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Blood First Edition Paper, prepublished online September 12, 2007; DOI 10.1182/blood-2007-03-078543
Effect of single nucleotide polymorphisms on expression of the gene encoding
thrombin activatable fibrinolysis inhibitor: a functional analysis
Short title: Polymorphisms and TAFI gene expression
Michael B. Boffa1, Deborah Maret1, Jeffrey D. Hamill1, Nazareth Bastajian1, Paul Crainich3,
Nancy S. Jenny3, Zhonghua Tang3, Elizabeth M. Macy3, Russell P. Tracy3,4, Rendrik F. Franco5,
Michael E. Nesheim1,2, and Marlys L. Koschinsky1
From the Department of 1Biochemistry and 2Medicine, Queen’s University, Kingston, Ontario,
Canada; Department of 3Pathology and 4Biochemistry, College of Medicine, University of
Vermont, Colchester, Vermont, USA; 5Fleury Research Institute, São Paulo, Brazil
Supported by a grant (to M.L.K.) from the Canadian Institutes for Health Research (# MOP36491). M.L.K. is a Career Investigator of the Heart and Stroke Foundation of Ontario.
Corresponding Author: Michael B. Boffa, Ph.D. Department of Biochemistry, Room 253
Botterell Hall, Queen’s University, Kingston, Ontario, K7L 3N6, Canada; Tel: 613-533-2990;
Fax: 613-533-2987; e-mail: [email protected]
Copyright © 2007 American Society of Hematology
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Abstract
Thrombin-activable fibrinolysis inhibitor (TAFI) is a plasma zymogen that acts as a molecular
link between coagulation and fibrinolysis. Numerous single nucleotide polymorphisms (SNPs)
have been identified in CPB2, the gene encoding TAFI, and are located in the 5’-flanking region,
in the coding sequences, and in the 3’-untranslated region (UTR) of the CPB2 mRNA transcript.
Associations between CPB2 SNPs and variation in plasma TAFI antigen concentrations have
been described but the identity of SNPs that are causally linked to this variation is not known. In
the current study, we investigated the effect of the SNPs in the 5’-flanking region on CPB2
promoter activity and SNPs in the 3’-UTR on CPB2 mRNA stability. Whereas the 5’-flanking
region SNPs (with 2 exceptions) did not have a significant effect on promoter activity, either
alone or in haplotypic combinations seen in the human population, all of the 3’-UTR SNPs
substantially affected mRNA stability. We speculate that these SNPs, in part, contribute to
variation in plasma TAFI concentrations via modulation of CPB2 gene expression through an
effect on mRNA stability.
2
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Introduction
Thrombin-activable fibrinolysis inhibitor (TAFI), also known as procarboxypeptidase U or R or
plasma procarboxypeptidase B, is a human plasma zymogen that may play a role in mediating
the balance between blood coagulation and fibrinolysis.1 Upon cleavage of TAFI by thrombin2,
thrombin-thrombomodulin3, or plasmin4, an enzyme is formed (TAFIa) that possesses basic
carboxypeptidase activity. TAFIa has been demonstrated to attenuate plasminogen activation,
and thus fibinolysis, by removing from partially degraded fibrin the carboxyl-terminal lysine
residues that mediate positive feedback in the fibrinolytic cascade.5 In addition, TAFIa has been
shown to remove the carboxyl-terminal arginine residues from bradykinin and the
anaphylatoxins C3a and C5a, thereby implicating the TAFI pathway as a link between
coagulation and inflammation.6-9
Plasma concentrations of TAFI vary significantly in the human population.10,11 The vast
majority of individuals have TAFI antigen levels between 50% and 150% of the mean population
value12,13, thereby ranging from approximately 100 to 200 nM. Importantly, these concentrations
of TAFI fall below the Km for activation of TAFI by thrombin or thrombomodulin (1 µM)3,
indicating that individuals with higher plasma TAFI concentrations would exhibit a higher rate
of TAFIa production following a procoagulant stimulus. Indeed, variation in “functional” TAFI
concentrations has been observed using a clot lysis assay of plasma samples.14 Therefore, it is
reasonable to consider the gene encoding TAFI (CPB2) as a candidate gene for thrombotic
disorders. Indeed, elevated concentrations of TAFI have been shown to be a mild risk factor for
first venous thrombosis15 as well as recurrent venous thrombosis16, and to be more common in
carriers of Factor V Leiden with venous thromboembolism than in asymptomatic carriers.17 High
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functional TAFI concentrations have been found to be associated with an increased risk for
ischemic stroke.18 While a possible association between plasma TAFI concentrations and
coronary artery disease and coronary events has been a point of controversy19-24, an association
between plasma TAFI antigen concentrations and restenosis following percutaneous coronary
interventions has been reported.25
It has been demonstrated that there is virtually no association between plasma TAFI
