Journal of Renin-Angiotensin-Aldosterone System http://jra.sagepub.com/ Angiotensin-converting enzyme gene insertion/deletion polymorphism in Egyptian patients with myocardial infarction Ahmad Settin, Rizk ElBaz, Amr Abbas, Ayman Abd-Al-Samad and Ahmed Noaman Journal of Renin-Angiotensin-Aldosterone System 2009 10: 96 DOI: 10.1177/1470320309105198 The online version of this article can be found at: http://jra.sagepub.com/content/10/2/96 Published by: http://www.sagepublications.com On behalf of: Frequently Asked Questions Additional services and information for Journal of Renin-Angiotensin-Aldosterone System can be found at: Email Alerts: http://jra.sagepub.com/cgi/alerts Subscriptions: http://jra.sagepub.com/subscriptions Reprints: http://www.sagepub.com/journalsReprints.nav Permissions: http://www.sagepub.com/journalsPermissions.nav Citations: http://jra.sagepub.com/content/10/2/96.refs.html >> Version of Record - Jun 5, 2009 What is This? Downloaded from jra.sagepub.com by guest on June 9, 2014 Paper Angiotensin-converting enzyme gene insertion/deletion polymorphism in Egyptian patients with myocardial infarction Ahmad Settin,* Rizk ElBaz,* Amr Abbas,† Ayman Abd-Al-Samad,# Ahmed Noaman^ Key words: angiotensinconverting enzyme, Egypt, gene polymorphism, ischaemic heart disease, myocardial infarction * Department of Genetics, Faculty of Medicine, Mansoura University, Mansoura, Egypt. † Department of Medical Physiology, Faculty of Medicine, Mansoura University, Mansoura, Egypt. # Department of Cardiology, Faculty of Medicine, Mansoura University, Mansoura, Egypt. ^ Department of Zoology, Faculty of Science, Mansoura University, Mansoura, Egypt. Correspondence to: Prof Dr Ahmad Settin Prof of Pediatrics and Genetics, Qassim University, PO Box 6655, Buraydah 51452, Qassim, Saudi Arabia. Tel: +966 6 380 0050/2523 Fax: +966 6 380 1228 Email: settin60@gmail. com Journal of the ReninAngiotensinAldosterone System (Including other Peptidergic systems) Abstract Introduction. This work aimed to test the association of the angiotensin-converting enzyme gene insertion/deletion (I/D) polymorphism with myocardial infarction. Subjects and methods. This study comprised 79 Egyptian myocardial infarction cases with a mean age of 54.4±9.9 years including 60 males and 19 females, plus 238 healthy unrelated individuals of nearly matched age and sex as a control group. For all subjects, DNA testing for the angiotensinconverting enzyme gene I/D polymorphism was done using PCR amplification for detection of both the D and I alleles followed by a second run PCR specific for the I allele for samples typed as DD in the first run. Results. Cases had a higher frequency of DD (29.1%) and ID (62.0%) genotypes than II (8.9%) genotype, with a higher frequency of D allele than I allele (64.4% vs. 33.6%). Compared to controls, cases had a significantly higher frequency of ID genotype (62.0% vs. 47.5%, p<0.05). This was more apparent among cases in the low risk group (p=0.002) than in the high risk group (p=0.041). Conclusion. The angiotensin-converting enzyme gene I/D polymorphism is probably a risk factor for ischaemic heart disease among Egyptian cases, particularly if integrated with other environmental and genetic risk factors. Introduction Coronary artery disease (CAD) is a multifactorial disease influenced by environmental and genetic factors. Family history of premature CAD in addition to other risk factors, such as smoking, obesity, diabetes, and dyslipidaemia, are all interactive factors contributing to the occurrence of the disease.1 Although the role of these environmental factors in the development of myocardial infarction (MI) has been clearly established, the role of nonconventional risk factors remains undefined. In the last few years, great interest has been focused on genetic factors with the intention of finding common markers that could identify a subgroup of patients at higher risk of death or with a worse prognosis in which new therapeutic timings and interventions could be tested.2 In an enzymatic cascade, angiotensinogen is cleaved by renin to produce angiotensin I, which is further converted to the bioactive octapeptide angiotensin II (Ang II) through the action of angiotensin-converting enzyme (ACE), a membrane- bound, zinc metalloendopeptidase involved in the metabolism of many small peptides. ACE and angiotensinogen play an important role in blood pressure and blood volume homeostasis.3 