Association of PvuII polymorphism in the lipoprotein lipase gene with the coronary artery disease in Macedonian population.

Abstract

In the etiology of coronary artery disease there are many factors involved as a result of the complex interaction between genetic predisposition and environmental influences. The lipoprotein lipase (LPL) plays a very important role in lipid metabolism. It hydrolyzes the triglycerides in hylomicrones and very low density lipoproteins - VLDL. PvuII polymorphism in the LPL gene is a frequent variant and it increases triglyceride levels and the risk of the appearance of coronary arterial disease.
The aim of this work is to show LPL-PvuII polymorphism as an independent risk factor and also as a predictor of coronary arterial disease in the Macedonian population.
The study included 109 randomized patients with coronary artery disease (CAD) (83 males, 26 females), treated at the Cardiology Clinic. The stenosis of coronary arteries greater than 70% of the artery lumen was angiographically documented in the CAD group. The control group consisted of 32 patients (25 males, 7 females) with documented normal coronarographic findings. The patients' age ranged from 50 to 59; the mean age in the CAD group was 59.4 and the mean age in the control group was 57.9. LPL-PvuII polymorphism in the intron 6 in the CAD and control group was detected by PCR amplification and restriction enzyme digestion.
A statistically significant association between CAD and the control group was found regarding the presence of hyperlipidaemia (p < 0.001), diabetes (p < 0.05) and the use of antilipidaemic drugs (p < 0.049). The presence of LPL-PvuII polymorphism in both investigated groups does not represent a statistically significant risk factor for the appearance of coronary artery disease (p = 0.816). The PvuII + allele frequency of 0.495 and 0,469 was obtained in both the angiographically confirmed CAD and the control groups, respectively. This finding indicates no significant differences between the prevalence of the LPL-PvuII genotypes in both study groups, suggesting a lack of association of LPL-PvuII polymorphism with CAD. However, the homozygous genotype (PvuII +/+) was more prevalent in the CAD group (22.9%) in comparison with the control group (15.6%).
In our study LPL-PvuII polymorphism was not identified as an independent risk factor for the appearance of CAD.

Full text

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Prilozi, Odd. biol. med. nauki, MANU, XXIX, 2, s. 213‡225 (2008)
Contributions, Sec. Biol. Med. Sci., MASA, XXIX, 2, p. 213–225 (2008)
ISSN 0351–3254
UDK: 616.132.2-005.4-056.7







ASSOCIATION OF PvuII POLYMORPHISM IN THE LIPOPROTEIN
LIPASE GENE WITH THE CORONARY ARTERY DISEASE
IN MACEDONIAN POPULATION

Georgiev A.,1 Panov S.,2 Sadikario S.1

1Heart Diseases Institute, Clinical Centre, Medical Faculty,
Ss. Cyril and Methodius University, Skopje, R. Macedonia
2Molecular Biology Laboratory, Biology Institute, Natural Sciences Faculty,
Ss. Cyril and Methodius University, Skopje, R. Macedonia


Abstract: In the etiology of coronary artery disease there are many factors
involved as a result of the complex interaction between genetic predisposition and envi-
ronmental influences. The lipoprotein lipase (LPL) plays a very important role in lipid
metabolism. It hydrolyzes the triglycerides in hylomicrones and very low density
lipoproteins – VLDL. PvuII polymorphism in the LPL gene is a frequent variant and it
increases triglyceride levels and the risk of the appearance of coronary arterial disease.
Aim: The aim of this work is to show LPL-PvuII polymorphism as an indepen-
dent risk factor and also as a predictor of coronary arterial disease in the Macedonian
population.
Material and Methods: The study included 109 randomized patients with coro-
nary artery disease (CAD) (83 males, 26 females), treated at the Cardiology Clinic. The
stenosis of coronary arteries greater than 70% of the artery lumen was angiographically
documented in the CAD group. The control group consisted of 32 patients (25 males, 7
females) with documented normal coronarographic findings. The patients’ age ranged
from 50 to 59; the mean age in the CAD group was 59.4 and the mean age in the control
group was 57.9. LPL-PvuII polymorphism in the intron 6 in the CAD and control group
was detected by PCR amplification and restriction enzyme digestion.
Results: A statistically significant association between CAD and the control
group was found regarding the presence of hyperlipidaemia (p < 0.001), diabetes (p <
0.05) and the use of antilipidaemic drugs (p < 0.049). The presence of LPL-PvuII poly-
morphism in both investigated groups does not represent a statistically significant risk
factor for the appearance of coronary artery disease (p = 0.816).