concentrations and conventional cardiovascular risk factors26, which led to the suggestion that
plasma TAFI concentrations are largely under genetic control. Further studies identified
numerous single nucleotide polymorphisms (SNPs) in the 5’-flanking, protein-coding, and 3’untranslated coding regions of CPB2.27,28 Early studies determined that virtually all of these
SNPs were strongly associated with plasma TAFI antigen concentrations27,28, and that two SNPs
in CPB2 accounted for approximately 60% of variation in plasma TAFI antigen concentrations.27
However, it was subsequently determined that the ELISAs used in many of these studies were
sensitive to the Thr/Ile polymorphism at position 325 and thus the extent of variation in plasma
TAFI antigen concentrations, the association between individual SNPs and TAFI levels, and the
extent to which the SNPs explained TAFI antigen variation was overestimated.12,13,29 The most
recent analyses indicate that less than 20% of the variation in plasma TAFI antigen
concentrations can be attributed to SNPs.13,30 However, there is extensive linkage disequilibrium
between all of the SNPs,27,28,30 complicating the identification of SNPs that directly influence
plasma TAFI concentrations, such as by altering expression of the gene encoding TAFI.
Therefore, in the current work, we have assessed the effect of polymorphisms in the CPB2 5’flanking region and 3’-untranslated region on promoter activity and mRNA stability,
respectively.
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Materials and Methods
Work on human DNA and plasma samples was conducted at the University of Vermont and the
University of Sao Paolo. Patients gave informed consent for donation of blood samples and the
protocols were approved by the IRB's at these respective locations.
Reporter Plasmids and Mutagenesis
The parental luciferase reporter plasmid used for the mutagenesis was TAFI[-2699]-luc,
described by Boffa and coworkers.31 This construct consists of the CPB2 5’-flanking region
spanning the EcoRI site at -2699 to the HindIII site at +21 inserted into pGL3 Basic (Promega).
The initiator methionine codon immediately upstream of the HindIII site was mutated to TTG.
Nucleotide numbering is as described31 where the +1 nucleotide is the first nucleotide of the
cDNA reported by Eaton and coworkers.32 The sequence of the genomic insert is identical to that
described by Boffa.31
All of the reported single nucleotide polymorphisms27,28 as well as an additional novel
SNP -298G/A in the CPB2 5’-flanking region were introduced into the TAFI[-2699]-luc plasmid
by site directed mutagenesis using either the GeneEditor (Promega) or QuikChange (Stratagene)
kits according to the manufacturers’ recommendations. Nucleotide numbering is as in the
corresponding first reports of the SNPs27,28 and in the original description of the sequence of the
CPB2 5’-flanking region.31 The corresponding rs numbers for each SNP are presented in the
Table. Oligonucleotides used in the mutagenesis reactions were purchased from Cortec DNA
Service Laboratories (Kingston, ON, Canada). The presence of the appropriate mutations was
verified by DNA sequence analysis. Where multiple substitutions were necessary, as in the
construction of the different haplotypes, mutagenesis reactions were carried out sequentially.
Reporter plasmid DNA was purified using QIAGEN Maxi-Prep kits. The DNA concentration
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was determined by ultraviolet absorbance spectroscopy. All preparations were subjected to
restriction enzyme digestion followed by agarose gel electrophoresis to assess their quality.
The parental fusion mRNA reporter plasmid used for mutagenesis was βG-TAFI/12731819 (ref. 33), which was constructed in the context of the pC7βG vector described by Wilson
and Deeley.34 This plasmid features a 460 bp segment of the rabbit β-globin cDNA whose
expression is driven by the cytomegalovirus promoter, and also contains Epstein-Barr virus
genomic sequences and a hygromycin resistance cassette to allow for stable episomal
maintenance of the plasmid in mammalian cells.34 A segment of the CPB2 cDNA between
+1273 (the first nucleotide after the stop codon) and +1819 (the 3’-most polyadenylation site)
was inserted downstream of the β-globin sequences. Using site-directed mutagenesis,
substitutions corresponding to the SNPs in the CPB2 3'-UTR were incorporated either alone or in
the combinations potentially observed in the human population. Site-directed mutagenesis was
carried out using the QuikChange Site-Directed Mutagenesis Kit (Stratagene), according to the
manufacturer’s specifications. In addition to the +1542 C/G and +1583 T/A SNPs identified by
Henry and coworkers27, we also considered a novel SNP +1344 G/A. The corresponding rs
numbers for each SNP are presented in the Table.
