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). 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