Вы находитесь на странице: 1из 5

PAI-1 Gene 4G/5G Genotype: A Risk Factor for

Thrombosis in Vessels of Internal Organs


Gunay Balta,* Cigdem Altay, and Aytemiz Gurgey
Hacettepe University, Faculty of Medicine, Department of Pediatrics, Institute of Child Health and Pediatric Hematology Unit,
Ankara, Turkey
Although the common 4G/5G polymorphism in the promoter of the PAI-1 gene was sug-
gested to be a risk factor for some of the thrombotic disorders, its significance in the
development of thrombosis is still controversial. This study presents the data on a total
of 357 patients with different types of thrombosis and 281 unrelated healthy controls. It
was found that the 4G/4G genotype is associated with a higher risk of thrombosis (OR,
1.7; 95% CI, 1.12.5). Patients were divided into five distinct groups according to the site
of thrombosis. Both 4G/4G and 4G/5G genotypes were associated with a higher risk of
thrombosis development in a group of 69 patients with internal organ thrombosis (OR,
6.35; 95% CI, 2.516.1 and OR, 4.85; 95% CI, 2.012.1, respectively). Interestingly, this
association was even stronger in a subgroup of 33 patients with portal vein thrombosis
(PVT) and 4G/4G and 4G/5G genotypes conferred more than 10- and 6-fold increases in
the risk of developing PVT (95% CI: 2.347.1 and 1.428.8), respectively. No statistically
significant association was found between 4G/4G genotype and the groups of deep vein
thrombosis (126 patients), cerebral thrombosis (80 patients), retinal thrombosis (72 pa-
tients), and purpura fulminans (16 patients). Factor V Leiden or prothrombin G20210A
mutations did not emerge as additional risk factors for thrombosis in any of the groups
studied. To conclude, this study suggests that there may be an association between
4G/4G and 4G/5G genotypes and the thrombosis in vessels of internal organs especially
in the portal veins. Am. J. Hematol. 71:8993, 2002. 2002 Wiley-Liss, Inc.
Key words: PAI-1 gene; 4G/5G polymorphism; thrombosis; internal organ vessels; ge-
netic risk factor
INTRODUCTION
Plasminogen activator inhibitor-1 (PAI-1) is the major
inhibitor of tissue type plasminogen activator (tPA). Re-
duced fibrinolytic capacity due to increased plasma
PAI-1 levels was postulated to play an important role in
the pathogenesis of disorders associated with thrombosis
[1]. Recently, a common functional deletion/insertion
polymorphism (4G/5G) in the promoter of the PAI-1
gene located 675 bp upstream from the transcription start
site was reported to result in the elevated expression of
PAI-1 gene [2]. Individuals homozygous for the 4G al-
lele had increased plasma PAI-1 concentrations com-
pared to the ones with 5G allele [3]. To date, this poly-
morphism has been studied extensively, and in some
studies, the prevalence of 4G allele was found to be
higher in disorders like coronary artery disease, menin-
gococcal septic shock, osteonecrosis, severe pre-eclamp-
sia, type 2 diabetic nephropathy, pulmonary thromboem-
bolism (PTE), and arterial thrombosis associated with
hereditary protein S deficiency [1,48]. Other studies
have failed to show such an association [9,10]. The pur-
pose of this study was to elucidate the significance of this
polymorphism in development of thrombosis in various
Contract grant sponsor: Hacettepe University; Contract grant number:
9902101003.
*Correspondence to: Gunay Balta, Ph.D., Hacettepe University, Fac-
ulty of Medicine, Institute of Child Health and Pediatric Hematology
Unit, 06100, Ankara, Turkey. E-mail: gbalta@hacettepe.edu.tr
Received for publication 15 April 2002; Accepted 15 June 2002
Published online in Wiley InterScience (www.interscience.wiley.
com). DOI: 10.1002/ajh.10192
American Journal of Hematology 71:8993 (2002)
2002 Wiley-Liss, Inc.
internal organ vessels, deep, cerebral, retinal veins, and
purpura fulminans.
PATIENTS AND METHODS
A total of 357 unrelated consecutive Turkish patients
(211 males/146 females) who were admitted to the
Hacettepe University Hospitals in Ankara, between April
1997 and September 2000 for evaluation of a recent
thrombotic event and 281 unrelated healthy controls
without family history of thrombosis who were sampled
at the same time period were the subjects of this study.
