Original Article
www.advmolmed.com
No association between paraoxonase 192 and 55
polymorphism and increasing risk of coronary artery
disease in Turkish patients
Bedia A¤açhan1, C. Selim ‹sbir2, H. Arzu Ergen1, Hülya Y›lmaz1, ‹lhan Yayl›m1, Ümit Zeybek1,
O¤uz Öztürk1, Turgay ‹sbir*1
1Department
2Department
of Molecular Medicine, Institute of Experimental Medical Research, ‹stanbul, Turkey
of Cardiovascular Surgery, Marmara University School of Medicine, ‹stanbul, Turkey
Objectives: Paraoxonase (PON1) has been emerged as an independent risk factor for cardio-
vascular disease. As there are no existing data for the Turkish population, we investigated the
effect of paraoxonase (PON1) 192 glutamine (A)/argenine (B) and PON1 55 methione
(M)/leucine (L) polymorphisms on serum PON1 activity and lipid profiles in 117 patients with
coronary artery disease (CAD) and 110 healthy controls in Turkish subjects.
Methods: Polymerase Chain Reaction (PCR), Restriction Fragment Length Polymorphism (RFLP),
and agarose gel electrophoresis techniques were used to determine the PON1 genotypes.
Serum lipid levels were measured enzymatically. PON1 activity was measured by spectrophotometric assay of p-nitrophenol production following addition of paraoxon.
Results: Distribution of PON1 192 AA, BB, AB and PON1 55 LL, MM, LM genotypes among the
patients with CAD were 0.32, 0.15, 0.52 and 0.39, 0.05, 0.55; among the control subjects, they
were 0.32, 0.09, 0.58 and 0.46, 0.06, 0.47 respectively. Mean serum paraoxonase activities
ware significantly higher in control group compared with the CAD patients (345.76±192.21
U/ml vs 225.09±155.84 U/ml, p<0.01). In CAD patients, serum PON1 activity was significantly
higher in PON1 192 BB genotypes than AA and AB genotypes (p<0.0001). Although serum
PON1 activity was higher in PON1 55 LL genotypes than MM genotypes, the results were not
statisticaly significant (p=0.077). The PON1 55 and 192 polymorphisms consistently not influenced the serum lipid profiles in either population.
Conclusions: In conclusion, our results suggest that the paraoxonase activities are influenced by
PON1 genetic variability but there is no association between the PON1 192 and PON1 55 genotypes and it is not a factor for coronary artery disease in Turkish patients.
Key words: Paraoxonase, polymorphism, activity, lipid, coronary artery disease, Turkish
Adv Mol Med 2005; 1(2): 92-96
Introduction
Coronary artery disease (CAD) is a multifactorial disease in which genetic and environmental factors play
an important role. These factors may differ in each race
and ethnic group.1-3 The serum activity of human
paraoxonase (PON1), which varies among individuals,
might have been involved in atherosclerosis.4 The
paraoxonase limits the LDL peroxidation by preventing
Adv Mol Med 2005; 1(2)
the transformation of LDL into biologically active
atherogenic particles4 PON1 activity towards paraoxon
is genetically determined by two polymorphisms with*Correspondence to: Turgay ‹sbir, PhD
Department of Molecular Medicine, Institute of Experimental
Medical Research, Çapa 34390 ‹stanbul, Turkey.
e-mail: [email protected]
Fax: +90 212 635 19 59
Accepted: April 3, 2005
No association between PON 192 and 55 polymorphism and increasing risk of CAD in Turkish patients
in the alleles acting at a single autosomal locus. The
first of these pairs reported involved an amino acid
substitution at position 192, giving rise to two alloenzymes with low activity (glutamine at position 192, A
allele) and high activity (arginine at position 192, B
allele) towards paraoxon.5,6 There also exists a second
polymorphism of the human PON1 gene affecting
amino acid 55, giving rise to a leucine (L-allele) substitution for methionine (M-allele).7,8 Recent case control
studies have indicated the PON1 192 polymorphism is
related to coronary artery disease (CAD)9-11 while others have not shown this relationship [12,13]. One study
has suggested that the PON1 55 L allele is a risk factor
for CAD in non-insulin dependent diabetes mellitus.7
Given the growing importance of paraoxonase as a
cardiovascular disease risk factor and its potential
involvement in protection against oxidative stress, we
examined the polymorphism at position 55 and 192
and effects of serum paraoxonase activity and serum
lipid profiles in Turkish coronary artery disease patients
and healthy controls.
