gene expression in patients with venous thromboembolic disease. The study population comprised 205 patients and 394 healthy subjects of similar age and sex distribution. No polymorphisms and only 1 point mutation (G-33A) were found. The G-33A mutation was present at the heterozygous state in 2 patients and in 1 control. Being more frequent in the patients (0.97%) than in the controls (0.25%), the G-33A mutation might be a risk factor for venous thrombosis. To investigate the effect of this mutation on the thrombomodulin promoter activity, the proximal promoter region of the gene (bearing or not bearing the G-33A mutation) was inserted into a promotorless expression vector, upstream of the firefly luciferase gene, and transiently transfected into EA.hy926 endothelial cells. Under the conditions of the assay, the G-33A mutation mildly decreased the promoter activity. This study confirms that abnormalities of the thrombomodulin proximal promoter are not frequent in patients with venous thromboembolism.
Key Words: thrombomodulin • thrombosis • coagulation • gene
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Introduction |
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Thrombomodulin (TM) is an endothelial cell surface glycoprotein receptor that forms a high-affinity complex with thrombin. The thrombin-TM 1:1 complex rapidly activates protein C (PC), which, helped by its cofactor protein S (PS), in turn degrades the clotting cofactors activated (a) factors (F) V and VIII. Moreover, thrombin bound to TM loses all its procoagulant activities such as fibrinogen clotting, activation of FV and FVIII, and activation of platelets. Thus, TM plays an important role in converting thrombin from a procoagulant to a physiological anticoagulant factor.1
Thrombophilia is considered to be a multifactorial disorder in that genetic and acquired risk factors often act together in the pathogenic process. Strong evidence of the physiological importance of the PC anticoagulant system has been demonstrated by the thrombotic risk associated with inherited deficiencies of PC, PS, or resistance to activated PC (APCR) linked to the FV Leiden.2 3 4 By analogy with other abnormalities in the PC anticoagulant system, an impaired TM cofactor function could be an additional risk factor for venous or arterial thromboembolic disease (TED). Indeed a point mutation in TM that eliminates the generation of activated PC and inhibition of thrombin does generate a prethrombotic state in homozygous mutant mice.5
Although TM is expressed constitutively on the surface of vascular endothelial cells, its expression can be altered by a variety of modulators. TM biosynthesis is decreased by tumor necrosis factor (TNF),6 7 8 9 10 11 12 13 14 15 interleukin-1,11 16 endotoxin,17 hypoxia,18 and transforming growth factor ß (TGFß).19 Agonists, including retinoic acid,20 21 22 cAMP,10 11 23 24 25 26 27 28 29 histamine,30 forskolin,23 25 phorbol esters,11 23 31 32 vascular endothelial growth factor (VEGF),33 and thrombin,34 enhance TM transcription in different cell types. Heat shock of vascular endothelial cells induces an upregulatory transcriptional response that abrogates the suppressive effect of TNF.35 Finally, posttranslational modifications have also been implicated in the regulation of TM expression.36 37 38
The gene for human TM has been cloned, sequenced, and mapped to
chromosome 20.39 40 41 42 The coding sequence contains no
intron. Recent studies have identified TM proximal promoter regions
that are important transcriptionally, providing positive and negative
regulatory elements for constitutive and modulated
expression.12 13 22 35 43 These elements consist of a TATA
box located between nucleotides (nt) -26 and -22 upstream
of the transcription initiation site described by Yu et
al,12 a CAAT box (GCAATC) (nt -110 to -105), 4 possible
Sp1 binding sites (nt -12 to -123, -140 to -135, -206 to -201,
and -269 to -264), a region responsive to heat shock (nt -77 to
-47), and a PyPu box (nt -76 to -56). Actual positive functions for
the activity of the TM promoter have been demonstrated for the CAAT
box, 2 of the 4 Sp1 binding sites (-206, -140),43 and
the nt -74 to 20 region. The PyPu box contains Ets core motifs able to
mediate in endothelial cells both specific positive
activation and, together with the TATA box, the TNF-
repression of
the TM promoter. A silencer element,43 a shear
stress–responsive element,44 and 4 retinoic
acid–response elements22 have been identified farther
upstream.