Luciferase Reporter Gene Assays
HepG2 cells (human hepatocellular carcinoma)35, were grown in MEM containing 10% fetal calf
serum (ICN) and 1% penicillin-streptomycin-Fungizone (PSF) (Life Technologies). Cells were
maintained in a humidified 37°C incubator under a 95% air/5% CO2 atmosphere. Transient
transfection of the cells was performed in 6-well tissue culture dishes (Corning) using the
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method of calcium phosphate co-precipitation.36 Cells were seeded into the wells approximately
18 hours prior to transfection at a density of 20% confluence. Each well received 5 µg of
luciferase reporter plasmid and 2 µg of the internal control plasmid RSV-βgal which contains the
E. coli β-galactosidase gene under the control of the Rous sarcoma virus promoter.37 After a sixhour exposure to the precipitate, the cells were washed three times with phosphate-buffered
saline (PBS) and fresh complete medium was added to each well. Forty-eight hours later,
cytoplasmic extracts were prepared by first washing each well three times with ice-cold PBS,
then lysing the cells using ice-cold lysis buffer (25 mM glycylglycine pH 7.8, 15 mM MgSO4, 4
mM EGTA, 1% (v/v) Triton X-100). Extracts were clarified by centrifugation at 6000 × g for 5
minutes at room temperature.
Luciferase and β-galactosidase activities in the cell extracts were measured using a
MicroLumat Plus plate-reading luminometer (EG&G Berthold) running WinGlow v. 1.24
software and a SpectraMax Plus plate reading spectrophotometer (Molecular Devices) running
SoftMax Pro v. 3.1.1 software, respectively. To assay luciferase activity, 10 µL of extract was
placed in the wells of a white 96-well plate and 50 µL luciferase assay buffer (Promega) was
injected sequentially; relative light units were measured 0.1 seconds after injection. To assay βgalactosidase activity, 10 µL of extract was placed in the wells of a 96-well plate and 40 µL of
water and 50 µL of β-galactosidase assay buffer (1.33 mg/mL O-nitrophenyl-β-Dgalactopyranoside (ONPG)) (as substrate), 200 mM sodium phosphate pH 7.3, 2 mM MgCl2,
100 mM β-mercaptoethanol. Absorbance at 405 nm was measured every 30 seconds at 37°C; the
β-galactosidase activity was taken to be the rate of increase of absorbance at 405 nm. Relative
luciferase activity was calculated as the luciferase activity per unit β-galactosidase activity per
unit volume of extract.
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For each reporter plasmid, four independent transfection experiments were performed. In
each independent experiment, triplicate wells were transfected, and luciferase and βgalactosidase activities for each well were determined in duplicate. The promoter activity of
constructs containing polymorphisms was compared to that of the wild-type construct using
paired t-tests.
mRNA Stability Assays
In order to construct HepG2 cells lines stably expressing the β-globin/TAFI 3’-UTR fusion
mRNAs, dishes of cells were stably transfected with the respective plasmids using FuGENE 6
Transfection Reagent (Roche Diagnostics). After a 48 h recovery period, selection of positive
clones was performed by adding hygromycin B (Roche) to a final concentration of 300 µg/ml
and changing the media every 2 to 3 days to remove dead cells. After approximately two weeks,
hygromycin resistant colonies were pooled and grown in MEM containing 10% fetal calf serum
and 1% PSF, while maintaining hygromycin B at 300 µg/ml.