All of the subjects were coming from Ankara and its
surroundings and were suitable for inclusion to the study.
The study was approved by the Hacettepe University
ethics committee on human research, and informed con-
sent was received from all of the subjects participating to
the study.
The peripheral and internal organ thrombi were diag-
nosed by Doppler ultrasonography, computed tomogra-
phy, and magnetic resonance imaging; and retinal
thrombi were diagnosed by fundoscopic examination.
Patients who had a history of an in-dwelling umbilical
artery catheter were excluded from the study.
Patients were divided into four groups according to the
site of the thrombosis: group Ainternal organ throm-
bosis (IOT), 69 patients (33 M/36 F) with hepatic,
splenic, cardiac, portal, renal, and pulmonary vein throm-
bosis; group Bdeep vein thrombosis (DVT), 126 pa-
tients (73 M/53 F) with thrombosis at femoral/popliteal,
mesenteric veins, vena cava superior/inferior, and DVT
combined with other thrombotic events like pulmonary
vein thrombosis; group Ccerebral thrombosis (CT), 80
patients (49 M/31 F) with cerebral infarct, cerebral, and
sinovenous thrombosis; group Dretinal thrombosis
(RT), 72 patients (45 M/27 F) with mostly retinal vein
and few retinal artery thrombosis; group Epurpura ful-
minans (PF), 16 patients including two newborn babies
(8 M/8 F).
High molecular weight DNA was extracted from pe-
ripheral blood by standard procedures. Part of the PAI-1
gene promoter flanking 4G/5G polymorphism was am-
plified by using the primers described earlier [9]. The
PCR products of 163-bp DNA fragments were digested
with BseL1 (MBI Fermentas) at 55C overnight. Diges-
tion products were subjected to electrophoresis on 3%
agarose gel. The presence of 107- and 56-bp DNA frag-
ments indicated the 4G allele, while the 74-, 56-, and
34-bp fragments indicated the 5G allele. Patients were
also screened for Factor V Leiden and prothrombin
G20210A mutations as described before [11,12].
The statistical significance of differences between
each of the patient groups and the control group were
estimated by logistic regression analysis on SPSS statis-
tical package (version 10). 5G/5G was taken as reference
category and odds ratios (ORs) of 4G/4G or 4G/5G ver-
sus 5G/5G were calculated and are given within 95%
confidence intervals (CI). Statistical analyses were also
performed after FV Leiden and prothrombin G20210A-
positive patients were eliminated from the study. The
possible association of 4G/5G genotype with FV Leiden
and prothrombin G20210A mutations in the generation
of thrombosis within each group of patients was analyzed
by the
2
test (2-sided).
RESULTS
Of the 357 patients [age 25 19 (mean SD) years at
sampling] that were included in this study, 32% had the
4G/4G, 43% the 4G/5G, and 25% 5G/5G genotype. On
the other hand, of the 281 healthy controls 26% had
4G/4G, 40% 4G/5G, and 34% 5G/5G genotype. The dif-
ference in the frequencies of the 4G/4G genotype was
statistically significant, and the presence of 4G/4G was
associated with an increased risk of thrombosis (OR, 1.7;
95% CI, 1.12.5) (Table I). As expected, the prevalence
of the 4G allele was higher among patients (53%) than
controls (46%).
The results of the statistical analysis of each group are
given in Table II. Of the 69 patients (mean age 23 19
years) with internal organ thrombosis in group A, 29
(42%) had the 4G/4G, 34 (49%) 4G/5G genotypes, while
only 6 (9%) had the 5G/5G genotype (Table II). The
allele frequency of 4G was 67%, while that of 5G was
33% for this group. The frequencies of both the 4G/4G
and the 4G/5G genotypes were significantly higher in
this group compared to controls, and the presence of
these genotypes showed an association with an increased
risk of thrombosis in vessels of internal organs (OR, 6.4;
95% CI: 2.516.1 and OR, 4.9; 95% CI, 2.012.1, re-
spectively).
Thirty-three of 69 patients with thrombosis in vessels
of internal organs had portal vein thrombosis (mean age
25 18 years; age range 264 years; 13 M/20 F); 16 of
these patients (48%) had the 4G/4G, 15 (45%) the 4G/
5G, and only 2 (6%) had 5G/5G genotype (Table III).