Analysis of the proximal promoter region of the TM gene to search for gene variations that could influence the TM function in patients with TED has been previously performed by 2 groups (for review, see References 45 and 4645 46 ). The proximal promoter was normal in more than 300 patients with TED and in 70 controls studied by Ohlin et al.45 Three different mutations (C-133A, G-33A, and GG-9/-10AT) were found by Ireland et al46 in, respectively, 1, 1, and 3 of 104 patients with myocardial infarction. These patients were matched by age, sex, and race to controls. One control carried the G-33A mutation. Controls did not carry the 2 other mutations. Taken together, these data do not show a strong association of TM gene defects to an increased thromboembolic risk.
The present study was designed to analyze the TM proximal promoter region to search for polymorphisms that could modify the expression of the gene in patients with venous TED and to study their effects on the TM promoter activity in a transient transfection system.
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Methods |
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Subjects, Blood Collection, and DNA Preparation
Two hundred five patients with venous TED were recruited in a university hospital vascular medicine department between November 1995 and November 1997 and were included in the study if they were younger than 61 years and had had at least one objectively diagnosed episode of deep venous thrombosis (DVT) (compression ultrasonography or venography) or pulmonary embolism (perfusion and ventilation lung scan, conventional pulmonary angiography, or computed tomographic angiography). Three hundred ninety-four healthy subjects aged from 20 to 60 years were recruited between May and September 1996 from a health center specializing in the prevention of cardiovascular disease, to which they had been referred for a routine check-up. Subjects with a history of arterial disease (stroke, myocardial infarction, angina, or peripheral vascular disease), venous TED, or known malignancies were excluded on the basis of a medical questionnaire. The place of birth of the cases and controls was recorded, but there was no geographic or ethnic criteria for eligibility. The study was approved by the local ethics committee and all the subjects gave their informed consent.
Venous blood was collected onto 0.129 mol/L trisodium citrate (1:10). DNA was isolated by the method of Miller et al47 and stored at 4°C.
Genomic DNA Studies
DNA was screened for the FV Q506 allele after polymerase
chain reaction (PCR) amplification of exon 10 and restriction enzyme
digestion.48 The prothrombin gene G20210A transition was
identified after amplification with primers A
(5'-TTACAAGCCTGATGAAGGGA-3') and B
(5'-CCATGAATAGCACTGGGAGCATTGAAGC-3'). The PCR mixtures contained 25
pmol of each primer, 200 µmol/L dNTPs (Pharmacia Biotech), 300
ng of genomic DNA, and 1x PCR buffer (10 mmol/L Tris-HCl, 20
mmol/L KCl, pH 8.3) with 1.5 mmol/L MgCl2
and 0.25 U of Taq polymerase (Super Taq, ATGC
Biotechnologie) in a final volume of 50 µL. The thermal profile
consisted of a 5-minute denaturation at 94°C, followed by 35 cycles
consisting of a 1-minute denaturation at 94°C, a 1-minute annealing
at 56°C, and a 1-minute extension at 72°C. The amplified fragments
were digested by HindIII (New England Biolabs), giving 2
fragments of 413 and 73 bp for the G20210 allele or 3 fragments of
384, 73, and 29 bp for the A20210 allele.
The TM promoter region spanning nt -293 to -12 was analyzed by using denaturing gradient gel electrophoresis (DGGE) as described by Attree et al,49 after amplification of 2 overlapping fragments of 387 and 303 bp. The design of the primers and the choice of the electrophoretic conditions (6 and 4 hours, respectively, at 160 V in 6.5% polyacrylamide gel containing 40% to 100% denaturant gradient [100% denaturant, 7 mol/L urea and 40% formamide in TEA buffer {2 mol/L Tris, 50 mmol/L EDTA, 1 mol/L sodium acetate, pH 7.6}]) were done to allow detection of abnormalities in the promoter regions spanning from nt -148 to nt -12 and nt -293 to -96, respectively.
Identification of the G-33A mutation was performed by sequencing. The nt -313 to +74 fragment of the TM promoter region was cloned into the pT7Blue Vector using the pT7Blue T-Vector kit (Novagen). Sequencing reactions were performed on plasmid DNAs using the Sequenase 2.0 DNA sequencing kit (USB) with R-20-mer and U-19-mer primers (Novagen) as sequencing primers.
Verification of the G-33A mutation identified by sequencing in 1 patient and screening for this mutation in other subjects were performed by using restriction site analysis with StuI (New England Biolabs).
The positions of the primers used for amplifications of genomic DNA to
screen for TM promoter variations by DGGE or to identify mutations by
sequencing or restriction site analysis are shown in Figure 1
. The sequences of the primers and the
PCR conditions are given in Tables 1 and 2. The whole PCRs were performed on
mixtures containing 1 µg genomic DNA, 200 µmol/L dNTP
(Pharmacia Biotech), and 1 U of Taq polymerase (ATGC
Biotechnologie) in 1x PCR buffer in final volumes of 100 µL.
