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The C4280A (rs5705) gene polymorphism of the renin (REN) gene is associated with risk of developing coronary artery disease, but not with restenosis after coronary stenting
Experimental and Molecular Pathology 99 (2015) 128–132
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The C4280A (rs5705) gene polymorphism of the renin (REN) gene is associated with risk of developing coronary artery disease, but not with restenosis after coronary stenting Jose Manuel Fragoso a, Edith Alvarez-León a, Hilda Delgadillo-Rodríguez b, Marva Arellano-González a, Filogonio Caín López-Pacheco a, David Cruz-Robles a, Marco Antonio Peña-Duque b, Oscar Pérez-Méndez a, Marco Antonio Martínez-Ríos b, Gilberto Vargas-Alarcón a,⁎ a b
Department of Molecular Biology, Instituto Nacional de Cardiología Ignacio Chávez, Mexico City, Mexico Department of Interventional Cardiology, Instituto Nacional de Cardiología Ignacio Chávez, Mexico City, Mexico
a r t i c l e
i n f o
Article history: Received 25 May 2015 Accepted 18 June 2015 Available online 20 June 2015 Keywords: Coronary artery disease Polymorphisms Renin–angiotensin-system (RAS) Restenosis
a b s t r a c t The aim of the present study was to evaluate the role of AGT and REN gene polymorphisms as susceptibility markers for coronary artery disease (CAD) and/or restenosis after coronary stent placement in a group of Mexican patients. Five polymorphisms of the AGT (rs699, rs4762, rs5051, rs5049, rs5046) and two of the REN (rs5707, rs5705) genes were analyzed by 5′ exonuclease TaqMan genotyping assays in 240 patients with CAD who underwent coronary artery stenting (76 with restenosis and 164 without restenosis). A group of 610 individuals without clinical and familial antecedents of cardiovascular diseases were included as controls. The results showed that the distribution of AGT and REN polymorphisms were similar in patients with and without restenosis. However, when the whole group of patients (with and without restenosis) was compared to healthy controls, under co-dominant, dominant, heterozygous and additive models, the REN A4280C (rs5705) polymorphism was associated with increased risk of CAD (OR = 1.76, PCo-dom = 0.006, OR = 1.81, PDom = 0.001, OR = 1.75, PHet = 0.003 and OR = 1.59, PAdd = 0.003, respectively). All models were adjusted for age, gender, diabetes, dyslipidemia, hypertension and smoking habit. The TC haplotype of the REN gene was associated with increased risk of CAD (OR = 1.53, P = 0.014). The data suggest that the REN C4280A (rs5705) polymorphism plays an important role in the risk of developing CAD with the highest risk for C allele, but do not support its role as a risk factor for developing restenosis after coronary stenting. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Coronary artery disease (CAD) is a complex, multifactorial disease, influenced by pathophysiologic conditions as well as by genetic and environmental factors. The treatment strategies for this disease are coronary artery bypass grafting, percutaneous transluminal coronary angioplasty (PTCA), and intracoronary stent. However, after PTCA, restenosis occurs in about 30 to 32% of patients and after intracoronary stent placement in 12 to 32% of patients (Kuchulakanti et al., 2006; Lee et al., 2011; Hamasaki and Tei, 2011; Latib et al., 2011). The restenosis is the arterial wall's healing response to mechanical injury and comprises two main processes, neointimal hyperplasia (i.e., smooth muscle migration/proliferation, extracellular matrix deposition) and vessel remodeling (Costa and Simon, 2005). In the literature, there has been evidence
⁎ Corresponding author at: Department of Molecular Biology, Instituto Nacional de Cardiología “Ignacio Chávez”, Juan Badiano No. 1, Tlalpan 14080, Mexico D.F., Mexico. E-mail address: [email protected] (G. Vargas-Alarcón).
http://dx.doi.org/10.1016/j.yexmp.2015.06.012 0014-4800/© 2015 Elsevier Inc. All rights reserved.
