pai-1 gene 4g-5g genotype

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PAI-1 Gene 4G/5G Genotype: A Risk Factor forThrombosis in Vessels of Internal Organs

Gunay Balta,* Cigdem Altay, and Aytemiz GurgeyHacettepe 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 ofthrombosis 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/insertionpolymorphism (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: [email protected]

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.


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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 theHacettepe 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

withBse

L1 (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) 1.0a

Total 357 281

aReference category 5G/5G.

90 Balta et al.


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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 showany 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 Organsa

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) 1.0b

Othersc

36 4G/4G 13 (36) 4.3 1.313.74G/5G 19 (53) 4.1 1.312.4

5G/5G 4 (11) 1.0b

aControl group given in Table I is used in the statistical analysis.bReference category 5G/5GcIncludes hepatic, splenic, cardiac, renal, and pulmonary thrombosis.

TABLE II. Distribution of PAI-1 4G/5G Polymorphism in

Various Groupsa

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 2.012.15G/5G 6 (9) 1.0b

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) 1.0b

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) 1.0b

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) 1.0b

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) 1.0b

aControl group given in Table I is used in the statistical analysis.bReference category 5G/5G.

4G/5G Polymorphism of PAI-1 Gene and Thrombosis 91


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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.

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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/totala Pb 2 bProthrombin

G20210A Het/total a Pb 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 1.2A. 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 2.0D. 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

aThe variations in the total number of samples were due to unsuccessful genotyping of some individuals for the mutations

tested.bStatistical 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.


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4G/5G Polymorphism of PAI-1 Gene and Thrombosis 93

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