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Georgiev A., Panov S. et al.
The PvuII + allele frequency of 0.495 and 0,469 was obtained in both the an-
giographically confirmed CAD and the control groups, respectively. This finding indi-
cates no significant differences between the prevalence of the LPL-PvuII genotypes in
both study groups, suggesting a lack of association of LPL-PvuII polymorphism with
CAD.
However, the homozygous genotype (PvuII +/+) was more prevalent in the
CAD group (22.9%) in comparison with the control group (15.6%).
Conclusion: In our study LPL-PvuII polymorphism was not identified as an
independent risk factor for the appearance of CAD.

Key words: LPL-PvuII polymorphism, coronary artery disease.


Introduction

Coronary artery disease (CAD) is a leading cause of exceptionally high
invalidity, mortality and morbidity in the world. Atherosclerosis is a complex
process, and several major risk factors and a growing number of new risk mar-
kers have been reported which contribute to an understanding of the etiopatho-
genetic mechanisms of CAD [14]. Thus, when evaluating the individual risk
concerning coronary artery disease, the determination of the genotype for cer-
tain mutations and polymorphism has an important role, besides the relevant
phenotype measurable laboratory and clinical parameters. In the greatest num-
ber of cases of coronary artery disease, specific polymorphism or mutations in
certain genes can be found [24, 28].
The association between polymorphisms and mutations in the lipo-
protein lipase (LPL) gene has been the subject of numerous studies [6]. The
LPL gene is located on the short branch of the 8p22 chromosome and extends to
a length of 30 kb. It consists of 10 exons with high evolutionary conservation
(homology between the species). The complementary DNA (cDNA) codes a
protein with 475 amino acid residues including a 27 amino acids signal peptide.
The protein product of this gene is the LPL enzyme. It is a multifunctional pro-
tein which hydrolyzes the triglycerides from the circulating hilomicrones and
from the cholesterol complexes with very low density (VLDL). The liberated
triglycerides are metabolized in hepathocytes or converted into LDL particles
by the hepatic lipase. During this process, the free cholesterol and phospholipids
transfer into HDL particles thus increasing the concentration of HDL-choleste-
rol. The catalytic centre of the enzyme is formed by three amino acid residuals,
namely serin-132, asparagine acid-156 and histidin-241. About 100 mutations
and single nucleotide polymorphisms (SNPs) in the human LPL gene have been
described so far. The missense mutations dominate (61) by substitution of one
into another amino acid residual. The greatest number of them is located in the
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Association of PvuII polymorphism…
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exons 5 and 6. The nonsense mutations (12) follow and they lead to a shortened
protein product. At least ten mutations have been identified in the promoter re-
gion of the LPL gene. Small deletions or insertions lead to the frame shift. The
frequency of mutations in the LPL gene varies significantly among different po-
pulations. In patients with a certain polymorphism of LPL, the gene effects are
only faintly expressed and environmental factors determine the intensity and
result of the clinically manifested arteriosclerosis [10].
Numerous studies have been conducted in which candidate genes were
investigated for the putative association with CAD [19, 20]. Only a few gene
polymorphisms and mutations were found to correlate with the risk of CAD or
the clinically manifested disease, but the reports were often giving contradictory
results. There are several reasons for these apparent inconsistencies that include,
but are not restricted to, genetic heterogeneity, environmental factors, statistical
methods, etc. [9, 13, 18]. It is believed that the presence of polymorphism in se-
veral genes at the same time (as those for the enzymes involved in the lipid me-
tabolism, coagulation factors, inflammatory mediators and other proteins), is the
basis for the multi-gene nature of arteriosclerosis and CAD [8, 34].
The LPL gene contains the polymorph sequence CAG↓CTG in intron 6
that can be recognized by the PvuII endonuclease restriction enzyme. LPL-
PvuII polymorphism is one of the genetic variants that are described in the LPL
gene that is associated with CAD appearance and progression in numerous
studies [3, 5, 35–37]. On the other hand, other authors did not find a significant
connection to CAD [1, 34, 26, 36].
Given the importance of LPL as a candidate gene for cardiovascular
risk, we evaluated an independent, angiographically controlled Macedonian po-
pulation to determine whether LPL- PvuII polymorphism was associated with
defined CAD [2, 21, 27]. Therefore, the aim of this study is to show the possible
associations of LPL-PvuII polymorphism and clinically verified coronary artery
disease in the Macedonian population.