that the activation of the renin–angiotensin system (RAS) induces the production of pro-inflammatory cytokines, proliferation and vasoconstriction on the vasculature, and in consequence can contribute to initiation and progression of atherosclerosis (Chamarthi et al., 2011; Mehta and Griendling, 2007; van Thiel et al., 2015; Pacurari et al., 2014). In addition, RAS has been implicated in the pathogenesis of neointimal hyperplasia, and a role for angiotensin II in the migration and proliferation of vascular smooth muscle cells in restenosis has been reported (Pacurari et al., 2014; Valente et al., 2012; Yao et al., 2012). Renin and angiotensinogen are encoded by the REN and AGT genes located in chromosome 1 position q32 and q42, respectively (Qin et al., 1993; Gaillard-Sanchez et al., 1990). The renin is a key enzyme produced in the juxtaglomerular apparatus of the kidney and in many other cells that cleave angiotensinogen to produce angiotensin I, the first step in generation of the angiotensin II through angiotensinconverting enzyme. In addition, the renin at the cellular level is amplified by a receptor-mediated binding process, which leads to a further increase in angiotensin II generation in many tissues (Nguyen et al., 2002; Paul et al., 2006). On the other hand, the angiotensinogen is a key
J.M. Fragoso et al. / Experimental and Molecular Pathology 99 (2015) 128–132
component of RAS, it is cleaved by renin to produce angiotensin I. AGT gene presents five important single nucleotide polymorphisms (SNPs), three in the promoter region [− 6 ANG (rs5051), − 217 GNA (rs5049) and − 532 CNT (rs5046)] and two in the exon 2 [C4072T (rs699) and C3889T (rs4762)]. These SNPs have been associated with risk of developing hypertension, acute myocardial infarction, vascular disease, coronary artery disease, atherosclerosis, and coronary artery bypass graft in several populations (Inoue et al., 1997; Karayannis et al., 2010; Ragia et al., 2010; Mehri et al., 2011; Dzielinska et al., 2011; Abd El-Aziz et al., 2012; Al-Najai et al., 2013; Brugts et al., 2011). On the other hand, the REN gen presents two relevant SNPs, one in the intron 4 [G5795T (rs5707)] and one in the exon 2 [C4280A (rs5705)]. The rs5707 has been associated with acute pancreatitis (Skipworth et al., 2015), with blood pressure levels and with the risk of having hypertension (Mansego et al., 2008). Considering the prominent role of the angiotensinogen and renin as key component of RAS, and the effect of RAS in the cardiovascular system, the aim of this study was to analyze whether AGT and REN gene polymorphisms are associated with risk of developing CAD and/or restenosis after coronary stent placement in a group of Mexican patients. 2. Materials and methods 2.1. Patients and controls The study included 240 Mexican Mestizo patients with symptomatic coronary artery disease (CAD) who underwent coronary stent implantation at our institution during the period between October 2008 and October 2014 and went to follow-up coronary angiography because of symptoms of ischemia documented in a myocardial perfusion imaging test. Basal and procedure coronary angiographies were analyzed for angiographic predictors of restenosis, and follow-up angiography was performed to screen for binary restenosis. Using a N50% stenosis at follow-up (50% reduction in the luminal diameter of the stenosis compared with the coronary angiography findings immediately following angioplasty) as the criterion to define restenosis, there were 76 patients with restenosis and 164 without restenosis. Also, neither a group of 610 healthy controls individuals with neither symptoms nor previous diagnosis of cardiovascular or systemic disease was studied as the control group. All subjects were ethnically matched, and we considered as Mexican Mestizos only those individuals who for three generations, including their own, had been born in Mexico. The Institutional Ethics and Research Committee approved the study, and all subjects signed informed consent.
on a 7900HT Fast Real-Time PCR system according to manufacturer's instructions (Applied Biosystems, foster City, USA) (Table 1).
2.3. Statistical analysis We used SPSS version 18.0 (SPSS, Chicago, IL) statistical package. Means, standard deviations and frequencies of baseline characteristics were calculated. Comparison of numerical variables, such as age and body mass index, between both groups was performed with Mann Whitney U test; data are presented as median (25th–75th percentile). Categorical variables were analyzed with Chi2 tests or Fisher's exact test, as needed, and presented as absolute frequencies and proportions. Logistic regression analysis was performed to look for associations of polymorphisms with CAD using the following models: codominant (major allele homozygotes vs. heterozygotes and major allele homozygotes vs. minor allele homozygotes), dominant (major allele homozygotes vs. heterozygotes + minor allele homozygotes), recessive (major allele homozygotes + heterozygotes vs. minor allele homozygotes), heterozygous (homozygote for the minor allele + homozygote for the major allele vs heterozygote) and log-additive (major allele homozygotes vs. heterozygotes vs. minor allele homozygotes). The inheritance models were adjusted for gender, age, hypertension, dyslipidemia, type II diabetes mellitus, and smoking habit. SNPstats (http://bioinfo.iconcologia.net/custom.php) software for Windows® were used to analyze the genetic frequencies and to evaluate the linkage disequilibrium (LD, D′) among polymorphisms, as well as to construct haplotypes. Hardy–Weinberg equilibrium was evaluated by Chi2 test.