For the mRNA stability assays, actinomycin D (Sigma) was added to 90% confluent
stably-transfected HepG2 cells, to a final concentration of 5 µg/mL. Cells were maintained
without hygromycin B during the course of these experiments. Incubation was continued for
different times up to 8 h, at which point total RNA was harvested using TriZOL reagent
(GIBCO) as recommended by the manufacturer. Poly(A)+RNA was prepared using Nucleotrap
mRNA purification kits (Clontech). Poly(A)+ RNA (approximately 2 µg/lane) (in 50% (v/v)
formamide, 10 mM MOPS pH 7.0, 2.2 M formaldehyde) was incubated at 65°C for 15 min,
quenched on ice, and then fractionated on a 1% (w/v) agarose gel containing 10 mM MOPS pH
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7.0, 2.2 M formaldehyde for Northern blot analysis. The RNA was blotted onto a nylon
membrane (Hybond-XL, Amersham Pharmacia Biotech) via capillary action in 20X SSC (1X
SSC is 15 mM trisodium citrate pH 7, 150 mM NaCl). RNA was crosslinked to the membrane
using Spectrolinker XL-1000 UV Crosslinker and blots were hybridized with radiolabeled
probes corresponding to the coding region of the rabbit β-globin cDNA. In order to correct for
differences in RNA loading and transfer, blots were stripped with boiling 0.5% SDS and
hybridized with radiolabeled probes corresponding to the glyceraldehyde-6-phosphate
dehydrogenase (GAPDH) cDNA. All probes were prepared using [α-32P]-dATP and the Prime-It
II random primer labeling kit (Stratagene). Hybridization was carried out at 68°C for 1 h in
ExpressHyb solution (Clontech). The blots were then washed at room temperature in 1X SSC,
0.1% (w/v) SDS and then at 50°C in 0.2X SSC, 0.1% (w/v) SDS. Blots were exposed to a
phosphor screen (Kodak) and band intensities were quantitated using a Molecular Imager FX
phosphorimager (BioRad). The amount of fusion RNA present at each time point was calculated
as previously described by Wilson and Deeley34, with the half-life of GAPDH mRNA assumed
to be 8 h.38 The results shown are the mean of three independent actinomycin D time courses for
each construct.
Results
Effect of 5’-flanking region SNPs on CPB2 promoter activity
To date, nine single nucleotide polymorphisms in the CPB2 5’-flanking region have been
described (Figure 1).27,28,39 In order to assess the effect of each SNP on CBP2 promoter activity,
SNPs were individually introduced, using site-directed mutagenesis, into the 5’-flanking region
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in the context of luciferase reporter plasmids. All reporter plasmids contained genomic sequence
spanning from -2699 to +21 of CPB2; the sequence of the genomic DNA in the parental reporter
plasmid was that reported by Boffa and coworkers.31 The resultant reporter plasmids were then
transiently transfected into HepG2 (human haptocellular carcinoma) cells, which are an excellent
model for CPB2 gene expression as the gene is expressed endogenously in these cells.40 An
internal control plasmid containing the β-galactosidase gene under the control of the RSV
promoter was included in each transfection in order to control for differences in transfection and
harvesting efficiency. When evaluated individually, none of the SNPs had a significant impact
on CPB2 promoter activity, with the exception of the -298 A and -152 G substitutions; each of
these only increased promoter activity by less than 20% (Figure 2).
There is substantial linkage disequilibrium between all CBP2 SNPs identified to date.27,28
As such, there is a limited number of haplotypes observable in the population. Henry and
coworkers described four main haplotypes (H1-4) involving -2599 C/G, -2345 1G/2G, -1690
A/G, -1102 G/T, -1053 C/T, and -438 G/A in the 5’-flanking region.27 Accordingly, we
constructed haplotypes H2, H3, and H4 by mutagenesis of the wild-type (H1) reporter plasmid
(Figure 1). When the promoter activity of these four contructs was compared in HepG2 cells, we
observed no significant differences between the haplotypes (Figure 3).
Franco and coworkers reported a separate block of 3 SNPs in the CPB2 5’-flanking
region (-1925 TC, -530 C/T, and -152 A/G).28 These SNPs are in virtually complete linkage
disequilibrium, and so the corresponding substitutions were introduced as a unit into the four
main haplotypes to construct H1*, H2*, H3*, and H4* (Figure 1). Once again, there was no
difference in promoter activity between any of the haplotypes (Figure 3).
