Association of the plasminogen activator inhibitor-1(PAI-1) gene 4G/5G promoter polymorphism in Buerger's disease (Tromboangiitis obliterans)

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Vol.2, No.5, 454-457 (201

doi:10.4236/health.2010.25068

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0) Health

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Association of the plasminogen activator

inhibitor-1(PAI-1) gene 4G/5G promoter polymorphism

in Buerger’s disease (Tromboangiitis obliterans)

Sinasi Manduz1, Nurkay Katrancioglu1, Oguz Karahan1, Oztürk Ozdemir2*

1Department of Cardiovascular Surgery, Faculty of Medicine, Cumhuriyet University, Sivas, Turkey

2Department of Medical Genetics, Faculty of Medicine, Cumhuriyet University, Sivas and University of COMU, Faculty of Medicine,

Canakkale, Turkey; *Corresponding Author: [email protected]

Received 3 January 2010; revised 26 January 2010; accepted 27 January 2010.

ABSTRACT

Thromboangiitis obliterans (TAO) is an unusual

tobacco-associated vasculopathy that is a non-

atherosclerotic inflammatory disorder of unkn-

own etiology that affects small and medium-

sized vessels of the extremities. The single

guanosine nucleotide deletion/insertion polym-

orphism (4G/5G) at -675 bp in promoter region

of the PAI-1 gene is the major genetic determi-

nant of PAI-1 expression. Plasma PAI-1 level is

higher in people with the homozygous 4G gen-

otype than in those with the 5G/5G genotype

and renders higher transcription activity. The

aim of this study was to determine the status

and the role of PAI-1 gene 4G/5G promoter pol-

ymorphism in patients with Buerger’s disease

(Thromboangiitis obliterans—TAO). The current

case-control study included 30 consecutive pat-

ients with Buerger’s disease (mean age 42.9 ±

14.3 years, 28 men and 2 women), and 30 heal-

thy volunteers (mean age 40.9 ± 4.79 years, 27

men and 3 women) between January 2006 and

September 2009. Patients and control cases were

genotyped for the 4G/5G polymorphism using

the multiplex PCR based stripassay reverse

hybridisation technique. It was found that hete-

rozygote PAI-1 gene polymorphisms (p < 0.05)

was significantly more frequent in patients with

TAO in the current results. There was a signif-

icant difference in genotype distribution between

the two groups (P < 0.001). The 4G allele occur-

red more frequently in the patient group of

heavy smokers (P = 0.05). The current study sh-

ows the higher prevalence of of 4G allele in

TAO patients in Sivas population means gene

may predispose to TAO.

Keywords: Thromboangiitis Obliterans; Genetic

Polymorphisms; PAI-1

1. INTRODUCTION

In 1908 Buerger suggested the name thromboangiitis

obliterans (TAO) for a disease of the peripheral arteries

which he had studied in eleven lower limbs amputated for

“presenile spontaneous gangrene” [1]. TAO (Buerger’s

syndrome) is an unusual tobacco-associated vasculopa-

thy that is a nonatherosclerotic inflammatory disorder of

unknown etiology that affects small and medium-sized

vessels of the extremities [1-3]. Etiopathogenesis of dis-

ease could not be evenly revealed. Researchers think that

tobacco is rolled for starting and progressing of disease

and by the way genetic factors, hipercoagulability, en-

dothelium function and immune mechanisms etc. are

suspicious factors that enrolled such as tobacco [1,2,4,5].

For example HLA-9 and HLA-B5 was excessively doc-

umented in patients. This may sign that genetic factors

likely play a role [5].

Highly cellular thrombus occludes much of the lumen

and lymphocytes, giant cells, fibrous hyperplasia, inti-

mae thickness have seen around vaso-vasorums in his-

tological investigation of vessel that affected by TAO [5].

A highly cellular thrombus occludes much of the lumen.

A multinucleate giant cell and microabscess are present

within the thrombus [5].

Both genetic and thrombotic events, reminds that

prothrombotic gene deletions might act a part in this

disorder. Prothrombotic genes detected significantly in

some studies about TAO [6]. Plasminogen is one of me-

diators of fibrinolysis that synthesized in the liver and

then circulates in plasma and other extracellular fluids.