2.4. Functional prediction analysis Two in silico programs were used [FastSNP (http://fastsnp.ibms. sinica.edu.tw) (Yuan et al., 2006) and SNP Function Prediction (http:// snpinfo.niehs.nih.gov/snpfunc.htm)] to predict the potential effect of the REN polymorphisms. These programs analyzes the location of the SNP (eg. 5′ upstream, 3′ untranslated region, intronic) and possible functional effects such as amino acid changes in protein structures, transcription factor binding sites in promoter or intronic enhancer regions, and alternative splicing regulation by disrupting exonic splicing enhancers or silencers.
Table 2 Clinical and angiographic characteristics of CAD patients with and without restenosis.
2.2. Genetic analysis The T4272C (rs699), C3889T (rs4762), −6 ANG (rs5051), −217 ANG (rs5049), −532 CNT (rs5046), G5795T (rs5707), and C4280A (rs5705) SNPs were genotyped using 5′ exonuclease TaqMan genotyping assays
Table 1 AGT and REN gene polymorphisms tested. Gene
Polymorphisma
dbSNPb
Chromosome position
Location in gene
AGT
C4272T (Met 235 Thr) C3889T (Thr 174 Met) A −6C G −217A C −532T G5795T A4280C (Thr68Thr)
rs699 rs4762 rs5051 rs5049 rs5046 rs5707 rs5705
1q42 1q42 1q42 1q42 1q42 1q32 1q32
Exon 2 Exon 2 5′ near region 5′ near region 5′ near region Intron 4 Exon 2
REN a b
Given name according to NCBI. SNP ID in database dbSNP.
129
Mena Blood pressurea (mm Hg) Type II diabetes mellitusa Hypercholesterolemiaa Smokinga Unstable anginaa Stable anginaa Statin therapya DESa BSMa Diameter smallera 2.5 mm Stent lengtha (mm) Bifurcationa
Agea (years) a
With restenosis (n(%))
Without restenosis (n(%))
P
61 (80) 48 (63)
129 (79) 91 (55)
NS NS
33 (43) 41 (54) 48 (63) 26 (34) 8 (10) 59 (78) 20 (26) 52 (68) 23 (30)
61 (37) 97 (59) 101 (62) 43 (26) 36 (22) 137 (83) 98 (59) 61 (37) 31 (19)
NS NS NS NS 0.03 NS b0.0001 b0.0001 0.04
31 (41) 19 (25) Median (percentile 25–75) 59.9 (54–67)
71 (43) 40 (24) Median (percentile 25–75) 58.6 (53–65)
NS NS
NS
(n(%)) number and proportion of subjects with the clinical and angiographic characteristics in both groups. Abbreviations: BMS = bare metal stent; DES = drug-eluting stent.
130
J.M. Fragoso et al. / Experimental and Molecular Pathology 99 (2015) 128–132
Table 3 Baseline clinical characteristics of the studied individuals (patients with CAD and healthy controls). Clinical characteristics
CAD patients (n(%)) Healthy controls (n(%)) P (n = 240) (n = 610)
Mena High blood pressure (mm Hg)a Type II diabetes mellitusa Dyslipidemiaa Smokinga
190 (79) 139 (58)
292 (48) 269 (44)
b0.001 0.003
94 (39) 138 (57) 149 (62) Median (percentile 25–75) 59 (53–66)
92 (15) 449 (74) 346 (57) Median (percentile 25–75) 56 (51–62)
b0.001 b0.001 NS
Age (years) a
NS
(n(%)) number and proportion of subjects with the clinical characteristic in both groups.
