Association of MMP-3 5A/6A gene polymorphism with susceptibility
to carotid atherosclerosis
Tamara Djurića,⁎, Maja Živkovića, Djordje Radakb, Djole Jekićc, Sandra Radakb,
Ljiljana Stojkovića, Ranko Raičevićc, Aleksandra Stankovića, Dragan Alavantića
aVINČA Institute of Nuclear Sciences, Laboratory for Radiobiology and Molecular Genetics, P.O. Box 522, 11001 Belgrade, Serbia
bCardiovascular Institute Dedinje, Vascular Surgery Clinic, Belgrade, Serbia
cMilitary Medical Academy, Department of Neurology, Belgrade, Serbia
Received 3 July 2008; received in revised form 22 August 2008; accepted 25 August 2008
Available online 4 September 2008
Objectives: Stromelysin-1 (MMP-3) as a key member of metalloproteinase family could have an important role in atherogenesis. The 5A/6A
polymorphism in the promoter of MMP-3 gene affects the level of MMP-3 gene expression. We assessed whether the MMP-3 promoter low- and
high-activity genotypes are related to susceptibility for carotid atherosclerosis (CA) in Serbian population.
Design and methods: The study group of case-control design consisted of 515 participants. The 265 patients with ultrasonographic evidence
of carotid plaque presence were recruited for the study. The 5A/6A polymorphism was typed by RFLP-PCR.
Results: There was significantly higher prevalence of genotypes containing 6A allele in the patients with CA compared to controls (pb0.05).
The model of inheritance with the dominant effect of 6A allele gave elevated and significant OR for carotid atherosclerosis (adjusted OR 2.35,
Conclusions: Subjects carrying genotypes with 6A allele had significantly higher susceptibility to carotid atherosclerosis.
© 2008 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Keywords: MMP-3; 5A/6A; Gene polymorphism; Carotid atherosclerosis; Human
The matrix metalloproteinases (MMPs) are a family of
peptidase enzymes responsible for the degradation of extra-
cellular matrix (ECM), clotting factors, lipoproteins, latent
growth factors, and chemotactic and cell adhesion molecules
[1–3]. Alterations in the structure and composition of the ECM
play one of the key roles in the atherogenic process. It is now
widely accepted that deregulation of the MMPs system plays a
pivotal role in vascular remodeling which is recognized as a
determinant of major vascular pathologies including athero-
sclerosis and restenosis . Data accumulated in the last decade
indicate contribution of MMPs gene polymorphisms to the inter-
individual differences in susceptibility to development of
atherosclerosis or future cardiovascular events (reviewed in ).
Stromelysin-1 (MMP-3) as a key member of metalloprotei-
nase family is involved in the turnover of the number of ECM
components which have important role in atherogenesis,
including types II, IV, and IX collagen, proteoglycans, laminin,
fibronectin, gelatins  and also activates some other members
of the matrix metalloproteinase family . The common 5A/6A
polymorphism in the promoter of this gene has been shown in
vitro  and under in vivo conditions  to affect the level of
MMP-3 gene expression. The 5A allele was associated with
higher and the 6A allele with lower transcriptional activity [7,8].
Up to date, the 6A/6A genotype was associated with greater
progression of coronary artery disease (CAD) in men [9,10] and
women , and with the greater number of coronary arteries
with significant stenosis [12,13], but not with susceptibility to
CHD [12,14]. Data considering association of MMP-3 5A/6A
polymorphism with carotid atherosclerosis (CA) were predomi-
nantly focused on investigation of intima media thickness (IMT)
or percent of carotid artery stenosis as phenotypes of interest.
Again, the 6A/6A genotype was associated with greater left,
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Clinical Biochemistry 41 (2008) 1326–1329
⁎Corresponding author. Fax: +381 11 244 74 85.
E-mail addresses: email@example.com (T. Djurić), firstname.lastname@example.org
0009-9120/$ - see front matter © 2008 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
common carotid artery (CCA) and burification IMT [15–18], as
well as with both the bilateral internal carotid artery (ICA)
stenosis and stenosis N70% . A cross-sectional study of 1111
randomly selected both, male and female community subjects
from Australia did not show an association of this polymorphism
with either IMTor with the presence of plaque .