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Plasmid Constructions
Two fragments of the TM gene promoter (spanning nt -294 to +11
or nt -154 to +11) were cloned into the pGL3 Enhancer Vector
(Promega). Primers TM PROM F, TM PROM G, and TM PROM H modified to
introduce cleavage sites for the restriction enzymes SacI,
SacI, and NheI, respectively (New England
Biolabs) (Table 1
), were used to amplify genomic DNA of both a
patient with the G-33A mutation and a normal subject. PCR
amplifications and enzymatic digestions were performed as described in
Table 3. The SacI
NheI fragments were electrically eluted after
electrophoresis in a 6% polyacrylamide gel then ligated into
the pGL3 Enhancer Vector digested with the same enzymes. The resultant
plasmids, pTM-294 mut or pTM-294wt and pTM-154 mut or pTM-154wt, were
transformed into Escherichia coli DH5 cells and purified
using Qiagen plasmid midi kits (Coger).
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Using the pTM-154wt construct as a template, constructs carrying
mutations of the CAAT box -pTMCAATmut- (nt -109 to -106, CAAT changed
to TCGA), or the TATA box sequence -pTMTATAmut- (nt -26 to -22, TATAA
changed to GGACC) were prepared using the PCR megaprimer-based
mutagenesis method described by Picard et al50 with
pGL3Enh A and B as flanking primers and TMSalI or TMAvaII respectively
as mutagenic primers (Table 1). To exclude artifactual errors
introduced during PCR or cloning, the sequence of each construct was
verified by sequencing.
Different batches of plasmid DNA were used in the transient transfection experiments to eliminate the possibility of bias originating from DNA preparation.
Cell Culture, Transfection Experiments, and Reporter Gene
Assays
The endothelial cell line, EA.hy926, was kindly
provided by Dr. C.J.S. Edgell (University of North Carolina, Chapel
Hill). Culture conditions were as described by Suggs et
al.51 Cells were cotransfected with the pGL3 construct
carrying the firefly luciferase as a reporter and a renilla luciferase
expression plasmid (pRL-TK, Promega) used as an internal control in
transcription efficiency. All the transfection experiments were
performed in triplicate. Cells were plated at
5x105 cells in 60-mm Petri dishes with 3 mL
complete medium and grown overnight at 37°C in 5%
CO2/95% air to obtain 50% to 70% semiconfluent
cultures. Cells were then transfected using lipofectin (Gibco-BRL)
according to the manufacturer's instructions with a mixture containing
8 µg of lipofectin reagent; 1 µg of TM promoter firefly luciferase
construct or control plasmid, pGL3 enhancer vector, which lacks any
promoter, or pGL3 control vector (Promega), which contains an efficient
promoter (SV40) upstream of the firefly luciferase gene; and 0.1 µg
of pRL-TK renilla luciferase plasmid. The culture medium was changed
after incubating for 3 hours at 37°C. Levels of renilla and firefly
luciferase activities in cell extracts, prepared 48 hours after
transfection, were measured by using the Dual Luciferase Reporter Assay
System (Promega) according to the manufacturer's instructions. Five to
9 independent experiments were performed for each construct.
Statistical Analysis
Mean age in the case and control groups was compared by using
the Student's t test. The statistical significance of the
clinical differences between the two groups (sex, oral contraception)
was calculated by using a 
2 test. FV and
prothrombin genotype frequencies were compared between cases
and controls by using a 2 test.
The nonparametric Wilcoxon's test was used to compare the promoter activities of the different constructs. Differences with probability value of 0.05 or less were considered significant.
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Results |
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Characteristics of the Study Population
Demographic data are shown in Table 4
. The case and control groups did not
differ significantly in terms of age or sex. Most of the cases and
controls were born in France. Subjects born outside Europe were
similarly distributed between the cases and controls (12% versus
15%).
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DVT was recurrent in 27.1% of the cases and pulmonary embolism occurred in 37.6%. Women on oral contraception, a known risk factor for venous thromboembolism (VTE), were more frequent among the cases than the controls. Thrombosis occurred in the absence of acquired risk factors (oral contraceptives, recent surgery, recent trauma, pregnancy or childbirth, and immobilization) in 38.5% of the cases, and 34.9% of the patients were on anticoagulants at the time of blood sampling.