3. Results 3.1. Characteristics of the study population
observed. In this case, we estimated the risk according to five inheritance models adjusted for age, gender, diabetes, dyslipidemia, hypertension and smoking habit (Table 4). Under co-dominant, dominant, heterozygous and additive models, the C4280A (rs5705) polymorphism was associated with increased risk of developing CAD (OR = 1.76, 95% CI: 0.74– 4.20, PCo-dom = 0.006, OR = 1.81, 95% CI: 1.26–2.60, PDom = 0.001, OR = 1.75, 95% CI: 1.21–2.54, PHet = 0.003 and OR = 1.59, 95% CI: 1.17–2.15, PAdd = 0.003, respectively). 3.3. Linkage disequilibrium analysis The REN polymorphisms (rs5707 and rs5705) were in high linkage disequilibrium (D′ N 0.8 and r2 N 0.9) and 4 different haplotypes (TA, GA, TC and GC) were observed. The TC haplotype was significantly associated with increased risk of developing CAD (OR = 1.53, 95% CI = 1.11–2.10, P = 0.014) (Table 5). 3.4. Functional prediction
The clinical and angiographic characteristics of CAD patients with and without restenosis are shown on Table 2. There were no significant differences between patients with or without restenosis with regard to age, gender, type II diabetes, hypertension, dyslipidemia, unstable angina, statin therapy, stent length, and bifurcation. Nonetheless, patients who underwent coronary bare-metal stent (BSM) implantation develop more restenosis (68%) than those patients who underwent drug-eluting stent (DES) implantation (26%) (P b 0.0001). Also, those lesions treated with stents with a diameter b 2.5 mm presented more restenosis (P = 0.04). When we analyzed the baseline characteristics of the CAD patients and healthy controls included in the study, there were significant differences between patients with CAD and healthy controls with regard to gender, type II diabetes, hypertension, and dyslipidemia, but not with smoking habit and age (Table 3).
3.2. Allele and genotype frequencies Observed and expected frequencies in the AGT and REN gene polymorphisms were in Hardy–Weinberg equilibrium. The allele and genotype distribution of the AGT and REN polymorphisms were similar in patients with and without restenosis (data no shown). The analysis made comparing the whole group of CAD patients (with and without restenosis) and healthy controls, showed similar distribution of AGT polymorphism in both groups (data not shown), whereas, an increased frequency of the C allele of the REN C4280A (rs5705) polymorphism in CAD patients (25.6%) when compared to healthy controls (17%) was
The functional prediction analysis obtained by FastSNP (http:// fastsnp.ibms.sinica.edu.tw) and SNP Function Prediction (http:// snpinfo.niehs.nih.gov/snpfunc.htm) bioinformatics tools showed that the presence of the C allele of the C4280A (rs5705) polymorphism produces a motif binding for the SF2/ASF2 protein. 4. Discussion It has been suggest that the RAS is implicated in the pathogenesis of neointimal hyperplasia, and migration and proliferation of vascular smooth muscle cells in restenotic lesions (Pacurari et al., 2014) and could contribute to initiation and progression of atherosclerosis (van Thiel et al., 2015; Sheppard and Schiffrin, 2013; Pacurari et al., 2014). Polymorphisms in the genes that encode these molecules have been associated with risk of developing inflammatory diseases, such hypertension, atherosclerosis, vascular disease, coronary artery disease, blood pressure, coronary artery bypass graft, preeclampsia, and acute pancreatitis (Mansego et al., 2008; Ragia et al., 2010; Dzielinska et al., 2011; Abd El-Aziz et al., 2012; Al-Najai et al., 2013; Brugts et al., 2011; Wang et al., 2014; Skipworth et al., 2015; Mirsha et al., 2012). In the present work, we studied seven polymorphisms, five located in the AGT gene and two located in the REN gene. None of the five polymorphisms of the AGT gene was associated with restenosis or CAD in our group of patients. The association of AGT gene polymorphisms with several diseases is controversial with positive and negative results. The AGT rs699 polymorphism was not associated with CAD in
Table 4 Distribution of REN polymorphisms in CAD patients and healthy controls. SNP
Genotype frequency n (%)
REN G5795T rs5707 Control (n = 610)
TT 346 (0.570)
TG 218 (0.360)
GG 46 (0.075)
0.254
158 (0.658)
71 (0.296)
11 (0.046)
0.193
AA 421 (0.690)
AC 170 (0.278)
CC 19 (0.031)
0.170
129 (0.537)
99 (0.412)
12 (0.050)
0.256
CAD (n = 240) REN A4280C rs5705 Control (n = 610) CAD (n = 240)
MAF
Model
OR (95%CI)
P
Co-dominant Dominant Recessive Heterozygous Log-additive
0.54 (0.24–1.19) 0.72 (0.50–1.03) 0.59 (0.27–1.29) 0.80 (0.55–1.17) 0.74 (0.55–1.00)
0.14 0.072 0.17 0.25 0.05
Co-dominant Dominant Recessive Heterozygous Log-additive
1.76 (0.74–4.20) 1.81 (1.26–2.60) 1.44 (0.61–3.38) 1.75 (1.21–2.54) 1.59 (1.17–2.15)
0.006 0.001 0.42 0.003 0.003
CAD, coronary artery disease; MAF, minor allele frequency; OR, odds ratio; CI, confidence interval. The P-values were calculated from logistic regression analysis and the ORs were adjusted for gender, age, diabetes, hypertension, dyslipidemia and smoking habit. Bold numbers indicate significant associations.