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Effect of 3’-UTR SNPs on CPB2 mRNA Stability
To investigate whether SNPs in the 3’-UTR can act to modulate CPB2 gene expression through
an effect on mRNA stability, we used a β-globin reporter mRNA system in which HepG2 cells
are stably transfected with plasmids expressing fusion mRNA species consisting of the CPB2 3’UTR fused to rabbit β-globin mRNA.33 These RNA species can be accurately quantitated (and
distinguished from the endogenous CPB2 mRNA in these cells) by Northern blot analysis using
β-globin as a probe. The parental plasmid (Figure 4A) represents the 3’UTR sequence originally
reported by Eaton and coworkers32, and contains CPB2 cDNA sequence downstream of the stop
codon and encompasses all three potential polyadenylation sites identified in the 3’-UTR.31,33
Using this system, we have found that cis-acting sequences specifically localized to the 3’-UTR
determine the stability of the CPB2 mRNA.33
Nucleotide substitutions corresponding to the SNPs were incorporated into the cDNA
segment encoding the 3’-UTR in combinations potentially observed in the human population. To
determine whether the SNPs in the 3’-UTR have an effect on CPB2 mRNA stability, we carried
out mRNA decay assays using HepG2 cell lines stably transfected with the respective fusion
mRNA reporter plasmids. Transcription was arrested using actinomycin D, and RNA was
harvested at different times after addition of this drug. The remaining fusion mRNA at each time
point was quantified by Northern blot analysis (Figure 4B,C). Compared to the “wild type”
haplotype (H2), mRNA corresponding to the H1/H3 haplotype has an increased stability (halflife of 4.5 h versus 3.2 h) while the H4 haplotype has a ~ 2-fold decreased stability (1.6 h). The
+1344 A substitution has a general destabilizing effect, as each haplotype containing this
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substitution (denoted with the daggers in Figure 4) had a decreased stability relative to the
variant containing a G at this position.
Discussion
While it is clear that SNPs in CPB2 (the human gene encoding TAFI) influence plasma
concentrations of TAFI antigen, the molecular basis of this relationship remains to be defined
and the identity of the particular SNP(s) directly responsible is not known.
SNPs could influence plasma TAFI concentrations through a variety of mechanisms.
SNPs in the 5’-flanking region could influence gene transcription by altering the binding of
transcription factors to promoter or enhancer elements, or by altering the local chromatin
architecture. SNPs in the 3’-UTR could affect CPB2 mRNA abundance by influencing the
stability of the CPB2 transcript or polyadenylation site selection. SNPs in the coding regions of
the gene could influence the residence time of the TAFI polypeptide in plasma. Furthermore, the
functional SNPs could be outside the CPB2 locus itself, albeit strongly linked to certain of the
SNPs. Thus, analysis of the direct effect of individual SNPs on factors that influence CPB2 gene
expression is a key aspect of the search for the functional SNPs.
The high degree of linkage disequilibrium that exists between the different SNPs
complicates identification of the SNPs that directly influence plasma concentrations of TAFI.
Genome-wide scans have confirmed that the CPB2 locus itself (or a locus in close proximity to
CPB2) explains virtually all the genetic variation in plasma TAFI antigen levels.41 A recent study
examining the association between CPB2 genotype and plasma TAFI antigen concentrations
using assays insensitive to the Thr-Ile polymorphism at position 325 suggest that two SNPs
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(possibly -1102 G/T and +1583 T/A) underlie the variability in concentrations.13 That these
SNPs are in fact functional could not be ascertained from this type of analysis, and their linkage
disequlibrium with other SNPs prevented an entirely conclusive demonstration of their direct
effects on variability. A recent trans-ethnic haplotype analysis suggested the presence of three
quantitative trait nucleotides (QTN’s; functional SNPs): +1583 T/A, and possibly -2599 C/G and
-2345 1G/2G.30
The data presented in the present work indicate that, with two exceptions, none of the
SNPs in the CPB2 5’-flanking region influence promoter activity when examined individually.
Furthermore, none of the haplotypic combinations examined influenced promoter activity.