Thus, it is not surprising that the genes coding for this system are being investigated in relation with MI.2 Concentrations of plasma and tissue ACE are determined by the ACE gene located on chro mosome 17q23. This gene manifests a 287-bp repeated Alu sequence insertion (I) or deletion (D) polymorphism in intron 16.4 The homozygous DD genotype, which is associated with a two- to threefold increase in levels of ACE, may cause a variety of adverse cardiovascular effects.5 The increased risk of MI associated with the ACE D allele is graded, with low risk for ACE II, intermediate risk for ACE ID, and high risk for ACE DD genotypes, which suggests codominant inheritance.6,7 It is hypothesised that in subjects with the ACE DD genotype, higher levels of ACE may contribute to coronary thrombogenesis.8 Recently, it has been reported that changes in pulse pressure and cardiac remodelling were detected among cases of MI having DD and ID genotypes rather than the II genotype.9,10 Month June 2009 2009 Volume X 10 Number X 2 SAGE Publications 2009 Los Angeles, London, New Delhi and Singapore 10.1177/1470320309105198 96 96 Downloaded from jra.sagepub.com by guest on June 9, 2014 Paper Reports on the associations of the ACE gene I/D polymorphism, in addition to being controversial in various Arabic and Mediterranean countries, are relatively lacking among Egyptian patients with MI. Therefore, this work was planned to investigate this association among cases from the Nile Delta region of Egypt. Subjects and methods Subjects This study comprised 79 cases with MI taken randomly from those admitted in the Intensive Care Unit of the Cardiology Department, Internal Medicine Specialised Hospital, Mansoura University, Mansoura, Egypt. Their mean age was 54.4±9.9 years with an age range of 25–75 years. There were 60 (75.9%) males and 19 (24.1%) females. Of them, 23 (29.1%) were smokers, 21 (26.6%) had a positive family history of MI, 25 (31.6%) were diabetic, and 16 (20.3%) were hyperlipidaemic. Regarding risk factors, cases were classified into high risk group with two or more risk factors and low risk group with no or only one risk factor. For comparison, 238 healthy individuals of nearly matched age and sex and with no history of any cardiac diseases or evident cardiovascular risk factors were taken as a control group. Informed consent was taken from all subjects in addition to an approval from the University Ethical and Scientific Committees. Journal of the ReninAngiotensinAldosterone System (Including other Peptidergic systems) June 2009 Volume 10 Number 2 Determination of the ACE gene I/D polymorphism From each subject, 3 ml venous blood was collected in a polyethylene tube containing EDTA solution as an anticoagulant and kept frozen until used for DNA extraction using the Generation DNA Purification Capture Column Kit (Gentra Systems Inc., MN, USA). PCR amplification was done with the respective fragments from intron 16 of the ACE gene according to the method described by Lindpaintner et al.11 Briefly, 20 µl of a PCR master mix containing 1 mM primers, 200 mM deoxynucleotide triphosphates, 1.3 mM magnesium chloride, 50 mM potassium chloride, 10 mM TRIS-hydrochloric acid (pH 8.4 at 25ºC), 0.1% Triton X-100, and 0.35 unit of Taq polymerase was added. Optimised primer pair was used to amplify the D and I alleles, resulting in 319-bp and 597-bp amplicons, respectively (5’-GCC CTG CAG GTG TCT GCA GCA TGT-3’ and 5’-GGA TGG CTC TCC CCG CCT TGT CTC-3’). The thermocycling profile (Genius; Techne, UK) consisted of denaturation at 94ºC for 30 seconds, annealing at 56ºC for 45 seconds, and extension at 72ºC for 2 minutes, repeated for Figure 1 Panel A (PCR products, first run) shows positive bands for both the D (319 bp) and I (597 bp) alleles, i.e. ID genotype in lanes 3, 5, 7, and 8, and only one band for the D allele, i.e. DD genotype in lanes 2, 4, and 6. Panel B (PCR products, second run) shows a positive band for the I allele (335 bp) in lanes 2, 3, 5, 7, and 8. This denotes that the sample in lane 2 is actually an ID genotype. D = deletion; I = insertion. 