J.M. Fragoso et al. / Experimental and Molecular Pathology 99 (2015) 128–132
populations could help define the true role of this polymorphism as risk factor for developing CAD.
Table 5 Frequencies (%) of REN haplotypes in patients with CAD and healthy controls.
Haplotype H1 (TA) H2 (GA) H3 (TC) H4 (GC)
CAD (n = 240)
Controls (n = 610)
Hf 0.554 0.190 0.252 0.004
Hf 0.582 0.248 0.164 0.006
OR
95%CI
131
P
Author disclosure statement NS NS 1.53 NS
No competing financial interests exist. 1.11–2.10
0.014
Acknowledgments
Abbreviations: Hf = haplotype frequency, CAD = coronary artery disease, P = P value, OR = odds ratio, 95%CI = confidential interval. The order of the polymorphisms in the haplotypes is according to the positions in the chromosome (rs5705 and rs5707). The OR was adjusted for gender, age, diabetes, hypertension, dyslipidemia and smoking habit. The significant P value is in bold.
This work was supported in part by grants from the Consejo Nacional de Ciencia y Tecnología (Project number 182962), Mexico City, Mexico. The authors are grateful to the study participants. References
Korean population (Ryu et al., 2002). Similar data were obtained by Renner et al., who reported no association of two AGT polymorphisms (rs699 and rs4762) with blood pressure and CAD in German population (Renner et al., 2005). On the other hand, two AGT polymorphisms studied by Ragia et al., were not associated with CAD and coronary artery bypass grafting in Greek population (Ragia et al., 2010). In contrast to these results, Al-Najai et al., reported that the rs5051, and rs699 polymorphisms were associated with risk of developing myocardial infarction in native Saudi population (Al-Najai et al., 2013). Similar data were obtained by Mehri et al., who reported the association of the AGT rs699 polymorphism with CAD and acute myocardial infarction in a Tunisia population (Mehri et al., 2011). On the other hand, in our study, the REN rs5707 (G5795T) polymorphism was not associated with risk of developing restenosis or CAD. Similar data were obtained by Wang et al., who studied this polymorphism and reported no association with hypertension in Han Chinese population (Wang et al., 2014). In contrast with these data, the rs5707 polymorphism has been associated with increased risk of developing hypertension and acute pancreatitis in European populations (Mansego et al., 2008; Skipworth et al., 2015). In our study, the REN rs5705 (C4280A) polymorphism was associated with increased risk of developing CAD. Association studies of this polymorphism with some pathologies are very scarce and controversial. Sun et al., reported association of this polymorphism with hypertension in a Caucasian population (Sun et al., 2011), whereas Vefring et al., did not detect association with preeclampsia in mother–father–child triads of preeclampsia pregnancies in a Norway population (Vefring et al., 2010). The two REN polymorphisms were in high linkage disequilibrium and one haplotype (TC) was associated with increased risk of developing CAD. Sun et al., studied two [rs5705 (C4280A) and rs10900555 in the intron 1] REN gene polymorphisms and reported the association of the “AA” haplotype with risk of hypertension (Sun et al., 2011). Unfortunately, in the literature there is no evidence of functional studies of the REN polymorphisms. Nonetheless, we determined the potential effect of the polymorphism associated with CAD (rs5705) using bioinformatics tools, as FastSNP and SNP function prediction database. This analysis showed that the presence of the C allele of the C4280A (rs5705) polymorphism produces a motif binding for the SF2/ASF2 protein. These proteins belong to family of SR proteins that regulate alternative splicing (Sureau et al., 2001). Individuals with this allele would have increased binding of the SF2/ASF2 proteins and, in consequence, would have increased expression of the renin isoforms. However, the predictions of functional consequences of this polymorphism need further experimental testing. In summary, our data suggest that the REN C4280A (rs5705) polymorphism plays an important role in the risk of developing CAD with the highest risk for C allele, but do not support its role as a risk factor for developing restenosis after coronary stenting. In our study, it was possible to identify one risk haplotype (TC) for CAD. Our data is still preliminary due to the study sample size and additional studies in other