Collectively, these data suggest against a significant direct functional role of 5’-flanking region
SNPs in determining CPB2 gene expression and thus plasma concentrations of TAFI. It is
possible that the two putative 5’-flanking region QTN’s described above are in linkage
disequilibrium with the true QTN’s30; indeed, the sample size in this study may have been too
small to unequivocally demonstrate an independent association between the presence of these
SNPs and plasma TAFI concentrations.30
Both the -298 A and -152 G substitutions resulted in a modest but significant increase in
promoter activity. The -298 G/A SNP is relatively rare in the population (allele frequency of
~0.017)42 and in a sample of 359 healthy elderly individuals, no difference in plasma TAFI
antigen concentrations was observed between the -298 GG and -298 GA genotypes (data not
shown). No difference in plasma TAFI antigen levels was observed in individuals with the -1925
TT/-530 CC/-152 AA versus the -1925 TC/-530 CT/-152 AG genotype.28 A recent transethic
study has shown that the -1925 T/C, -1053 C/T, -152 A/G block of SNPs is present in African
subjects, but not Europeans, and is in complete linkage disequilibrium in the former population.30
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However, the two haplotypes that differ at those positions did not have significantly different
plasma TAFI concentrations.30 It is possible that these particular SNPs are too rare for their
effects on CPB2 gene expression to be observed in the relatively small samples analyzed thus
far. Interestingly, in recent studies from our laboratory which used DNase I footprinting to
identify transcription factor binding sites in the CPB2 promoter, both the -298 G/A and -152 A/G
SNPs were located between, rather than within, transcription factor binding sites, in keeping with
their relatively modest effect on CPB2 promoter activity.43
In contrast to the results observed for the 5’-flanking region SNPs, all of the SNPs in the
CPB2 3’-UTR influenced mRNA stability. The combinations of SNPs introduced into the fusion
transcripts (see Figure 4) are based on the pattern of major haplotypes reported by Henry and
coworkers.27 Accordingly, there are three combinations with respect to +1542 C/G and +1583
T/A, specifically +1542 C/+1583 T (H1/H3 haplotype), +1542 C/+1583 A (H2), and +1542
G/+1583 T (H4), with the H2 haplotype corresponding to the cDNA cloned by Eaton and coworkers.32 It has very recently been demonstrated that an allele containing +1542 G/+1583 A
exists in an African population, albeit at the low frequency of 0.04.30 The +1344 G/A SNP is
relatively rare, with an allele frequency of ~0.05. Inspection of genotyping data from 127
unrelated individuals (data not shown) reveals the presence of either +1344A or +1344G with
two of the three combinations; the +1344 A/+1542 C/+1583 A haplotype could not be
conclusively identified in this small sample. Thus, the panel of 3’-UTR model alleles that we
have constructed likely corresponds to all the major haplotypes present in the population.
The +1583 T SNP is associated with lower plasma TAFI antigen levels.13,19,30 However,
the +1344 G/+1542 C/+1583 T (H1/H3 haplotypes)13,30 transcript was more stable than the
+1344 G/+1542 C/+1583 A (H2 haplotype) transcript (Figure 4C). On the other hand, when
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combined with +1542 G (+1344 G/+1542 G/+1583 T; H3 haplotype), the +1583 T SNP resulted
in a 2-fold decrease in transcript stability. These data are consistent with recent studies in which
the +1542 G/+1583 T haplotype was associated with the lowest plasma TAFI antigen
concentrations and the +1542 C/+1583 A haplotype with the highest.19,30 However, it is clear that
additional factors beside an effect of the 3’-UTR SNPs on mRNA stability must be at play, since
the H1 and H3 haplotypes feature the same mRNA stability yet are associated with different
plasma TAFI antigen levels.19,30
The +1344 A SNP had a general destabilizing effect, with the +1344 A/+1542 G/+1583 T
(H4†) transcript being the least stable of all the transcripts tested (Figure 4C). Moreover, the
+1344 A/+1542 C/+1583 T (H1†/H3†) transcript was less stable than the +1344 G/+1542
C/+1583 T (H1/H3) transcript. Despite the marked effect of this SNP on mRNA stability, there
was no significant association between the novel +1344 G/A SNP and TAFI antigen
concentrations in a sample of 127 unrelated individuals, although there was a trend towards
lower TAFI antigen concentrations with the A allele (Joost C.M. Meijers, personal
communication). The relatively low frequency of this SNP indicates that larger sample sizes
would be required to detect associations with TAFI concentrations. Indeed, power calculations
reveal that in order to have an 80% chance of rejecting the null hypothesis of no association
between +1344 G/A SNP and TAFI concentrations (SD = 10%, α = 0.05), the sample size would
have to increase to almost 1000.