35 cycles, followed by a final extension at 72ºC for 7 minutes. The amplification products of the D and I alleles were identified by electrophoresis on a 2% agarose gel and visualised on a 300-nm ultraviolet transilluminator. Because the D allele in heterozygous samples is preferentially amplified, each sample found to have the DD genotype was subjected to a second, independent PCR amplification with a primer pair that recognises an insertion-specific sequence (5’-TGG GAC CAC AGC GCC CGC CAC TAC-3’ and 5’-TCG CCA GCC CTC CCA TGC CCA TAA-3’), with identical PCR conditions except for an annealing temperature of 67ºC. The reaction yields a 335-bp amplicon only in the presence of an I allele, and no product in samples homozygous for DD (figure 1). Statistical analyses Data were processed and analysed using the Statistical Package of Social Science (SPSS, version 10.0). The frequency of studied allelic polymorphisms among cases was compared to that of controls and tested for positive association using chi-square (c2), Fisher’s exact tests and odds ratio (OR) with 95% confidence interval (95% CI). A minimum level of significance was considered if p was ≤ 0.05. Furthermore, the distribution of alleles in studied groups was tested for fitting to the Hardy-Weinberg equilibrium assuring no SAGE Publications 2009 Los Angeles, London, New Delhi and Singapore Downloaded from jra.sagepub.com by guest on June 9, 2014 97 Paper Table 1 Distribution of the ACE gene I/D polymorphism in a sample of patients with acute MI compared to controls. Cases (n=79) Controls (n=238) Fisher p OR (95% CI) Genotypes DD 23 (29.1%) 113 (47.5%) 0.0057** ID 49 (62.0%) 113 (47.5%) 0.027* II 7 (8.9%) 12 (5.0%) 0.27 Alleles (n=158) (n=476) D 95 (64.36%) 339 (71.20%) 0.01* I 63 (33.64%) 137 (28.78%) 0.01* 0.45 (0.26-0.79) 1.81 (1.07-3.04) 1.80 (0.69-4.83) 0.61 (0.42-0.88) 1.64 (1.13-2.39) Key: * p<0.05 and ** p<0.01 (significance level testing each group versus controls). ACE = angiotensin-converting enzyme; D = deletion; I = insertion; MI = myocardial infarction; OR (95% CI) = odds ratio (95% confidence interval). Table 2 Distribution of ACE I/D genotypes among low risk and high risk subjects compared to controls using c2 test of trend. Studied groups Controls (n=238) Total cases (n=79) Low risk cases (n=30)# High risk cases (n=44) ACE genotypes DD n (%) ID n (%) 113 (47.5) 23 (29.1) 6 (20.0) 13 (29.5) 113 (47.5) 49 (62.0) 20 (66.7) 28 (63.6) II n (%) c2 p 12 (5.0) 7 (8.9) 8.28 0.004** 4 (13.3) 9.58 0.002** 3 (6.8) 4.18 0.041* Key: # Five cases dropped from the analysis with non-informative history to be assigned to particular risk group. * p<0.05 and ** p<0.01 (significance level testing each group versus controls). ACE = angiotensin-converting enzyme; D = deletion; I = insertion. significant difference between observed and expected frequencies using the c2 test. Results The distribution of the ACE gene I/D poly morphism in studied cases of MI and controls (table 1) showed that cases had a higher frequency of DD and ID genotypes than II genotype (29.1%, 62.0%, and 8.9%, respectively). Compared to controls, cases had a significantly lower frequency of the DD genotype (29.1% vs. 47.5%, p<0.05), but with a significantly higher frequency of the ID genotype (62.0% vs. 47.5%, p<0.05). On the other hand, cases showed a higher frequency of the II genotype than controls, but this was statistically non-significant (8.9% vs. 5.0%, p>0.05). Journal of the ReninAngiotensinAldosterone System (Including other Peptidergic systems) June 2009 Volume 10 Number 2 using c2 test of trend (table 2) showed that low risk cases had a higher frequency of ID (66.7%) than controls (47.5%), which was statistically significant (p=0.002). The same was observed in the high risk group, but with a lower level of significance (63.6% vs. 47.5%, p=0.041). On the other hand, comparing ACE I/D genotype frequencies in cases-subgroups related to each single risk factor, such as age of onset, sex, hyperlipidaemia, smoking, positive family history, diabetes, and obesity, revealed no significant difference (data not shown). Regarding allele frequency, the D allele was the predominant allele in all cases (64.4% vs. 33.6% for the I allele). However, when compared to controls, the D allele frequency was found to be significantly lower among cases than controls (64.4% vs. 71.2%, p<0.05). Discussion CAD continues to be the main cause of death in developed countries. During the last decade, there has been a growing interest in the study of the ACE gene I/D polymorphism as a potential risk factor for conditions like MI.12 However, despite the large number of studies with different designs and populations, the role of the ACE gene I/D polymorphism on MI is still controversial.13 Comparison of the ACE I/D genotypes