This respect presents in all tissues [7]. Urokinase-type

plasminogen activator (uPA) and the tissue-type PA (tPA)

are two regulative enzymes that directed the modulation

of plasminogen to plasmin. The enzymatic activity of

uPA can be inhibited plasminogen activator inhibitor

S. Manduz et al. / HEALTH 2 (2010) 454-457

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(PAI) -1 and PAI-2 [8]. PAI-1 is a major inhibitor of the

fibrinolytic system. The PAI-1 gene polymorphism is

known as a deletion/insertion of G base (5G/4G) in

codon -675 and the major genetic determinant of PAI-1

expression. The 4G allele renders higher transcription

activity than the PAI-1 promoter with the 5G allele in

stimulated MCs [9]. The polymorphic PAI-1 gene is a

member of plasminogen cascade with an inhibitory role

in plasmin activation by response to cytokines, hor-

mones and many growth factors. Isordila et al., claimed

that the 4G allele is an independent risk factor for acute

myocardial infarction in young patients, as are smoking,

hypertension and a family history of inherited cardio-

vascular disease [9]. It was aimed to find out the possi-

ble role of polymorphic 5G/4G alleles of PAI-1 gene in

TAO in the current results.

2. MATERIAL AND METHOD

Thirty patients diagnosed with TAO at 1998 to 2009 in

our clinic were included the study. Total genomic DNA

from peripheral blood samples were used for each patient

and control for 4G/5G polymorphism in PAI-1 gene.

Control group was composed of healthy individuals who

had no vascular complaints.

Clinical diagnosis of TAO was made according to cri-

teria of Shionoya and defined as fallows; a history of

smoking, disease on set before the age of 50 years, in-

frapopliteal arterial occlusions, upperlimb involvement

or phlebitis migrans, and the absence of atherosclerotic

risk factors other than smoking. In Doppler ultrasound of

cases, the decreased arterial flow pattern was also re-

vealed. Because of this arteriography was applied all

cases. In arteriography, arterial occlusion and corkscrew

collaterals revealed typically.

Mutation Analysis: Two ml of peripheral blood-EDTA

samples were used for total genomic DNA isolation (In-

vitek, Berlin, Germany). Multiplex PCR-based reverse

hybridisation stripAssay technique (Vienna Lab, Labor-

diagnostika GmbH, and Vienna, Austria) was used for

mutation analysis (ProfiBlot T48, Tecan, Switzerland).

Genotyping of PAI-1 gene was made and compared to

the control group individuals.

Statistical Analysis: The SPSS software (ver; 14.0)

was used for evaluation of data, χ²-test (chi-square) was

used to compare the significance of the differences be-

tween groups. The test of importance between the mean of

two groups was used for accordance of two groups and

margin of error was approved to as 0.05. Furthermore

appropriateness of groups was evaluated with importance

test of difference of between two groups.

3. RESULTS

The informed consents of the patients were also obta-

ined. 30 TAO patients (28 males, 2 females and mean

ages was 42.9 ± 14.3 years) and 30 healthy controls (27

males, 3 females and mean ages was 40.9 ± 4.79 years)

were included in the current study. Two groups were

similar regarding age and sex distribution [(t = 0.69); P =

0.487; p > 0.05]. Twenty nine (97%) cases had a history

of smoking in TAO group and 22 (73%) cases had a his-

tory of smoking in control group. Detection of etiopa-

thogenesis is important for meticulous care of these pa-

tients. Homozygous 4G/4G profile was 7% for TAO and

4% for control individuals. The homozygous wide type

5G/5G genotype was 10 % in patients with TAO and

53% in healthy controls (Table 1). Heterozygote muta-

tion of 4G/5G profile was detected in 25 (83.3%) pa-

tients with TAO and 13 (43.3%) control subjects. The

current results indicate that the 4G/5G gene polymor-

phism of PAI-1 is significantly associated with TAO

while 4G/4G type may probably be an important heredi-

tary risk factor as well. We found that heterozygote PAI

gene polymorphisms (p < 0.05) were significantly more

frequent in patients with TAO. We detected PAI-1 5G/

4G polymorphism was significantly higher in TAO

group (p = 0.001) (Table 1 and Figure 1). Most possibly

the 4G/5G gene polymorphism for PAI-1 may be an ac-

ceptable risk factor of TAO.