The data considering the association of carotid athero-
sclerosis with MMP-3 5A/6A gene polymorphism are still
sporadic and controversial. Therefore, we investigated whether
the MMP-3 promoter low- and high-activity genotypes are
related to susceptibility to carotid atherosclerosis in Serbian
Study group consisted of 515 participants. All of them were
Caucasians with European descent, from Serbia. The 265
patients were recruited among subjects consecutively admitted
for carotid endarterectomy to the Vascular Surgery Clinic,
Dedinje Cardiovascular Institute, Belgrade, Serbia from 2004 to
2005 year and from Military Medical Academy, Department of
2006 with evidence of carotid plaque presence either on CCA or
ICA. Exclusion criteria for all patients were history of previous
carotid endarterectomy (possible restenosis), tumors, chronic
inflammatory diseases, autoimmune disease or renal failure.
None of the subjects were receiving statins before or at the time
of enrolment in the study.
check-up at Occupational Medical Center, VINČA Institute,
Belgrade, Serbia, without clinical and radiological evidence of
any chronic inflammatory disease, renal failure or diabetes
mellitus were recruited as controls. For all individuals enrolled in
the study, a complete medical history was collected and included
smoking and drinking habits, presence of diabetes, coronary
artery disease, peripheral arterial occlusive disease and drug
treatment. Hypertension was defined as a systolic blood
pressure≥140 mm Hg, a diastolic blood pressure≥90 mm Hg,
or current treatment with an antihypertensive drug.
The study was approved by the local research ethics
committee and each participant gave their written informed
consent to participate in the study.
Ultrasound assessment of the carotid arteries
Ultrasound assessment of carotid atherosclerosis was
performed by high-resolution B-mode ultrasound (Toshiba,
PowerVision 6000, 7.5 MHz, Riverside, CA). The scanning
protocol entails the longitudinal scanning of the near and far
wall of both carotid arteries at the distal CCA, carotid
bifurcation, and proximal ICA. The IMT was defined as the
distance from the leading edge of the lumen–intima interface to
the leading edge of the media–adventitia interface. Carotid
plaque was defined as an echogenic thickening of intimal
reflection that encroaches on the arterial lumen with minimal
IMTN1.2 mm .
Lipid concentrations were determined in the fresh sera, after
overnight fasting. The total plasma cholesterol (TC) and
triglyceride (TG) levels were determined on Monarch Plus
apparatus (Instrumentation Laboratory, Lexington, USA) using
enzymatic colorimetric methods. High-density lipoprotein
(HDL) cholesterol was determined after dextran sulfate-Mg2+
precipitation of VLDL and low-density lipoprotein (LDL)
cholesterol; using CHOD-PAP method. LDL cholesterol was
calculated according to the Friedwald's formula  for
participants with TG levelsb4.5 mmol/L. All reagent kits were
provided by Instrumentation Laboratory (Lexington, USA).
Genomic DNA was isolated from whole blood samples
extractionmethod.The5A/6A polymorphismwastyped by
polymerase chain reaction (PCR) in ABI 9700 (Applied
Biosystems, USA) as previously described .
Statistical analysis was performed using Statistica Version 5,
software package (StatSoft Inc, 1997). In all tests, differences
with two-tailed alpha-probability (p)≤0.05 were considered
significant. The allelic frequencies and genotype distribution
were estimated by gene counting method. Differences in allele
frequencies and genotype distribution between the cases and
controls as well as deviation from Hardy–Weinberg equilibrium
were estimated by chi-square (χ2).
Means of normally distributed continuous variables were
compared by unpaired t-test and means of skewed continuous
variables with nonparametric Mann–Whitney U test. Multi-
variate logistic regression analysis was performed in order to
asses weather MMP-3 6A containing genotypes are
Characteristics of patients with carotid atherosclerosis and healthy controls
Gender, n (m/f)
Hypertension, n (%)
Smokers, n (%)
Values are mean±SD, for age, BMI, TC, LDLC, HDLC and TG; p — Students'
t-test was used to compare values between controls and patients with carotid
atherosclerosis; * — Mann–Whitney U test; ns — non significant.