The intrinsic stability of mRNA species is determined by cis-acting sequences located
within the mRNA (often in the 3’-UTR), as well as trans-acting RNA binding proteins.44 As
such, the nucleotide substitutions in the 3’-UTR that arise from the SNPs may alter the binding
of these trans-acting factors, and thus influence mRNA half-life. One possibility is that the SNPs
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are located within the binding sites for these factors. Alternatively, the SNPs may alter the
secondary structure of the 3’-UTR which is an important determinant of protein binding site
formation. Alterations in secondary structure may also influence the rate of endonucleolytic
cleavage of the mRNA. Secondary structure analysis using RNA folding software (MFOLD;
Michael Zuker, Rensselaer Polytechnic Institute) predicts that the region spanning the three
SNPs in the CPB2 3’-UTR can form extensive stem loop structures through internal basepairing. More specifically, in the H2 sequence (+1344 G/+1542 C/+1583 A), +1344 G is
involved in base-pairing at the base of an internal loop, +1542 C is located in a terminal loop,
and +1583 A is involved in base pairing. The +1344 A variant results in disruption of secondary
structure in the surrounding region, placing +1344 A in a relatively large terminal loop
consisting of approximately 20 bases. Therefore it is no longer involved in base pairing, perhaps
rendering the transcript more susceptible to endonucleolytic cleavage in this region. In addition,
+1344 is in a region that is A/U rich, and it is possible that substitution of a G for an A at this
position results in targeting the transcript for rapid degradation, which is commonly observed in
transcripts with A/U rich sequence elements.45,46 Substitution of a C for a G at position +1542
results in the involvement of this nucleotide in base pairing; the nucleotide at position +1583
remains involved in base pairing when an A is substituted for a T at this position. Finally, the
H4† (+1344 A/+1542 G/+1583 T) haplotype, which resulted in a profound effect on mRNA
stability (Figure 4C), corresponds to a marked change in predicted secondary structure. In this
context, there are a greater number of relatively large terminal loops formed which may increase
the potential for degradation of this transcript.
A precedent for the effect of a 3’-UTR SNP on mRNA abundance and thus plasma
concentrations of a hemostatic factor can be found in the +20210G/A SNP in the F2 gene
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encoding human prothrombin. This SNP results in more efficient processing of the 3’-end of the
mRNA, through increased endocytic cleavage site recognition, with the effect of increasing the
abundance of the mRNA encoding prothrombin.47 The F2 SNP is located downstream of the
polyA signal sequence but upstream of the U-rich region; since none of the CPB2 SNPs
investigated here are located in the regions where processing of the pre-mRNA occurs48, we
would argue that an analogous mechanism is not at work in the case of the CPB2 mRNA.
Although our fusion transcript system is not appropriate for examining the effects of SNPs on the
efficiency of polyA site selection or mRNA 3’-end processing, owing to the presence of the
strong SV40 polyA signal in the plasmid constructions33, it must be pointed out that the SNPs are
in the region common to all 3 polyadenylated forms, and thus the effects of the SNPs on mRNA
stability would impact all three forms of the transcript. We have previously demonstrated that
our fusion mRNA reporter system accurately models the effect of instability elements in the
CPB2 3’-UTR.
33
In fact, our previous studies had shown that the CPB2 3’-UTR acts as an
autonomous regulatory element that strongly specifies transcript stability even in the presence of
the SV40 sequences.
Our studies represent the first demonstration of a direct effect of SNPs on CPB2 gene
expression. These findings will assist in the interpretation of association studies aimed at
identifying functional quantitative trait loci both within and outside of the CPB2 gene.
Moreover, our findings underscore the idea that such analyses, coupled with epidemiological
studies aimed at uncovering associations between CPB2 genotype and the occurrence of vascular
disease, should take into account the haplotypic combinations of the 3’-UTR SNPs.
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Acknowledgements
Author contributions were as follows: Michael B. Boffa: designed research, analyzed data, wrote
paper; Deborah Maret: performed research; Jeffrey D. Hamill: performed research; Nazareth
Bastajian: performed research; Paul Crainich: performed research; Nancy S. Jenny: performed
research; Zhonghua Tang: performed research; Elizabeth M. Macy: performed research; Russell
P. Tracy: supervised research; Rendrik F. Franco: performed research; Michael E. Nesheim:
supervised research; Marlys L. Koschinsky: supervised research. We thank Dr. Joost Meijers
(Academic Medical Centre, University of Amsterdam) for performing ELISA assays and Dr.
Roger Deeley (Queen’s University) for providing the pC7βG plasmid.
The authors have no competing financial interests to disclose.
18
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26
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Table. SNPs investigated in this study. The SNPs are presented according to published
numbering schemes27,28,31 along with the corresponding rs numbers (acquired from the dbSNP
database).