Table 1. The percantages of polymorphic alleles (5G/4G) for

PAI-1 gene in the current TAO and control groups.

GROUP ALTERNATIVE GENOTYPES FOR PAI-1

GENE

4G/4G 4G/5G 5G/5G Total

(n-%)

TAO

(n-%) 2–7 25–83 3–10 30–100

Control

(n-%) 1–4 13–43 16–53

*P 0.05 0.001 0.001

30–100

*p < 0.05 is significant

Figure 1. Shows the barr diagram of alternative 5G/4G alleles

of PAI-1 gene in TAO and control groups.

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4. DISCUSSIONS

Some association has been suggested between Buerger’s

disease (TAO) and other syndromes but there is still no

consensus about diagnostic criteria. TAO is a nonathero-

sclerotic inflammatory disorder of unknown etiology

that affects small and medium-sized vessels of the ex-

tremities [1-3]. It is usually present at less than 40 years

old in male sex. However, last studies show that this

disease can be appears in smoking females [5]. In our

study, there were 28 (93%) males, 2 (7%) females’ cases

in TAO group and the mean age was 42.9 ± 14.3 years.

On the other hand, control group was consisted of 27

(90%) males, 3 (10%) females. Additionally 29 (97%)

subjects had smoking history in TAO group and 22 (73%)

in control group. TAO is diagnosed and treated with dif-

ficulty because of elusive etiology (10]. There is no suf-

ficient knowledge about the other risk factors and pre-

disposing conditions in the literature [11,12]. It was

documented that there was a strongly relationship be-

tween TAO and HLA-9 and HLA-B5. This situation

remind to genetical predisposition can be rolled in back-

ground of this disorder [5]. The effected vessel was

filled with the thrombus formation in microscopical

scanning [5]. Prothrombotic risk factors were observed

in this disease because of genetical suspicions and

thrombotic findings [6]. The 4G/5G alleles in PAI-1 and

MTHFR C677T gene polymorphisms may help to iden-

tify the couples at risk for recurrent pregnancy loss [13].

Abboud et al. showed that the risk of myocardial infarc-

tion (MI) was notably high in 4G and -844A carriers

with elevated plasma PAI-1 and were associated with

reduced tPA levels [14]. PAI-1 concentration increases

because of the 4G deletion of 675th promoter region of

PAI gene. Therefore, fibrinolytic activity is reduces and

predisposing thrombotic events are increases [15]. It was

demonstrated that all alone or combined effect of this

gene deletion was impacted deep venous thrombosis

(DVT), in studies [16]. Choudhury and friends detected

that the free PAI-1 levels can be significant venous oc-

clusion in TAO patients [17]. This gene mutation ac-

cused in Turkish DVT patients [18]. A meta-analyze

signs to 4G/4G genotype was 20% folds increased of

myocardial infarction risk that have included 9 studies

[19]. Another meta-analyze points to each 4G allele was

slightly increased of myocardial infarction risk that have

included 37 studies [20]. Recently another meta-analyze

that included 17 studies points that there was average

relation ship between 4G allele and venous thrombo-

emboli in subjects which have no genetical and acquired

prothrombotic risk factors [21]. In our study we detected

PAI-1 4G/5G deletion in 25 (83%) subjects in TAO

group (Figure 1). Although there were 13 (43%) sub-

jects detected with PAI-1 4G/5G deletion in control

group. Evaluation of the difference between two groups

was statistically significant (p = 0.001) (Table 1). Anal-

ysis of etiopathogenesis of TAO disease has importance

for loss of extremity and labor force by reason of this

disease and these results should be verified by larger

population studies. In view of previous and current res-

ults, there is a clear need to redefine the diagnostic algor-

ithm and the criteria for diagnosing TAO (Buerger’s

disease).

In conclusion, our study revealed the presence of sus-

picious relationship between TAO and PAI-1 gene

polymorphism. However this is a pilot study including

limited number of subjects and it must be supported with

large cohort studies. In addition, it is believed that the

current results could be helpful for analysis of etiopa-

thogenesis of TAO in the future studies. Additionally,

analyses of etiopathogenesis of the TAO disease have

importance for prevention of potential losses of extrem-

ity and labor force caused by this disease.

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