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152 Original articles | March 2014 - Issue 3 Protective role of Lipoprotein-Associated Phospholipase A2 Gene (A379V) Polymorphism against Myocardial Infarction among Egyptians Ola Sharaki MD1, Mohamed Sobhi MD2, Doreen Younan MD1, Eman Elkemary MSc1. 1. Clinical Pathology Department, Faculty of Medicine, Alexandria University, Alexandria, Egypt. 2. Cardiology Department, Faculty of Medicine, Alexandria University, Alexandria, Egypt. Abstract Background: Oxidation of low density lipoproteins is an initial step of atherogenesis that generates pro-inflammatory phospholipids, including platelet-activating factor (PAF) and its analogs. Platelet-activating factor is degraded by lipoprotein associated phospholipase A2 (Lp-PLA2), also known as platelet-activating factor-acetylhydrolase (PAF-AH), a circulating enzyme having both pro and anti-inflammatory activities. Lipoprotein associated phospholipase A2 activity has been postulated to be a risk factor for acute coronary syndrome (ACS); however, whether Lp-PLA2 has a causal or protective role is still unclear. A large number of single nucleotide polymorphisms (SNPs) that affect Lp-PLA2 mass and activity in plasma have been described. Aim: The aim of the present work is to determine the prevalence of Lp-PLA2 gene A379V single nucleotide polymorphism (SNP) in Egyptians suffering from myocardial infarction (MI) in comparison to healthy controls and to correlate this genetic variant with different cardiovascular risk factors. Methods: Lp-PLA2 gene A379V polymorphism (rs1051931) was investigated in fifty patients having MI and fifty age and sex matched healthy controls using real-time PCR. Results: The homozygous CC genotype, coding for alanine at position 379 of Lp-PLA2 protein, had the highest frequency among patients (72%) compared with controls (46%) while heterozygous CT genotype had the highest frequency among controls (46%) compared with patients (24%) with a significant difference (p=0.033). The major “C” allele had the highest frequency among patients (84%) compared with controls (69%) while the minor “T” allele, coding for valine at the same position, had the highest frequency among controls (31%) compared with patients (16%) with a significant difference (p=0.012). Conclusion: The Lp-PLA2 A379V gene polymorphism was found to be less frequent in MI patients presented with ACS than in healthy controls, suggesting that this SNP might be protective against the development of MI. Key words: lipoprotein associated phospholipase A2, Myocardial infarction, platelet-activating factor-acetylhydrolase, single nucleotide polymorphism. Introduction The recognition that atherosclerosis has a strong inflammatory component has stimulated a great deal of research on the role of inflammatory mediators in the atherosclerotic disease process.1 Oxidation of low density lipoproteins (LDLs) is an initial step in atherogenesis, that generates a myriad of proinflammatory phospholipids, including platelet-activating factor (PAF) and its analogs,2, 3 which are implicated in signaling and activation of pro-inflammatory cells such as platelets, leukocytes and macrophages.4 Platelet-activating factor exerts its various effects via the G-protein-coupled PAF-receptor that binds PAF with high affinity.5 PAF and its biologically active analogs are degraded by lipoprotein-associated phospholipase A2 (Lp-PLA2), a circulating enzyme bound mainly to LDLs, and to a lesser extent to high density lipoproteins (HDLs).6, 7 Lipoprotein-associated phospholipase A2 is also known as PAF-acetylhydrolase (PAF-AH). Besides having an anti-inflammatory activity by degrading PAF, Lp-PLA2 may also exert a pro-inflammatory activity by massively hydrolyzing phospholipids to generate lyso-phosphatidylcholine (lyso-PC) and free oxidized fatty acids, both are pro-inflammatory mediators largely responsible for the pro-atherogenic activity of oxidized LDL.8 Lipoprotein associated phospholipase A2 is a member of the group VII family of PLA2 enzymes which are Ca2+-independent enzymes, consisting of 45.4 kDa polypeptide chains.9 With the classification of this enzyme as a positive risk factor in coronary heart disease, it has become a very attractive drug target. A specific inhibitor of this enzyme, Darapladib, was developed in 2003.10 This drug binds reversibly and noncovalently to human recombinant Lp-PLA2 and inhibits LpPLA2-mediated exogenous substrate hydrolysis in plasma and LDL in vitro.10 In vivo studies showed that darapladib treatment reduced