The presence of the 4G/4G and 4G/5G genotypes
showed an association with 10.5-fold (95% CI: 2.347.1)
and 6.4-fold (95% CI: 1.428.8) increased risk of devel-
TABLE I. Prevalence of the PAI-1 4G/5G Genotype in
Thrombotic Patients and Controls
Genotype
No. of patients
(%)
No. of controls
(%) OR 95% CI
4G/4G 114 (32) 73 (26) 1.7 1.12.5
4G/5G 152 (43) 112 (40) 1.4 1.02.1
5G/5G 91 (25) 96 (34) 1.0
a
Total 357 281
a
Reference category 5G/5G.
90 Balta et al.
oping PVT, respectively. The remaining 36 patients of
this group (mean age 21 20 years; age range 173
years; 19 M/17 F) also had a 4-fold increased risk of
thrombosis for both 4G/4G and 4G/5G genotypes (95%
CI: 1.313.7 and 1.312.4, respectively) (Table III).
The frequency of the 4G/4G genotype did not show
any significant difference in 126 patients (mean age 31
17 years) with deep vein thrombosis (group B), 80 pa-
tients (mean age 19 19 years) with cerebral thrombosis
(group C), 72 patients (mean age 32 16 years) with
retinal thrombosis (group D), 16 patients (mean age 4.5
4.7 years; age range 3 days13 years) with purpura
fulminans (group E) and the control group (Table II).
Although the number of patients in the PF group was too
small to obtain reliable results from the statistical analy-
sis, it is included here only to provide preliminary data.
The positive associations remained the same when
Factor V Leiden or prothrombin G20210A-positive cases
were eliminated from the study. The co-existence of ei-
ther Factor V Leiden or prothrombin G20210A mutation
with 4G/4G genotype was not found to be statistically
significant in any of the groups studied (P > 0.05 for all)
(Table IV).
DISCUSSION
Although there are many reports indicating that the
4G/5G polymorphism of the PAI-1 gene promoter is a
risk factor for some diseases related to thrombosis, the
exact role of this functional polymorphism in the devel-
opment of these disorders still remains to be controver-
sial [1]. In some studies, the 4G/4G genotype has been
shown to be associated with some thrombotic disorders;
in others, however, no association was found [8,10].
There are even reports on the selection of 5G/5G geno-
type in some of the thrombotic disorders [13,14].
The results of this study indicated that the difference in
the frequencies of the 4G/4G genotype is statistically
significant between the patients of all groups and the
control subjects (Table I). When the study groups were
analyzed separately, the presence of a significant asso-
ciation was shown between carriers of the 4G deletion
polymorphism in the PAI-1 gene promoter and the
thrombosis of various organ vessels (Table II). When the
subgroup of patients with portal vein thrombosis (PVT)
were analyzed as an independent group, the difference
between the patients with PVT and control group became
even more prominent (Table III). FV Leiden and pro-
thrombin G20210A mutations seem not to be additional
risk factors for development of thrombosis since the
positive associations were remained the same even after
positive cases for these mutations were eliminated from
the study. To date, there was no report on the role of
PAI-1 promoter polymorphism for the development of
portal vein thrombosis. In this respect, information given
about the presence of a possible association between car-
riers of the 4G deletion polymorphism and the risk of
development of PVT is important. However, since the
number of patients was quite small, further studies are
needed to confirm this association.
The lack of a difference in the frequencies of the 4G
genotypes between group of patients with DVT and the
control subjects in this study is consistent with the results
of previous studies [15,16]. However, absence of a sta-
tistically significant association between the 4G/4G ge-
notype and Factor V Leiden mutation contradicts to the
results of a previous study in this respect [15].
It was reported that PAI-1 4G/4G homozygotes had a
markedly reduced risk of cerebrovascular mortality com-
pared to 5G/5G homozygotes in older patients [14].
TABLE III. Frequencies of PAI-1 4G/5G Genotype in Portal
Vein Thrombosis and Thrombosis of Other Internal Organs
a
Thrombosis
site
Total
no. of
patients Genotype
No. of
patients
(%) OR 95% CI
Portal vein 33 4G/4G 16 (48) 10.5 2.347.1
4G/5G 15 (45) 6.4 1.428.8
5G/5G 2 (6) 1.0
b
Others
c
36 4G/4G 13 (36) 4.3 1.313.7
4G/5G 19 (53) 4.1 1.312.4
5G/5G 4 (11) 1.0
b
a
Control group given in Table I is used in the statistical analysis.
b
Reference category 5G/5G
c
Includes hepatic, splenic, cardiac, renal, and pulmonary thrombosis.