Aim

The aim of this study is to investigate the LPL-PvuII polymorphism as
an independent risk factor, but also as a predictor of coronary artery disease in
the Macedonian population.


Material and methods

The study is of a prospective character and it is randomized. Two insti-
tutions were involved, namely the Cardiology Clinic and the Molecular Biology
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Georgiev A., Panov S. et al.
Laboratory at the Natural and Mathematical Sciences Faculty at the Ss Cyril and
Methodius University in Skopje.
The study included 109 patients as the examined group (CAD group –
83 males, 26 females), treated at the Cardiology Clinic. It was angiographically
documented that the patients suffered from stenosis of the coronary arteries over
70% of the artery lumen. The control group consisted of 32 patients (25 males,
7 females) and they had normal coronarographic findings, which were also
angiographically documented.
The realization of the study was approved by the Ethical Committee of
the Medical Doctors' Chamber of the Republic of Macedonia.
The following data on the patients were analyzed: demographic data:
age, sex, place of living, and nationality; risk factors for CAD and their inci-
dence (according to Joint Task Force for Cardiac Prevention, 1998 and ATP,
2001): profession, family predisposition to CAD, information on myocardial
infarction, history of hyphercholesterolaemia, hypherlipidaemia, hypertension
or diabetes mellitus, smoker or non-smoker, data on physical activity, educa-
tion, use of antilipemics, use of alcohol, and BMI (Body-Mass Index).
The patients were chosen randomly, consecutively, according to the
time of the angiographic examination performed at the Cardiology Clinic, and
whether s/he satisfied the criteria for inclusion or exclusion from the study.

Sample Collection and DNA Extraction
Five millilitres of peripheral blood were aseptically collected in a test
tube (Vaccutainer®) with anticoagulant (EDTA disodium salt), following infor-
med consent, from all individuals who participated in this study. The standard
isolation of genomic DNA was performed on nucleated cells, using DNA
extraction with sodium chloride and chloroform, and ethanol-precipitation [23].

Detection of LPL-PvuII polymorphism
The LPL-PvuII polymorphism in intron 6 was identified by restriction
enzyme digestion of the PCR-amplified segment of the LPL gene [2, 21]. The
polymerase chain reaction (PCR) was performed with the primers: forward, 5'-
ATC AGG CAA TGC GTA TGA GGT AA-3'; reverse, 5'-GAG ACA CAG ATC
TCT TAA GAC-3'. Each PCR-amplification reaction was performed using 100–
250 ng genomic DNA; 10 pmol of each primer; 200 mmol/L each of dATP,
dCTP, dGTP, and dTTP; and 0.05 U of Taq polymerase in a total reaction volume
of 20 :L. Amplification was performed in a GeneAmp PCR System 2400 (Perkin
Elmer). The initial denaturation at 94ºC for 5 minutes was followed by 30 cycles
of denaturation at 94ºC for 1 minute, annealing at 57ºC for 1 minute, and exten-
sion at 72ºC for 1 minute, with final extension at 72ºC for 10 minutes.
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The LPL-PvuII polymorphism was identified by restriction fragment
length polymorphism (RFLP) analysis of the amplified fragments using PvuII
endonuclease (Sigma-Aldrich) digestion. The restriction products were resolved
by agarose electrophoresis, visualized under UV-illumination (312 nm) follo-
wing ethidium bromide staining and recorded by a digital camera (Canon A70).
Images were analyzed by GelPro software.

Statistical data processing
The statistical tests were calculated using SPSS 8.0 for MS Windows.
A p-value less than 0.05 was considered as significant.


Results

The study (CAD) group included 114 patients with angiographically
confirmed stenosis of the coronary arteries greater than 70% of the artery lu-
men. The control group consisted of 35 patients with angiographically normal
coronary arteries. PCR amplification was unsuccessful in 5 samples of the CAD
group and in 3 of the control group. LPL-PvuII polymorphism was identified by
restriction fragment length polymorphism (RFLP) of the amplified fragments
using PvuII endonuclease digestion (Figure 1).


Figure 1 – Genotyping of PvuII polymorphism in intron 6 of LPL gene
Slika 1 ‡ Genotipizacija na PvuII polimorfizmot vo intronot 6
od LPL genot

The LPL-PvuII genotype in this study was successfully determined in
109 patients (83 males and 26 females) of the CAD group included and 32
subjects (25 males and 7 females) of the control group.

Prilozi, Odd. biol. med. nauki, XXIX/2 (2008), 213–225