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The C4280A (rs5705) gene polymorphism of the renin (REN) gene is associated with risk of developing coronary artery disease, but not with restenosis after coronary stenting Jose Manuel Fragoso a, Edith Alvarez-León a, Hilda Delgadillo-Rodríguez b, Marva Arellano-González a, Filogonio Caín López-Pacheco a, David Cruz-Robles a, Marco Antonio Peña-Duque b, Oscar Pérez-Méndez a, Marco Antonio Martínez-Ríos b, Gilberto Vargas-Alarcón a,⁎ a b
Department of Molecular Biology, Instituto Nacional de Cardiología Ignacio Chávez, Mexico City, Mexico Department of Interventional Cardiology, Instituto Nacional de Cardiología Ignacio Chávez, Mexico City, Mexico
a r t i c l e
i n f o
Article history: Received 25 May 2015 Accepted 18 June 2015 Available online 20 June 2015 Keywords: Coronary artery disease Polymorphisms Renin–angiotensin-system (RAS) Restenosis
a b s t r a c t The aim of the present study was to evaluate the role of AGT and REN gene polymorphisms as susceptibility markers for coronary artery disease (CAD) and/or restenosis after coronary stent placement in a group of Mexican patients. Five polymorphisms of the AGT (rs699, rs4762, rs5051, rs5049, rs5046) and two of the REN (rs5707, rs5705) genes were analyzed by 5′ exonuclease TaqMan genotyping assays in 240 patients with CAD who underwent coronary artery stenting (76 with restenosis and 164 without restenosis). A group of 610 individuals without clinical and familial antecedents of cardiovascular diseases were included as controls. The results showed that the distribution of AGT and REN polymorphisms were similar in patients with and without restenosis. However, when the whole group of patients (with and without restenosis) was compared to healthy controls, under co-dominant, dominant, heterozygous and additive models, the REN A4280C (rs5705) polymorphism was associated with increased risk of CAD (OR = 1.76, PCo-dom = 0.006, OR = 1.81, PDom = 0.001, OR = 1.75, PHet = 0.003 and OR = 1.59, PAdd = 0.003, respectively). All models were adjusted for age, gender, diabetes, dyslipidemia, hypertension and smoking habit. The TC haplotype of the REN gene was associated with increased risk of CAD (OR = 1.53, P = 0.014). The data suggest that the REN C4280A (rs5705) polymorphism plays an important role in the risk of developing CAD with the highest risk for C allele, but do not support its role as a risk factor for developing restenosis after coronary stenting. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Coronary artery disease (CAD) is a complex, multifactorial disease, influenced by pathophysiologic conditions as well as by genetic and environmental factors. The treatment strategies for this disease are coronary artery bypass grafting, percutaneous transluminal coronary angioplasty (PTCA), and intracoronary stent. However, after PTCA, restenosis occurs in about 30 to 32% of patients and after intracoronary stent placement in 12 to 32% of patients (Kuchulakanti et al., 2006; Lee et al., 2011; Hamasaki and Tei, 2011; Latib et al., 2011). The restenosis is the arterial wall's healing response to mechanical injury and comprises two main processes, neointimal hyperplasia (i.e., smooth muscle migration/proliferation, extracellular matrix deposition) and vessel remodeling (Costa and Simon, 2005). In the literature, there has been evidence
⁎ Corresponding author at: Department of Molecular Biology, Instituto Nacional de Cardiología “Ignacio Chávez”, Juan Badiano No. 1, Tlalpan 14080, Mexico D.F., Mexico. E-mail address: [email protected] (G. Vargas-Alarcón).
http://dx.doi.org/10.1016/j.yexmp.2015.06.012 0014-4800/© 2015 Elsevier Inc. All rights reserved.