Materials and Methods
Subjects
PON1 gene polymorphisms were studied in 117
patients with CAD (44 (37.6%) women, 73 (62.4%)
men). The patients with severe coronary vascular disease were documented by angiography. Angiographic
inclusion criterias were; ≥ 50 % stenosis at least one
major coronary vessel because of atherosclerosis, and a
vascular event, defined as myocardial infarction, percutaneous transluminal coronary angioplasty, or coronary
artery bypass grafting. Patients were included irrespective of concomitant risk factors for atherosclerosis such
as smoking, arterial hypertension, hyperlipidemia,
increased body mass index, and diabetes mellitus
There were 56 (47.86%) smoker, 27(23.07%) hypertension, 9 (%7.69) left ventricul hypertrophy, 50 (42.73%)
non-insulin dependent diabetes mellitus and 14
(%11.96) obese patients with CAD.
Healthy persons (54 (%49.1) women, 56 (%50.9)
men) without any symptoms of CAD were selected for
the control group. Coronary angiography was not performed on these individuals, and therefore the presence of atherosclerotic coronary arteries could not be
excluded. However, none of these individuals had any
history of vascular event.
93
Biochemical determinations
Paraoxonase activities was measured according to
Furlong et al.14 The assay buffer contains 0.132 M Tris
HCl (pH 8.5), 1.32 m M CaCl 2 and 2.63 M NaCl.
Addition of 200 ul of 6 m M freshly prepared paraoxon
(0,0-diethyl-0-p-nitrophenylphosphate; Sigma, Poole,
UK) and 40Ìl of serum initiated the assay. The rate of
generation of p-nitrophenol was determined at 37°C,
with the use of a continuously recording spectrophotometer at 405 nm. A molar extinction coefficient of
18.05 x 103 was used for calculation using paraoxon as
substrate. Pararaoxonase activity is expressed as
units/liter (unit: Ìmol paraoxon hydrolyzed/min).
Fasting serum total cholesterol was measured by the
esterase oxidase method,15 High Density Lipoprotein
(HDL)-cholesterol was determined enzymatically following dextran sulfate magnesium precipitation,16 and
serum triglycerides by the enzymatic procedure of Mc
Gowan.17 Low Density Lipoprotein (LDL)-cholesterol
value was calculated with Friedewald equation [16]
when the triglyceride levels was less than 400 mg/dl.
Genotyping method of the Paraoxonase 55/192
Polymorphism: Blood specimens were collected in
tubes containing EDTA, and DNA was prepared from
leucoycte pellet by SDS lysis ammonium acetate extraction and ethanol precipitation.18
PON1 genotypes were determined following PCR
according to previously published protocols.5,6 For the
192 polymorphism sense primer 5' TAT TGT TGC TGT
GGG ACC TGA G 3' and antisense primer 5' CAC GCT
AAA CCC AAA TAC ATC TC 3' which encompass the
192 polymorphic region of the human PON1 gene were
used. For the 55 polymorphism sense primer 5' GAA
GAG TGA TGT ATA GCC CCA G 3' and antisense
primer 5' TTT AAT CCA GAG CTA ATG AAA GCC 3'
were used. The PCR reaction mixture contained 100 ng
DNA template, 0.5 M of each primer, 1.5 mM MgCl2,
200 M 4dNTP's and 1 U Taq DNA polymerase (MBI
Fermentas). After denaturing the DNA for 5 min at
94°C, the reaction mixture was subject to 35 cycles of
denaturing for 1 min at 95°C, 1 min annealing at 60°C
and 1 min extension at 72°C for the 192. The 99 bp PCR
product was digested with 8 U BspI restriction endonuclease (MBI Fermentas, Lithuania) overnight at 55°C
and the digested products separated by electrophoresis
on 4% metaphore agarose gel and visualised using
ethidium bromide. The B-genotype (arginine) contains
a unique BspI restriction site which results in 66 and 33
Adv Mol Med 2005; 1(2)
94
A¤açhan B et al
bp products and the A-genotype (glutamine) will not
cut allowing the PON1 192 genotype to be determined.5
For the PON1 55 polymorphism, PCR reaction and
cycling was the same as above. The PCR product (170
bp) was digested with Hsp192II (Promega, USA) in the
presence of BSA (0.1 µg/µL final concentration) (37°C,
overnight) and the digested products were separated
and identified as above. Allele L (leucine) did not contain the Hsp192II site whereas M (methionine) contained the Hsp192II site giving rise to 126 and 44 bp
products.6
Statistical analyses
Statistical analyses were performed using the SPSS
software package, revision 10.0. Clinical laboratory data
are expressed as means + SD. Mean values were compared between patients and control subjects by the
unpaired Student's t test.