the content of lyso-PC in pig atherosclerotic lesions, owing to inhibition of hydrolysis of endogenous phospholipids.11 The gene for Lp-PLA2, PLA2G7, has 12 exons and is located on chromosome 6p21.2 to 12. A large number of single nucleotide polymorphisms (SNPs) that affect Lp-PLA2 mass and activity in plasma have been described. Some variants are noted mainly March 2014 - Issue 3 | 153 Original articles Table 1. Clinical Characteristics of the two studied groups. Parameter (mean±SD) Patients (n=50) Controls (n=50) Age (years) 48.34 ± 7.67 45.16 ± 8.73 Male 23 (46%) 25 (50%) Female 27 (54%) 25 (50%) TG (mg/dl) 187.82 ± 92.50* 112.82 ± 29.73 Cholesterol (mg/dl) 228.26 ± 71.98* 165.88 ± 30.11 LDL (mg/dl) 152.32 ± 62.08* 85.80 ± 25.36 HDL (mg/dl) 38.06 ± 13.77* 56.18 ± 12.64 CK (U/L) 1654.46 ± 1390.38* 80.04 ± 32.39 CK-MB (ng/ml) 163.06 ± 185.24* 0.47 ± 0.32 Troponin I (ng/ml) 61.92 ± 74.46* 0.0 ± 0.0 AST (U/L) 324.42 ± 266.37* 23.22 ± 10.09 LDH (U/L) 1261.64 ± 965.39* 151.16 ± 30.61 Hs-CRP (mg/L) 138.48 ± 145.80* 8.54 ± 10.17 Glucose (mg/dl) 110.88 ± 33.02* 97.74 ± 16.41 TG: Triglycerides, LDL: Low denisty lipoprotein, HDL: High denisty lipoprotein, CK: Total creatine kinase, CKMB: Creatine kinase MB isoform, AST: Aspartate transaminase, LDH: Lactate dehydrogenase, Hs-CRP: High sensitivity C-reactive protein. *p value < 0.05 compared with controls. in certain ethnic groups. The most frequently studied SNPs are R92H (rs1805017), I198T (rs1805018), V279P and A379V (rs1051931).12-14 The missense mutation of the PLA2G7 gene, which results in alanine (ACG) to valine (ATG) transition at position 379 of Lp-PLA2 protein, A379V (rs 1051931) (46672943 C > T), has been observed in Caucasians, Chinese, Taiwanese and South Koreans.15, 16, 17, 18, 19 This polymorphism is thought to decrease the substrate affinity of Lp-PLA2, possibly prolonging the activity of PAF, which in turn is associated with many inflammatory diseases.13 Materials and Methods Study Population: This study was conducted on fifty Egyptian patients; 23 males (46%) and 27 females (54%), all suffering from MI which was confirmed by ECG changes (ST segment elevation) and elevation of cardiac enzymes (CK-MB and troponin). All patients were recruited from the Cardiology Department at Alexandria Main University Hospital and their ages ranged between 32-65 years with a mean of 48 years. Patients with inflammatory or liver diseases were excluded to eliminate the relationship between this gene polymorphism and diseases other than MI. Fifty healthy individuals, 25 males (50%) and 25 females (50%), whose ages ranged between 30-70 years with a mean of 45 years, were included as a control group. They had no history of hypertension, DM, atherosclerosis or cancer. Full history was taken from all participants; including smoking habits, physical activity, alcohol consumption, drug history and medical history for hypertension and DM. Also, supine blood pressure was measured for all participants. All subjects signed a written informed consent before enrollment in the study. Table 2. Clinical Characteristics of the two studied groups. Lipid Profile Genotype CC (n= 36) CT (n= 12) TT (n=2) TG (mg/dl) Mean ±SD. 191.44 ± 93.18 188.92 ± 97.42 116.0 ± 11.31 Cholesterol (mg/dl) Mean ±SD. 224.92 ± 77.55 240.0 ± 57.18 213.50 ± 68.59 LDL (mg/dl) Mean ±SD. 149.31 ± 63.09 163.25 ± 61.73 141.0 ± 74.95 HDL (mg/dl) Mean ±SD. 37.75 ± 15.13 37.17 ± 9.59 49.0 ± 4.24 Test of sig. p: p value for comparing between the three genotype KW: Kruskal Wallis test F: F test (ANOVA) *: Statistically significant at p ≤ 0.05 The study protocol conforms to the ethical guidelines of the 1975 Declaration of Helsinki and has obtained the approval of the Medical Ethics Committee of the Faculty of Medicine, Alexandria University Routine Laboratory Investigations: Three milliliters of whole blood were collected from every subject by aseptic veni-puncture in a plain red-topped vacutainer, left to clot slowly at room temperature for 15-30 minutes. The clot was removed by centrifugation at 1000-1200 g for 10 minutes, then the serum was used for measurement of lipid profile (triglycerides, total cholesterol, LDL and HDL), cardiac enzymes (CK- total, CK-MB, troponin, LDH, AST), hsCRP and fasting blood glucose. All parameters were measured by chemistry auto-analyzer Dimension RxL Max (Siemens Health Care Diagnostics, USA). Genomic Analysis for Detection of Lp-PLA2 A379V (rs 1051931) Gene Polymorphism by 5` Nuclease Allele Discrimination Assay using Real-Time PCR: 1- DNA Extraction: Another 2 