TABLE II. Distribution of PAI-1 4G/5G Polymorphism in
Various Groups
a
Patient
group
Thrombosis
site
Total
no. of
patients Genotype
No. of
patients
(%) OR 95% CI
A Internal organ 69 4G/4G 29 (42) 6.4 2.516.1
4G/5G 34 (49) 4.9 2.012.1
5G/5G 6 (9) 1.0
b
B Deep vein 126 4G/4G 35 (28) 1.4 0.82.4
4G/5G 57 (45) 1.4 0.92.4
5G/5G 34 (27) 1.0
b
C Cerebral 80 4G/4G 24 (30) 1.2 0.62.2
4G/5G 29 (36) 0.9 0.51.7
5G/5G 27 (34) 1.0
b
D Retinal 72 4G/4G 25 (35) 1.6 0.83.0
4G/5G 26 (36) 1.1 0.62.0
5G/5G 21 (29) 1.0
b
E Purpura 16 4G/4G 3 (19) 0.8 0.23.4
fulminans 4G/5G 8 (50) 1.4 0.44.3
5G/5G 5 (31) 1.0
b
a
Control group given in Table I is used in the statistical analysis.
b
Reference category 5G/5G.
4G/5G Polymorphism of PAI-1 Gene and Thrombosis 91
However, the results of this study indicated that the 4G/
5G polymorphism of the PAI-1 gene had no significant
contribution to the development or prevention of cerebral
thrombosis probably because the patients with younger
age range were used in this study.
The role of PAI-1 4G/5G genotype on development of
retinal thrombosis has not been reported previously. Al-
though a slightly higher frequency of 4G/4G genotype
was observed in patients (35%) than in controls (26%),
no statistically significant association could be found be-
tween PAI-1 genotype and retinal thrombosis in this
study (Table II).
Although no difference was observed in the frequency
of 4G/5G polymorphism between the purpura fulminans
and the control group, the number was too small to draw
any statistical conclusion. Therefore, this study would
only provide preliminary data for future studies on this
subject.
In conclusion, the results of this study suggest that 4G
allele of the PAI-1 gene promoter may be an increased
risk factor for development of thrombosis in the vessels
of several internal organs especially in the portal veins.
However, further studies are needed to draw a definite
conclusion.
ACKNOWLEDGMENT
We are grateful to Associate Prof. Dr. Osman Sarac-
basi for his valuable comments in the statistical analysis
of this study.
REFERENCES
1. Kohler HP, Grant PJ. Mechanisms of disease: plasminogen-activator
inhibitor type 1 and coronary artery disease. New Engl J Med 2000;
342:17921801.
2. Dawson SJ, Wiman B, Hamsten A, Green F, Humphries S, Hanney
AM. The two allele sequences of a common polymorphism in the
promoter of the plasminogen activator inhibitor-1 (PAI-1) gene re-
spond differently to interleukin-1 in HepG2 cells. J Biol Chem 1993;
25:1073910745.
3. Eriksson P, Kallin B, vant Hooft FM, Bavenholm P, Hamsten A.
Allele-specific increase in basal transcription of the plasminogen-
activator inhibitor 1 gene is associated with myocardial infarction.
Proc Nat Acad Sci USA 1995;14:18511855.
4. Westendorp RGJ, Hottenga JJ, Slagboom PE. Variation in plasmino-
gen-activator-inhibitor-1 gene and risk of meningococcal septic shock.
Lancet 1999;354:561563.
5. Glueck CJ, Fontaine RN, Gruppo R, Stroop D, Sieve-Smith L, Tracy
T, Wang P. The plasminogen activator inhibitor-1 gene, hypofibri-
nolysis and osteonecrosis. Clin Orthop 1999;366:133146.