Differences in the distribution of PON1 genotypes
or alleles between cases and controls were tested using
the Chi-square statistic, respectively. Allele frequencies
were estimated by gene counting methods. P<0.05
were considered statistically significant.
Statistical analysis for effects of PON1 alleles on
serum lipid profiles and PON1 activity in patients and
control subjects were made by one-way analysis of
variance (ANOVA).
Table 1
Characteristics of the study groups
Control
Gender (female/male) (n)
Age (years)
The demographic characteristics of the control and
CAD populations are given in Table 1. Body mass
index (BMI), Systolic blood pressure (SBP), Diastolic
blood pressure (DBP), glucose total cholesterol and
triglyceride levels were significantly higher in CAD
compared with the control subjects (p<0.05). Mean
serum paraoxonase activities was significantly higher in
control group compared with the CAD patients
(p<0.01).
The PON1 192 and PON1 55 genotypes and allele
freqencies for CAD patients and control subjects are
shown in Table 2. Frequencies of PON1 192 AA, BB
and AB genotypes among the patients with CAD were
0.32,0.15,0.52; and among the control subjects 0.32,
0.09,0.58, respectively. The gene frequency for the
PON1 192 polymorphisms in controls and CAD patients
was not significantly (P=NS, c2 test).
Adv Mol Med 2005; 1(2)
54/56
44/73
58.36±10.99
59.77±13.15
Body Mass Index (kg/m2)
24.37±3.56
27.68±4.46 ***
Systolic pressure (mmHg)
119.80±10.60
135.19±29.88**
Diastolic pressure (mmHg)
72.57±8.96
82.31±18.54 **
Glucose (mg/dl)
98.78±23.79
261.42±140.94***
PON activity (U/ml)
345.76±192.21**
225.09±155.84
164.98±32.21
205.13±50.86***
Triglyceride (mg/dl)
124.40±42.24
157.51±103.25*
LDL-cholesterol (mg/dl)
114.81±32.93
125.43±42.60
HDL-cholesterol (mg/dl)
40.45±15.42
39.11±13.82
VLDL-cholesterol (mg/dl)
28.25±15.32
32.32±24.37
Total-cholesterol (mg/dl)
n: number of individuals. The results are shown as mean±SD
*: p< 0.05, **: p< 0.01, ***: p< 0.001
Table 2
PON 192 and 55 genotype distribution and gene frequency in the
controls and CAD subjects
Control
CAD
n=110
n=117
AA
36 (32.7%)
38 (32.5%)
BB
10 (9.1%)
18 (15.4%)
AB
64 (58.2%)
61 (52.1%)
A
133 (62.14%)
81 (75.0%)
B
81 (37.85%)
27 (25.0%)
51 (46.4%)
46 (39.3%)
PON 192 genotypes
Alleles
PON 55 genotypes
LL
Results
CAD
MM
7 (6.4%)
6 (5.1%)
LM
52 (47.3%)
65 (55.6%)
L
153 (70.18%)
76 (70.37%)
M
65 (29.81%)
32 (29.62%)
Alleles
Frequencies of PON1 55 LL, MM, and LM genotypes
among the patients with CAD were 0.39, 0.05, 0.55 and
among the control subjects 0.46, 0.06, 0.47, respectively. The gene frequency for the PON1 55 polymorphisms in controls and CAD patients was not significantl (P=NS, c2 test).
The relationships between the PON1 192/55 genotypes and PON1 activity, and lipid profiles are shown
in Table 3. The PON1 192 polymorphism did have a
major effect on serum PON1 activity. In both populations PON1 activity was significantly higher in the BB
genotype, and lowest in the AA with the AB genotype
having an intermediate activity. In CAD patients serum
No association between PON 192 and 55 polymorphism and increasing risk of CAD in Turkish patients
95
Table 3
PON activity and lipid profiles in CAD populations according to their PON1 192/55 genotypes
PON1 192 genotypes
AA
PON1 55 genotypes
BB
AB
LL
MM
LM
220.49±132.11
PON1 Activity (U/ml)
172.40±100.98
559.96±76.50
206.41±126.00
260.54±188.01
104.25±65.70
Total cholesterol (mg/dl)
202.93±51.28
237.80±43.44
197.13±52.18
205.29±46.05
200.50±57.12
204.50±57.67
Triglyceride (mg/dl)
149.10±65.40
152.80±58.39
180.20±165.21
142.13±58.03
117.25±83.19
184.23±138.52
HDL-cholesterol (mg/dl)
38.81±9.23
41.40±10.36
41.93±19.30
41.67±13.30
36.50±6.19
39.23±14.12
LDL-cholesterol (mg/dl)
128.41±43.17
142.60±42.34
111.40±42.30
123.43±36.15
135.50±48.69
123.50±48.07
VLDL-cholesterol (mg/dl)
30.14±13.08
29.60±12.60
38.40±15.10
28.45±11.87
23.25±16.64
38.59±14.43
Values are mean±SD.