that the activation of the renin–angiotensin system (RAS) induces the production of pro-inflammatory cytokines, proliferation and vasoconstriction on the vasculature, and in consequence can contribute to initiation and progression of atherosclerosis (Chamarthi et al., 2011; Mehta and Griendling, 2007; van Thiel et al., 2015; Pacurari et al., 2014). In addition, RAS has been implicated in the pathogenesis of neointimal hyperplasia, and a role for angiotensin II in the migration and proliferation of vascular smooth muscle cells in restenosis has been reported (Pacurari et al., 2014; Valente et al., 2012; Yao et al., 2012). Renin and angiotensinogen are encoded by the REN and AGT genes located in chromosome 1 position q32 and q42, respectively (Qin et al., 1993; Gaillard-Sanchez et al., 1990). The renin is a key enzyme produced in the juxtaglomerular apparatus of the kidney and in many other cells that cleave angiotensinogen to produce angiotensin I, the first step in generation of the angiotensin II through angiotensinconverting enzyme. In addition, the renin at the cellular level is amplified by a receptor-mediated binding process, which leads to a further increase in angiotensin II generation in many tissues (Nguyen et al., 2002; Paul et al., 2006). On the other hand, the angiotensinogen is a key
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component of RAS, it is cleaved by renin to produce angiotensin I. AGT gene presents five important single nucleotide polymorphisms (SNPs), three in the promoter region [− 6 ANG (rs5051), − 217 GNA (rs5049) and − 532 CNT (rs5046)] and two in the exon 2 [C4072T (rs699) and C3889T (rs4762)]. These SNPs have been associated with risk of developing hypertension, acute myocardial infarction, vascular disease, coronary artery disease, atherosclerosis, and coronary artery bypass graft in several populations (Inoue et al., 1997; Karayannis et al., 2010; Ragia et al., 2010; Mehri et al., 2011; Dzielinska et al., 2011; Abd El-Aziz et al., 2012; Al-Najai et al., 2013; Brugts et al., 2011). On the other hand, the REN gen presents two relevant SNPs, one in the intron 4 [G5795T (rs5707)] and one in the exon 2 [C4280A (rs5705)]. The rs5707 has been associated with acute pancreatitis (Skipworth et al., 2015), with blood pressure levels and with the risk of having hypertension (Mansego et al., 2008). Considering the prominent role of the angiotensinogen and renin as key component of RAS, and the effect of RAS in the cardiovascular system, the aim of this study was to analyze whether AGT and REN gene polymorphisms are associated with risk of developing CAD and/or restenosis after coronary stent placement in a group of Mexican patients. 2. Materials and methods 2.1. Patients and controls The study included 240 Mexican Mestizo patients with symptomatic coronary artery disease (CAD) who underwent coronary stent implantation at our institution during the period between October 2008 and October 2014 and went to follow-up coronary angiography because of symptoms of ischemia documented in a myocardial perfusion imaging test. Basal and procedure coronary angiographies were analyzed for angiographic predictors of restenosis, and follow-up angiography was performed to screen for binary restenosis. Using a N50% stenosis at follow-up (50% reduction in the luminal diameter of the stenosis compared with the coronary angiography findings immediately following angioplasty) as the criterion to define restenosis, there were 76 patients with restenosis and 164 without restenosis. Also, neither a group of 610 healthy controls individuals with neither symptoms nor previous diagnosis of cardiovascular or systemic disease was studied as the control group. All subjects were ethnically matched, and we considered as Mexican Mestizos only those individuals who for three generations, including their own, had been born in Mexico. The Institutional Ethics and Research Committee approved the study, and all subjects signed informed consent.
on a 7900HT Fast Real-Time PCR system according to manufacturer's instructions (Applied Biosystems, foster City, USA) (Table 1).
2.3. Statistical analysis We used SPSS version 18.0 (SPSS, Chicago, IL) statistical package. Means, standard deviations and frequencies of baseline characteristics were calculated. Comparison of numerical variables, such as age and body mass index, between both groups was performed with Mann Whitney U test; data are presented as median (25th–75th percentile). Categorical variables were analyzed with Chi2 tests or Fisher's exact test, as needed, and presented as absolute frequencies and proportions. Logistic regression analysis was performed to look for associations of polymorphisms with CAD using the following models: codominant (major allele homozygotes vs. heterozygotes and major allele homozygotes vs. minor allele homozygotes), dominant (major allele homozygotes vs. heterozygotes + minor allele homozygotes), recessive (major allele homozygotes + heterozygotes vs. minor allele homozygotes), heterozygous (homozygote for the minor allele + homozygote for the major allele vs heterozygote) and log-additive (major allele homozygotes vs. heterozygotes vs. minor allele homozygotes). The inheritance models were adjusted for gender, age, hypertension, dyslipidemia, type II diabetes mellitus, and smoking habit. SNPstats (http://bioinfo.iconcologia.net/custom.php) software for Windows® were used to analyze the genetic frequencies and to evaluate the linkage disequilibrium (LD, D′) among polymorphisms, as well as to construct haplotypes. Hardy–Weinberg equilibrium was evaluated by Chi2 test.