6. Yamada N, Arinami T, Yamakawa-Kobayashi K, Watanabe H, Sohda
S, Hamada H, Kubo T, Hamaguchi H. The 4G/5G polymorphism of
TABLE IV. Assessment of the Effects of FV-Leiden and Prothrombin G20210A Mutations Within Each
PAI-1 Genotype of the Groups Studied
Group Genotype
FV-Leiden
Hom&Het/total
a
P
b

2 b
Prothrombin
G20210A Het/total
a
P
b

2 b
Total patients 4G/4G 23/114 4/113
4G/5G 40/151 9/146
5G/5G 29/90 0.15 3.9 6/90 0.55 1.2
A. Internal Organ 4G/4G 7/29 2/29
4G/5G 4/34 2/33
5G/5G 4/6 0.01 9.2 1/6 0.65 0.9
1. Portal Vein 4G/4G 1/16 1/16
4G/5G 1/15 1/14
5G/5G 1/2 0.12 4.3 0/2 0.93 0.2
2. Others 4G/4G 6/13 1/13
4G/5G 3/19 1/19
5G/5G 3/4 0.04 6.7 1/4 0.43 1.7
B. Deep vein 4G/4G 10/32 2/31
4G/5G 18/53 6/52
5G/5G 13/33 0.78 0.5 4/33 0.71 0.7
C. Cerebral 4G/4G 5/24 0/24
4G/5G 11/29 0/28
5G/5G 8/27 0.4 1.8 1/27 0.38 2.0
D. Retinal 4G/4G 0/25 0/25
4G/5G 4/26 1/24
5G/5G 2/21 0.14 4.0 0/21 0.39 1.9
E. Purpura fulminas 4G/4G 0/2 0/2
4G/5G 3/8 0/7
5G/5G 1/4 0.57 1.1 0/4
a
The variations in the total number of samples were due to unsuccessful genotyping of some individuals for the mutations
tested.
b
Statistical analysis was performed by
2
test in order to assess the significance of the co-existence of FV-Leiden or
prothrombin G20210A mutations with PAI-1 genotypes in each group.
92 Balta et al.
the plasminogen activator inhibitor-1 gene is associated with severe
pre-eclampsia. J Hum Genet 2000;45:138141.
7. Wong TY, Poon P, Szeto CC, Chan JC, Li PK. Association of plas-
minogen activator inhibitor-1 4G/4G genotype and type 2 diabetic
nephropathy in Chinese patients. Kidney Int 2000;57:632638.
8. Zoller B, Garcia de Frutos P, Dahlback B. A common 4G allele in the
promoter of the plasminogen activator inhibitor-1 (PAI-1) gene as a
risk factor for pulmonary embolism and arterial thrombosis in heredi-
tary protein S deficiency. Thromb Haemost 1998;79:802807.
9. Doggen CJM, Bertina RM, Manger Cats V, Reitsma PH, Rosendaal
FR. The 4G/5G polymorphism in the plasminogen activator inhibitor-1
gene is not associated with myocardial infarction. Thromb Haemost
1999;82:115120.
10. Hooper WC, Lally C, Austin H, Renshaw M, Dilley A, Wenger NK,
Philips DJ, Whitsett C, Rawlins P, Evatt BL. The role of the t-PA I/D
and PAI-1 4G/5G polymorphisms in African-American adults with a
diagnosis of myocardial infarction or venous thromboembolism.
Thromb Res 2000;1:223230.
11. Bertina RM, Koeleman BPC, Koster T, Rosendaal FR, Dirven RJ, de
Ronde H, van den Velden PA, Reitsma PH. Mutation in blood coagu-
lation factor V associated with resistance to activated protein C. Nature
1994;369:6467.
12. Poort SR, Rosendaal FR, Reitsma PH, Bertina RM. A common genetic
variation in the 3-untranslated region of the prothrombin gene is
associated with elevated prothrombin levels and an increase in venous
thrombosis. Blood 1996;88:36983703.
13. Luomala M, Elovaara I, Ukkonen M, Koivula T, Lehtimaki T. Plas-
minogen activator inhibitor 1 gene and risk of MS in women. Neurol-
ogy 2000;9:18621864.
14. Roest M, van der Schouw YT, Banga JD, Tempelman MJ, de Groot
PG, Sixma JJ, Grobbee DE. Plasminogen activator inhibitor 4G poly-
morphism is associated with decreased risk of cerebrovascular mor-
tality in older women. Circulation 2000;101:6770.
15. Akar N, Yilmaz E, Akar E, Avcu F, Yalcin A, Cin S. Effect of
plasminogen activator inhibitor-1 4G/5G polymorphism in Turkish
deep vein thrombotic patients with and without FV1691 G-A. Thromb
Res 2000;15:227230.
16. Segui R, Estelles A, Mira Y, Espana F, Villa P, Falco C, Vaya A,
Grancha S, Ferrando F, Aznar J. PAI-1 promoter 4G/5G genotype as
an additional risk factor for venous thrombosis in subjects with genetic
thrombophilic defects. Br J Hematol 2000;111:122128.
4G/5G Polymorphism of PAI-1 Gene and Thrombosis 93