PON1 activity were significantly higher in PON1 192 BB
genotypes than AA and AB genotypes (p<0.0001). In
both populations serum PON1 activity was higher in
the LL genotype and lowest in the MM with the LM
genotype having an intermediate activity . Serum PON1
activity were higher in PON1 55 LL genotypes than MM
genotypes but not statisticaly significant (p=0.077).
The PON1 192 and PON1 55 polymorphisms had no
major significant effect on plasma lipid levels either
groups.
Discussion
In this study we examined the effect of PON1 192
and PON1 55 polymorphisms on serum PON1 activity
and lipid profiles in CAD patients and healthy controls
in Turkish subjects.
There is increasing evidence showing that serum
PON1 is related to the prevention of CAD.9-11 The
alloenzymes of PON1 with B and L alleles have high
activity towards artificial substrate paraoxon contrary to
the alloenzymes with A and M alleles, which have the
low activity towards this substrate.19 In our sample, we
showed that the two polymorphisms were associated
with PON1 activity, which increased in the order of the
AA < AB < BB genotype in the PON1 192 polymorphism and MM < ML < LL genotype in the PON1 55
polymorphism.
Recently, several laboratories have reported the
results of case-control studies investigating the relationship between PON1 192 polymorphism and the presence of CAD.9-13,20-22 The results of these studies have
varied. In some studies, a positive association between
the B allele and the presence of disease was found,
while in others, no such association emerged. Some
contradictory results can be explained, at least partially, by admixture of genetically heterogeneous popula-
tions, but it cannot explain the differences found in
studies conducted in the same ethnic population.
However, the previous reports from Northern Europe,
including Finland, have found no association between
PON192 polymorphism and CAD.12,23
There is only limited information on the association
between the M/L55 locus and CAD. Previous retrospective case-control study from Japan found no association between M/L55 polymorphism and CAD.21 A
cross-sectional study suggested that the L allele increases the risk of CAD in patients with type 2 diabetes.7 In
line with this observation, it was also found that in
middle aged and elderly study population, the LL genotype is an independent predictor of carotid atherosclerosis and the PON1 192 polymorphism does not modulate the effect of the L allele.24 In addition, one study
found a marginal increase in the risk of myocardial
infarction in subjects with LL genotype.22
In the present study, we found no significant differences in genotype and allele frequencies for PON1
polymorphisms at positions 55 and 192 between control subjects and patients with CAD. The frequencies
were similar to those described for other Caucasian
populations.7,9,10,13 Recently, the BB genotype has been
found to be associated with an increased risk of CAD
in several studies [9-11], also we found that PON1 192
BB genotype frequencies higher in CAD groups than
controls (15.4% vs 9.1%) (p=NS, c2 test).
Low PON1 activity in patients who had suffered
from MI, a significant decrease in PON1 activity toward
paraoxon hydrolysis, has been shown in diseases with
accelerated atherogenesis, such as familial hypercholesterolemia24,26 and diabetes mellitus,25,27 In our study,
we observed that serum PON1 activity were significantly decreased in CAD patients than control groups
(p<0.01). Our findings are in agreement with most of
the earlier studies.7,25,9,11,27,28
Adv Mol Med 2005; 1(2)
96
A¤açhan B et al
In summary, patients have lower plasma paraoxonase activity than controls and paraoxonase activities
effected by PON1 genetic variability but there is no
association between the PON1 192 and PON1 55 genotypes and increased risk of CAD in Turkish coronary
artery disease patients.
Abbreviations:
PON1
CAD
PCR
RFLP
BMI
SBP
DBP
HDL
LDL
VLDL
: Paraoxonase
: Coronary artery disease
: Polymerase Chain Reaction
: Restriction Fragment Length Polymorphism
: Body mass index
: Systolic blood pressure
: Diastolic blood pressure
: High Density Lipoprotein
: Low Density Lipoprotein
: Very Low Density Lipoprotein
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