SNP
5’-flanking region SNPs
3’-UTR SNPs
rs number
-2599 C/G
rs34813434
-2345 1G/2G
rs35814191
-1925 T/C
rs17844145
-1690 A/G
rs9526146
-1102 G/T
rs7999168
-1053 C/T
rs9526144
-530 C/T
rs11574977
-438 G/A
rs2146881
-298 G/A
rs17843980
-152 A/G
rs11574980
+1344 G/A
rs1049669
+1542 C/G
rs940
+1583 A/T
rs1087
27
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Figure Legends
Figure 1. CPB2 5’-flanking region SNPs and haplotypes examined in this study. The H1 to
H4 haplotypes are those described by Henry and coworkers.27 The haplotypes denoted H1* to
H4* have the substitutions corresponding to a second block of SNPs (shaded) reported by Franco
and coworkers.28 The linkage relationship of the relatively infrequent -298 G/A SNP to the other
SNPs is not known. The H1 haplotype (wt) corresponds to the sequence of the 5’flanking region
originally reported by Boffa and coworkers.31
Figure 2. Effect of individual SNPs on CPB2 promoter activity. The indicated substitutions
were introduced into the wild-type (wt; H1 haplotype) sequence31 by site-directed mutagenesis,
in the context of the TAFI[-2699]-luc luciferase reporter plasmid.31 Reporter plasmids were
transiently transfected into HepG2 cells along with an internal control plasmid (RSV-βgal) to
control for differences in transfection and harvesting efficiency. Forty-eight hours after
transfection, cytoplasmic extracts were prepared for assay of luciferase and β-galactosidase
activities. The relative luciferase activity is defined as the luciferase activity per unit βglacatosidase activity per unit volume of extract, and is expressed at a percentage of the relative
luciferase acivity of the wt plasmid. The data shown are the means ± s.e.m. of four independent
experiments. The asterisks denote p < 0.05 compared to the wt plasmid.
Figure 3. Promoter activity of different CPB2 5’-flanking region haplotypes. Substitutions
were introduced into the wild-type (wt; H1 haplotype) sequence31 in the context of the TAFI[2699]-luc luciferase reporter plasmid in the haplotypic combinations shown in Figure 1.
28
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Transfections and reporter gene assays were performed as described in the legend to Figure 2.
The data shown are the means ± s.e.m. of four independent experiments.
Figure 4. Effect of CPB2 SNPs on mRNA stability. (A) The parental construct consists of the
CPB2 3’-UTR (nucleotides +1273 to +1819) inserted into pC7βG downstream of the rabbit βglobin cDNA.33 The construct also contains the cytomegalovirus promoter (PCMV), translation
termination cassette (TTC) and simian virus 40 polyadenylation sequences (SV40 PA). Using
site-directed mutagenesis, SNPs in the CPB2 3’-UTR, at positions +1344, +1542, and +1583,
were introduced either alone or in the haplotypic combinations observed in the human
population. The H1 to H4 haplotypes are those described by Henry and coworkers.27 The
haplotypes denoted H1† to H4† contain the +1344 A substitution. Also shown are the locations of
the three polyadenylation signal sequences present in the 3’-UTR at +1660, +1693, and + 1819.
(B) Representative Northern blots for each construct are shown, along with a representative blot
for the parental pC7βG plasmid (βG). In each case, the lanes correspond to mRNA harvested at
0, 1, 2, 4, 6, and 8 hours after the addition of actinomycin D. The full collection of blots is
available as a Data Supplement. (C) HepG2 cells stably transfected with the β-globin fusion
mRNA reporter plasmids indicated to the right of the graph, or with a reporter plasmid lacking
CPB2 3’-UTR sequences (βG), were treated with actinomycin D. RNA was harvested at various
times after the addition of the drug and subjected to Northern blot analysis using a probe specific
for rabbit β-globin. To control for differences in RNA loading and transfer, the blots were
stripped and hybridized with a probe specific for human GAPDH. Corrected fusion transcript
abundance was normalized to that observed before the addition of actinomycin D to obtain the
29
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decay curves shown. The data shown are the mean ± s.e.m. of three independent experiments.
The mean half-lives of the respective fusion transcripts are shown to the right of the graph.
30
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Prepublished online September 12, 2007;
doi:10.1182/blood-2007-03-078543
Effect of single nucleotide polymorphisms on expression of the gene
encoding thrombin activatable fibrinolysis inhibitor: a functional analysis
Michael B Boffa, Deborah Maret, Jeffrey D Hamill, Nazareth Bastajian, Paul Crainich, Nancy S Jenny,
Zhonghua Tang, Elizabeth M Macy, Russell P Tracy, Rendrik F Franco, Michael E Nesheim and Marlys L
Koschinsky
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