among low risk and high risk subjects with controls This study of the ACE gene I/D polymorphism among Egyptian controls showed an equal SAGE Publications 2009 Los Angeles, London, New Delhi and Singapore 98 Downloaded from jra.sagepub.com by guest on June 9, 2014 Paper frequency of ID and DD genotypes that was relatively high (47.4% each), with a lower frequency of the II genotype. The same finding has also been reported in Saudi Arabia (47.1% for DD and 41.0% for ID) and in Italy (44.5% and 42.8%, respectively).14,15 However, almost all other studies showed a higher frequency of the ID genotype among their control groups (ID genotype frequency was one and a half to two times as much as the DD). Examples include studies in Japan,16 South Asia,17 Chile,18 Australia,19 France,20 USA,11 and Germany.21 Regarding the ACE gene I/D polymorphism among Egyptian MI cases, this study showed a higher frequency of the ID than the DD genotype, with a lower frequency of the II genotype. The frequency of the D allele was also higher than the frequency of the I allele. This is in agreement with most studies, e.g. the ones in Colombia,22 India,23 Saudi Arabia,14 France,24 Poland,25 and Italy.2 However, compared to controls, this study showed a significantly higher frequency of the ID genotype among MI cases, but with a significantly lower frequency of the DD genotype. The same finding was reported by Dzimiri et al. in Saudi Arabia.14 Furthermore, in Turkey, Acarturk et al. reported that the ID genotype was the most frequent in all subjects who underwent diagnostic coronary angiography, although in cases found to have CAD, the DD genotype was higher compared to the controls.26 In Poland, Zak et al. reported that the D allele carriers (DD + ID genotypes) were more frequent in the CAD patients compared to the control group, whereas the familial CAD risk group showed the highest frequency of the ID genotype.25 Journal of the ReninAngiotensinAldosterone System (Including other Peptidergic systems) June 2009 Volume 10 Number 2 The lower frequency of the DD genotype observed among our cases may be due to the fact that our sample was restricted to new MI cases in the Intensive Care Unit rather than ambulant cases under maintenance treatment. Here we can speculate that the patients with DD genotype can manifest early with alarming signs of hypertension or angina and receive treatment as ambulatory cases. On the other hand, cases with ID genotype, who may have an intermediate elevation of ACE, may be neglected until coming under the effect of other risk factors that lead together to the outcome of acute MI. This is manifested by having 63% of our cases with two or more risk factors. In this respect, we recommend a bigger sample including both acute cases with MI in addition to ambulatory cases with ischaemic heart disease; considering at the same time the various risk factors pertaining to the disease. Studies stressing the possible role of ACE DD genotype as a risk factor in CAD related to disease prognosis in terms of disease onset and mortality include ones in France,6 New Zealand,27 UK,28 Colombia,22 Spain,29,30 and South Africa.4 However, in the Rotterdam Study (The Netherlands), Sayed-Tabatabaei et al. reported no association between the ACE gene I/D polymorphism and MI, although they observed an increased risk of cardiovascular mortality for carriers of the D allele among smokers in younger subjects and this diminished at later ages.31 On the other hand, Agrawal et al. in India23 and Franco et al. in Italy2 observed that the DD genotype was slightly more frequent in patients as compared to controls; however, the differences were not significant. From this study, we conclude that the ACE gene I/D polymorphism probably has some association with MI among Egyptian patients, particularly if integrated with other environmental and genetic risk factors. References 1. 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Sayed-Tabatabaei FA, Schut AFC, Arias Va´squez A et al. Angiotensin converting enzyme gene polymorphism and cardiovascular morbidity and mortality: the Rotterdam Study. J Med Genet 2005;42(1):26-30. Journal of the ReninAngiotensinAldosterone System (Including other Peptidergic systems) June 2009 Volume 10 Number 2 SAGE Publications 2009 Los Angeles, London, New Delhi and Singapore 100 Downloaded from jra.sagepub.com by guest on June 9, 2014
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