1327T. Djurić et al. / Clinical Biochemistry 41 (2008) 1326–1329
independently associated with susceptibility to carotid athero-
sclerosis. Variables with p≤0.05 in the univariate analyses
(age, BMI, hypertension status, HDLC and TG) were entered in
the multivariate logistic regression model. The p for model was
b0.00. Results were expressed in terms of crude and adjusted
odds ratios (OR) with their 95% confidence interval (CI).
The maincharacteristics of study group are shownin Table 1.
Patients with CA were older, had greater TG and lower HDLC
higher proportion of hypertensives was in the patients group.
Distribution of MMP-3 5A/6A genotype frequencies was in
the equilibrium was moved toward higher frequency of
heterozygotes and lower frequency of both homozygotes
compared to expected frequency distribution within Hardy–
There was significantly higher prevalence of genotypes
containing 6A allele in the patients with CA compared to
controls (Table 2). Patients carrying the 6A allele had 1.74 fold
homozygotes (crude OR 1.74, CI=1.0–3.0, p=0.045) (Table 3).
After adjustment for factors that significantly influenced suscept-
ibility to carotid atherosclerosis (age, BMI, hypertensive status,
HDLC and TG, p for model b0.00) the estimated OR for carotid
atherosclerosis was elevated and remained significant (adjusted
OR 2.35, CI=1.0–5.5, p=0.048) (Table 3).
MMP-3, together with some other MMPs, is expressed in the
atherosclerotic lesion, suggesting that it has a role in the
extracellular matrix remodeling associated with atherogenesis
[6,24]. Since MMP-3 has a broad spectar of substrates including
ECM proteins, bioactive molecules and other MMPs, it
represents a key regulator of extracellular matrix turnover.
Lower production of MMP-3 would consecutively lead to
greater accumulation of ECM, faster arterial wall thickening
and plaque growth .
The 5A/6A polymorphism in the promoter of the MMP-3
gene has been shown to have an effect on its expression in vitro
and in vivo and is associated with a number of cardiovascular
conditions [7,8,25]. The 6A allele was associated with lower
promoter activity [7,8]. In our study, subjects carrying
genotypes with one or more 6A alleles had significantly higher
odds ratio for development of CA compared to 5A/5A
homozygotes. This is in agreement with previous results of
several studies. So far, 6A6A genotype was associated with
greater progression of coronary atherosclerosis  and the
number of coronary arteries with stenosis N50% . Also, it
was associated with carotid stenosis N70 %  and greater
IMT [15,17,18]. Moreover, healthy male individuals homo-
zygous for the 6A allele showed increased wall thickness,
enlarged arterial lumen, and local reduction of wall shear stress,
which might predispose them to atherosclerotic plaque
Recent meta analysis which pooled together data from
seven studies considering association of this polymorphism
with MI has showed a moderate genetic influence of the 5A
allele as a plaque-disrupting risk factor . Their findings are
going along with the genetic model in which higher
expression of MMP-3 by 5A allele containing promoter and
higher protein activity could lead to atherosclerotic plaque
instability and possible rupture as a cause of cardio- and
cerebrovascular events. We could hypothesize that decrease in
frequency of 5A/5A genotype in our patients with advanced
atherosclerosis could be the consequence of the association of
this genotype with mortal clinical events, such as stroke or MI.
Still, it is not clear which of the cardiovascular risk factors are
the tissue and time specific switch for 5A or 6A dominant
effect on the level of MMP-3 gene transcription and
Our study has shown that MMP-3 5A/6A polymorphism is
significant and independent risk factor for carotid atherosclero-
sis in the Serbian population. Further research on elucidation of
different MMPs and their mutual and individual effects in
certain environment should resolve the complex role of MMPs
and their tissue inhibitors in atherosclerosis.
This work was supported by a Serbian Government Research
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