The population was screened for known genetic risk factors for thrombosis. The FV R506Q mutation was observed in 19.5% of cases and 3.5% of controls (P<0.001). The prothrombin gene G20210A mutation was observed in 10.2% of cases and 2.8% of controls (P<0.001).
TM Promoter Gene Polymorphisms Screening
The TM proximal promoter region (nt -293 to -12) was
analyzed by PCR-DGGE in 125 patients. The fragment of 387 bp
displayed an aberrant band pattern for only 1 patient (Figure 2). The fragment of 303 bp was normal in
all the patients. The mutation responsible for the abnormal pattern was
a G to A transition at position -33 present at the heterozygous
state. This mutation identified by sequencing was verified by
restriction site analysis after amplification with primers TM
PROM A and TM PROM Stu. TM PROM Stu was designed with
nucleotide substitutions at position -4 and -3 relative
to the 3' end of the primer to create a restriction site for the enzyme
StuI when the G-33A transition was present. The
amplified fragment corresponding to the normal allele (-33G) was
not digested (313 bp), whereas the amplified fragment corresponding to
the mutated allele (-33A) was digested into 2 fragments of 283 and
30 bp (Figure 3). The G-33A mutation was
searched for in the other 80 patients and in the 394 controls using
this technique. A second patient and 1 control were heterozygous for
this mutation. The G-33A mutation was therefore more frequent in the
patients than in the controls (0.97% [2/205] versus 0.25%
[1/394], respectively).
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The patients bearing the G-33A mutation were 2 women aged 45 and 39 years, born in France. The first one had suffered from 3 DVTs of the lower limb. The first thrombosis, which occurred at the age of 26 years, was spontaneous. The second thrombosis occurred in the postpartum, and the third thrombosis occurred after a long travel. This patient had no family history of thrombosis. The second 39-year-old patient had suffered 4 months after a childbirth, at the age of 22 years, from a DVT of the lower limb and from a pulmonary embolism. She had taken an oral contraceptive treatment for 1 month before the thrombotic event. Five family members (her father, a sister, a grandmother, an uncle, and an aunt) had suffered from VTE. No members of the 2 families were available for study. Other coagulation studies including antithrombin, PC, PS, APCR measurements, lupus anticoagulant, and the FV R506Q and prothrombin gene G20210A mutations screening were normal in both patients.
Effect of the G-33A Mutation on Gene Expression
Transient transfection experiments were performed in
EA.hy926 cells with the TM promoter constructs described above. These
constructs contained either fragments of the normal promoter (wt) or of
the promoter bearing the G-33A mutation (mut), a fragment of the
promoter bearing a CAAT box mutation previously demonstrated to
decrease the TM promoter activity,43 or a fragment of the
promoter bearing a TATA box mutation (the effects of the CAAT box and
TATA box mutations were studied to validate the expression system). The
results are reported in Figure 4. The
pGL3 enhancer vector, which does not contain promoter sequences, was
used as a negative control and displayed a very low activity
(2.5±0.7%). As expected, the activity of the construct bearing the
CAAT box mutation was reduced, to less than half the activity of the
wild-type promoter (51.0±7.5% versus 117.4±25.7%, respectively;
P=0.04). The activity of the construct bearing the TATA box
mutation was also significantly reduced (37.1±4.9%,
P=0.04). The activity of the constructs bearing the G-33A
mutation was slightly lower than the activity of the wild-type
constructs (181.9±24.5% versus 198.4±27% for pTM-294 and
98.9±27.5% versus 117.4±25.7% for pTM-154). The difference was
significant for the pTM-154 constructs (P=0.015) but not for
the pTM-294 constructs.