milliliters of whole blood were aseptically drawn into lavender-topped EDTA vacutainer. Genomic DNA was extracted from EDTA whole blood samples, using QIAGEN total DNA purification kit (QIAamp DNA blood mini kit, QIAGEN, Germany, cat. no. 51104) according to the manufacturer’s instructions. The DNA samples were stored at -20°C until use. 2- 5´Nuclease Allele Discrimination Assay using Real-Time PCR: Ready-made “TaqMan SNP Genotyping Assay” (Assay ID C_2032800_20, catalog # 4351379, Applied Biosystems, USA) was used to detect Lp-PLA2 A379V SNP (rs1051931). In Lp-PLA2 A379V polymorphism (46672943 C > T), alanine (ACG) is replaced with valine (ATG), with the C allele being the major allele (coding for alanine) and the T allele being the minor one (coding for valine). This assay kit contains primer/ probe mixes (40X); 2 unlabeled sequence-specific forward and reverse primers to amplify the sequence of interest harboring the polymorphism and 2 labeled TaqMan minor groove binder (MGB) probes for detecting both the major C and the minor T alleles. The first probe, labeled with FAM (green fluorescence) as the 154 Original articles Table 3. Relation between the different genotypes and patients’ cardiac enzyme levels. Cardiac Enzymes CK (U/L) Mean ±SD. CK-MB (ng/ml) Mean ±SD. TnI (ng/ml) Mean ±SD. AST (U/L) Mean ±SD. LDH (U/L) Mean ±SD. Genotype CC CT (n= 36) (n= 12) 1629.94 ± 1435.48 1929.67 ± 1296.0 Test of sig. TT (n=2) 444.50 ± 518.31 149.95 ± 130.17 222.49 ± 302.60 42.40 ± 57.28 63.03 ± 70.99 68.12 ± 89.58 4.86 ± 0.40 323.81 ± 255.50 353.0 ± 315.25 164.0 ± 193.75 1213.61 ± 818.83 1555.08 ± 1330.47 365.50 ± 74.25 KWp = 0.128 p: p value for comparing between the three genotypes. KW: Kruskal Wallis test *: Statistically significant at p ≤ 0.05 Table 4. Relation between the different genotypes and patients’ glucose and hs-CRP levels. Genotype Parameter CC (n= 36) CT (n= 12) TT (n=2) Genotype 112.72 ± 37.58 108.83 ± 15.99 444.50 ± 518.31 hs-CRP (mg/L) Mean ±SD. 161.93 ± 144.53 90.02 ± 143.96 MWp1 0.100 MWp2 p: p value for comparing between the three genotypes p1 : p value for comparing between CC with each of CT and TT p2 : p value for comparing between AG and AA MC: Monte Carlo test FE: Fisher Exact test KW: Kruskal Wallis test MW: Mann Whitney test *: Statistically significant at p ≤ 0.05 reporter dye at the 5’ end, detects the major C allele, present in alanine (ACG). (AGCTTTGTTGCTAAGATCAATAGC TGC ATTTGAATCTATGTCTCCCTTTAA). The second probe, labeled with VIC (yellow fluorescence) as the reporter dye at the 5’ end, detects the minor T allele, present in valine (ATG). (AGCTTTGTTGCTAAGATCAATAGC TAC ATTTGAATCTATGTCTCCCTTTAA). The PCR reaction mix was prepared. This 5´nuclease allele discrimination assay, using real-time PCR, was used to detect this genetic variant using the following thermal profile: holding at 95°C for 10 minutes followed by 40 cycles of denaturation (92°C for 15 seconds) and annealing/extension (60°C for 1 minute) in the Rotor Gene thermal cycler machine (serial no R0211172). A no template control (NTC) containing nucleasefree water, instead of DNA, was included in each run to exclude contamination. The fluorescence profile of each sample was measured by the Rotor Gene software which plots a graphic presentation of the fluorescence against the number of cycles. The plotted fluorescence signals indicate which alleles are in each sample. The threshold cycle (Ct): is the cycle at which the instrument can distinguish the amplification generated fluorescence as being above the background signal. Positive cases are 4.86 ± 0.40 0.049* 0.100 Test of sig. | March 2014 - Issue 3 those with a Ct before cycle 40, while cases in whom no Ct was detected were considered negative. Amplification plot curve for A379 (FAM labeled) was constructed (Figure1) and another for 379V (VIC labeled). 3- Statistical Analysis of the Data (20) Data were fed to the computer and analyzed using IBM SPSS software package version 20.0. (21) Qualitative data were described using number and percent. Quantitative data were described using mean and standard deviation, median, minimum and maximum. Comparison between different groups regarding categorical variables was tested using Chisquare test. When more than 20% of the cells have expected count less than 5, correction for chi-square was conducted using Fisher’s Exact test or Monte Carlo correction. The distributions of quantitative variables were tested for normality using Kolmogorov-Smirnov test, Shapiro-Wilk test and D’Agstino test, also Histogram and QQ plot were used