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Discussion |
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Polymorphisms in the TM proximal promoter region could be responsible for an impaired expression of TM contributing to thrombophilia, as previously described for polymorphisms or mutations in the PC gene promoter.52 53 54 55 56 We have therefore investigated 205 patients with VTE and 394 healthy subjects of similar age and sex distribution for the presence of such gene variations. The patient and control populations were not different from other European populations in term of genetic risk factors for thrombosis such as the FV R506Q mutation4 and the prothrombin gene G20210A mutation.57 58 59 60 61
First, we screened this TM promoter region in 125 patients by PCR-DGGE, a scanning strategy previously shown to be highly sensitive for the detection of point mutations in other genes.62 63 No polymorphisms and only 1 point mutation (G-33A) were found, confirming data showing that sequence variations of the proximal TM promoter are not frequent in patients with VTE.45 46
Later, the G-33A mutation, searched for in the other 80 patients and in the controls by restriction site analysis, was found in another patient and in 1 control. The prevalence of the mutation was slightly higher in the whole patient group (2/205) than in the control group (1/394) (0.97% versus 0.25%, respectively) suggesting that this rare mutation might be a risk factor for VTE. Unfortunately, because of the low prevalence of the mutation, statistical analysis of the results could not be performed to confirm this hypothesis. It must however be pointed out that in both patients carrying the mutation, the first thrombotic event had occurred before age 30, that both had suffered from recurrent thrombotic episodes, and that one of them had a strong family history of thrombosis. Taken together, these data suggest the possible presence of genetic risk factors for thrombosis in both patients.
The G-33A mutation is located 7 nt upstream of the TATA box,
within a promoter region important for basal TM gene transcriptional
activity12 13 43 and very near the putative TNF- and
heat shock responsive sequences.12 13 35 Moreover,
efficient transcription initiation of a human protein-encoding gene
requires assembly on the promoter DNA of a multiprotein complex
containing RNA polymerase II and 6 general transcription factors, and a
consensus sequence (G/C-G/C-G/A-CGCC) located immediately upstream of
the TATA element has recently been shown to affect the ability of one
of these general transcription factors to enter transcription complexes
and support transcription initiation.64 Being located in
this consensus sequence, the G-33A mutation might therefore induce a
downregulation of the TM promoter. This hypothesis was studied in vitro
in an experimental system consisting of EA.hy926 cells transfected with
promoter TM gene fragments (nt -294 to +11 or nt -154 to +11) cloned
into a luciferase reporter vector. The EA.hy926 cell line, which
results from the fusion of human umbilical vein
endothelial cells with the lung cancer A549 cell
line,65 has been previously demonstrated to express the TM
gene.66 67
The position of the TM promoter fragments was chosen according to the findings of Tazawa et al,43 who had previously demonstrated in transient transfection assays that the activity of a construct containing the nt -290 to +145 region is maximal and that a construct containing the nt -181 to +145 smaller region, thus lacking 2 Sp1 binding sites, displays about half of the peak activity. Our results showing that the pTM-294wt and pTM-154wt constructs exhibit 198% and 117%, respectively, of the activity of the pGL3 control vector are in accordance with these previous findings.
We have verified that the transient transfection assay system used in the present study was able to recognize an impaired transcription. For this purpose, the activity of pTM-154 constructs bearing a mutation of the TATA box or a mutation of the CAAT box was studied. As expected, the activity of these constructs was low (41.6% and 30.5% of the activity of the normal construct, respectively). The effect of the CAAT box mutation was similar to the effect previously described for a CAAT box mutation introduced in a -374 to +145 construct.43 The TATA box mutation also decreased TM promoter activity to less than half the activity of the wild-type promoter, demonstrating that the TATA box plays a critical role in transcriptional activity of the TM promoter.
Mean promoter activities of the constructs that bore the G-33A
mutation were slightly lower than activities of the wild-type
constructs (92% and 84% for pTM-294 mut and pTM-154 mut,
respectively). The difference was significant only for the pTM-154
construct (P=0.015). An explanation for this finding could
be the difference in the size of the promoter fragments. The pTM-294
fragment is 140 nt longer than the pTM-154 fragment. It has a stronger
transcriptional activity and comprises more regulatory elements,
particularly 2 additional Sp1 binding sites. The effect of one or
several regulatory elements located in this region could have offset
the effect of the G-33A mutation on promoter activity. On the whole,
these results do not clearly support the hypothesis of the G-33A
mutation being a risk factor for venous TED by lowering the expression
of the TM gene on vascular endothelial cells. However,
it must be pointed out that the effect of the G-33A mutation was
studied only in conditions of basal transcription. Different
modulators, for example, interleukin-1, TNF, or endotoxin, can
downregulate TM expression while in a concomitant manner favor tissue
factor expression and thereby induce a hypercoagulable
state.16 17 68 Being localized near the region
responsive to TNF- (nt -76 to -56), the G-33A mutation might
modify the response of the promoter in stimulated
endothelial cells.
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Acknowledgments |
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L.L.F. was a recipient of a grant from La Fondation pour la Recherche Médicale. We thank Dr. C.J.S. Edgell for kindly providing us the EA.hy926 cells.
Received August 8, 1998; accepted October 26, 1998.
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