for vision test. If it reveals normal data distribution, parametric tests were applied. If the data were abnormally distributed, nonparametric tests were used. For normally distributed data, comparison between two independent populations was done using independent t-test while more than two populations were analyzed F-test (ANOVA) to be used and Post Hoc test (Scheffe). For abnormally distributed data, comparison between two independent populations were done using Mann Whitney test while Kruskal Wallis test was used to compare between different groups. Significant test results are quoted as two-tailed probabilities. Significance of the obtained results was judged at the 5% level. Results Patients of both sexes were more often smokers, had a higher prevalence of hypertension, diabetes and a more unfavorable lipid profile compared with controls. The inflammatory marker, hs-CRP, was markedly increased in patients compared with controls. Also, CK-total, CK-MB, troponin, AST and LDH were markedly increased in patients compared with controls. (Table 1) Regarding the different PLA2G7 A379V genotype distributions between the 2 studied groups, we found that homozygous CC genotype had the highest frequency among patients (72%) compared with controls (46%), while we found that heterozygous CT genotype had the highest frequency among controls (46%) compared with patients (24%) with a statistically significant difference (p=0.033). (Figure 1) March 2014 - Issue 3 | 155 Original articles Figure Figure Percentage Percentage PrecentagePrecentage 80 CK-MB, troponin, AST, LDH, (Table 3)]. However, a statistically significant difference (p=0.043) was found between different genotypes regarding hs-CRP, (Table 4). Validity of Hardy-Weinberg equilibrium regarding the 3 genotypes of the Lp-PLA2 A379V in all the studied population: As shown in table 5, the incidence of the C allele (p) = [(2X59) + 35]/ 200= 0.765 and the incidence of the T allele (q) = [35 + (2X6)]/ 200 =0.235. {p+q=1}. The observed and expected values were found nearly identical. This means that the Egyptian population is in Hardy-Weinberg equilibrium for the Lp-PLA2 A379V gene variant. Patients 70 80 Controls 60 70 Patients Controls 50 60 40 50 30 40 20 30 10 20 0 10 CC TC TT Genotype Genotype 0 CC TC TT Genotype Genotype Percentage Percentage PrecentagePrecentage 90 Patients 80 90 70 80 60 70 50 60 40 50 30 40 20 30 10 20 0 10 Validity of Hardy-Weinberg equilibrium regarding the 3 genotypes of the Lp-PLA2 A379V gene among patients: As shown in table 6, the incidence of the C allele (p) = [(2X36) + 12] / 100 = 0.84 and the incidence of the T allele (q) = [12 + (2X2)]/ 100 = 0.16. {p+q=1}. The observed and expected values were found to be quite similar denoting that Egyptian patients having MI are in Hardy-Weinberg equilibrium for the Lp-PLA2 A379V gene variant. Controls Patients Controls C T Allele Allele 0 C T Allele Allele Validity of Hardy-Weinberg equilibrium regarding the 3 of the Lp-PLA2 A379V gene among healthy subjects: genotype As shown in table 7, the incidence of the C allele (p) = [(2X23) 24 + 23]/ 100 = 0.69 and the incidence of the T allele (q) = [23 + 24 (2X4)]/ 100 =0.31. {p+q=1}. The observed and expected values were found to be quite similar meaning that the controls are also in Hardy-Weinberg equilibrium for the Lp-PLA2 A379V gene variant. Figure 1: Comparison between the the two groups groups according to different genotype genotypes Comparison between twostudied studied distributions (p=0.033) and allele frequencies (p=0.012). Figure 1: according toFigure different genotype distributions (p=0.033) and 1: Comparison between the two studied groups according to different distributions (p=0.033) and allele frequencies (p=0.012). allele frequencies (p=0.012). Table 5. The observed and expected values of the Lp-PLA2 A379V genotype frequencies among the whole studied population, among patients and among controls. Genotype Observed Expected Difference CC 59 58.5 (p2X 100) 0.5 TC 35 35.9 (2pqX 100) 0.9 TT 6 5.5 In all subjects (q2X 100) 0.5 Total =100 Among patients CC 36 35.28 (p2X 50) 0.72 TC 12 13.44 TT 2 1.28 (q2X 50) (2pqX50) 1.44 0.72 (p2X 50) 0.8 (2pqX50) 1.6 Total =50 Among controls CC 23 23.8 TC 23 21.4 TT 4 4.8 (q2X 50) 0.8 Total =50 Discussion In our study, we found that homozygous CC genotype, coding for alanine at position 379 of Lp-PLA2 protein, had the highest frequency among patients compared with controls and was associated with increased incidence of MI, while we found that heterozygous CT genotype had the highest frequency among controls compared with patients and was associated with decreased incidence of MI, with a statistically significant difference (p=0.033) between patients and controls. The allelic frequencies for Lp-PLA2 A379V (46672943 C > T) SNP in our studied population did not show any deviation from HardyWeinberg equilibrium. Also, we found that the major “C” allele, coding for alanine, had the highest frequency among patients compared with controls and was associated with increased incidence of MI, while we found that the minor “T” allele, coding for valine, had the highest frequency among controls compared with patients and was associated with decreased incidence of MI. So, there is a significant difference (p=0.012) between patients and controls with predominance of C allele in patients and T allele in controls. Also, we found that the major “C” allele had the highest frequency among patients (84%) compared with controls (69%), while we found that the minor “T” allele had the highest frequency among controls (31%) compared with patients (16%) with a significant difference (p=0.012) between both groups with predominance of the “C” allele, coding for alanine, among patients and the minor “T” allele, coding for valine among controls. (Figure 1) In agreement with our study, Ninio E et al., 22 Abuzeid AM et al., 23 and Ling LC et al., 24 reported that the homozygous (TT) and heterozygous (CT) forms of 379V polymorphism were less frequent in MI patients than in controls, suggesting that this allele might be protective against the development of CAD while A379 variant was more prevalent among patients. Our results showed no difference in genotype distributions or allele frequencies among patients regarding their sex. Also, there was no statistically significant difference between the different genotypes regarding the patients’ lipid profile [TG, cholesterol, LDL, HDL, (Table 2)] or cardiac enzyme levels [CK, In contrast to our study, Liu PY et al.,25 and Casas JP et al.,16 reported that 379V gene variant was more prevalent in Taiwanese patients who presented with acute coronary syndrome (ACS) than in controls. Also, Sutton et al.,26 reported that 379V polymorphism was more prevalent among MI patients 156 than controls with a significant difference (p=0.002) which was against our results. This dissimilarity in results may be due to differences in ethnic groups, sample size and selection criteria of patients and controls. However, Wotton P et al.,27 reported absence of any significant association between this polymorphism and coronary heart disease complications. In a Chinese study, the risk of MI was found to be higher among cardiovascular patients harboring the minor T allele compared with the major C allele.17 In a Taiwanese study, the T allele (379V polymorphism) was associated with lower Lp-PLA2 activity and increased risk of MI.18 In contrast, a study of European Caucasians revealed that T allele was associated with reduced risk of MI.23 But other studies on European Caucasians reported no association with CHD risk.16, 28 In South Koreans, a similar lack of association between A379V and CVD was reported.19 Personalized medicine is of growing interest, with a number of pharmacogenetic drug examples, like clopedogril and warfarin, where genetic variants influence the rate of drug metabolism and efficacy.29 Among the limitations of our study are the relatively small sample size and the inability to correlate the studied polymorphism with enzyme activity or mass. It could be concluded from this study that the Lp-PLA2 A379V polymorphism was less frequent in Egyptians having MI than in healthy controls and was associated with a lower risk of cardiovascular events, suggesting that the minor T allele, coding for valine, might be protective against the development of MI while A379 variant was more prevalent among patients than controls, suggesting that the major “C” allele, coding for alanine could be used a risk factor for the development of MI. Moreover, there was no significant correlation between A379 and lipid profile, suggesting that the action of this enzyme is independent of other traditional risk factors. So, patients harboring the Lp-PLA2 A379 gene variant, or the C allele, might be candidates for specific Lp-PLA2 enzyme inhibitors, as darapladib. Correspondence to: Dr. Doreen Nazeih Assaad Younan Mobile: +2 012 222 82681 Tel: +2 03 582 2492 Fax: +2 03 582 4124 Postal Address: 596 Horreya Ave., Zizinya, Apt# 105, Alexandria, Egypt. 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