2.4. Functional prediction analysis Two in silico programs were used [FastSNP (http://fastsnp.ibms. sinica.edu.tw) (Yuan et al., 2006) and SNP Function Prediction (http:// snpinfo.niehs.nih.gov/snpfunc.htm)] to predict the potential effect of the REN polymorphisms. These programs analyzes the location of the SNP (eg. 5′ upstream, 3′ untranslated region, intronic) and possible functional effects such as amino acid changes in protein structures, transcription factor binding sites in promoter or intronic enhancer regions, and alternative splicing regulation by disrupting exonic splicing enhancers or silencers.
Table 2 Clinical and angiographic characteristics of CAD patients with and without restenosis.
2.2. Genetic analysis The T4272C (rs699), C3889T (rs4762), −6 ANG (rs5051), −217 ANG (rs5049), −532 CNT (rs5046), G5795T (rs5707), and C4280A (rs5705) SNPs were genotyped using 5′ exonuclease TaqMan genotyping assays
Table 1 AGT and REN gene polymorphisms tested. Gene
Polymorphisma
dbSNPb
Chromosome position
Location in gene
AGT
C4272T (Met 235 Thr) C3889T (Thr 174 Met) A −6C G −217A C −532T G5795T A4280C (Thr68Thr)
rs699 rs4762 rs5051 rs5049 rs5046 rs5707 rs5705
1q42 1q42 1q42 1q42 1q42 1q32 1q32
Exon 2 Exon 2 5′ near region 5′ near region 5′ near region Intron 4 Exon 2
REN a b
Given name according to NCBI. SNP ID in database dbSNP.
129
Mena Blood pressurea (mm Hg) Type II diabetes mellitusa Hypercholesterolemiaa Smokinga Unstable anginaa Stable anginaa Statin therapya DESa BSMa Diameter smallera 2.5 mm Stent lengtha (mm) Bifurcationa
Agea (years) a
With restenosis (n(%))
Without restenosis (n(%))
P
61 (80) 48 (63)
129 (79) 91 (55)
NS NS
33 (43) 41 (54) 48 (63) 26 (34) 8 (10) 59 (78) 20 (26) 52 (68) 23 (30)
61 (37) 97 (59) 101 (62) 43 (26) 36 (22) 137 (83) 98 (59) 61 (37) 31 (19)
NS NS NS NS 0.03 NS b0.0001 b0.0001 0.04
31 (41) 19 (25) Median (percentile 25–75) 59.9 (54–67)
71 (43) 40 (24) Median (percentile 25–75) 58.6 (53–65)
NS NS
NS
(n(%)) number and proportion of subjects with the clinical and angiographic characteristics in both groups. Abbreviations: BMS = bare metal stent; DES = drug-eluting stent.
130
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Table 3 Baseline clinical characteristics of the studied individuals (patients with CAD and healthy controls). Clinical characteristics
CAD patients (n(%)) Healthy controls (n(%)) P (n = 240) (n = 610)
Mena High blood pressure (mm Hg)a Type II diabetes mellitusa Dyslipidemiaa Smokinga
190 (79) 139 (58)
292 (48) 269 (44)
b0.001 0.003
94 (39) 138 (57) 149 (62) Median (percentile 25–75) 59 (53–66)
92 (15) 449 (74) 346 (57) Median (percentile 25–75) 56 (51–62)
b0.001 b0.001 NS
Age (years) a
NS
(n(%)) number and proportion of subjects with the clinical characteristic in both groups.
3. Results 3.1. Characteristics of the study population
observed. In this case, we estimated the risk according to five inheritance models adjusted for age, gender, diabetes, dyslipidemia, hypertension and smoking habit (Table 4). Under co-dominant, dominant, heterozygous and additive models, the C4280A (rs5705) polymorphism was associated with increased risk of developing CAD (OR = 1.76, 95% CI: 0.74– 4.20, PCo-dom = 0.006, OR = 1.81, 95% CI: 1.26–2.60, PDom = 0.001, OR = 1.75, 95% CI: 1.21–2.54, PHet = 0.003 and OR = 1.59, 95% CI: 1.17–2.15, PAdd = 0.003, respectively). 3.3. Linkage disequilibrium analysis The REN polymorphisms (rs5707 and rs5705) were in high linkage disequilibrium (D′ N 0.8 and r2 N 0.9) and 4 different haplotypes (TA, GA, TC and GC) were observed. The TC haplotype was significantly associated with increased risk of developing CAD (OR = 1.53, 95% CI = 1.11–2.10, P = 0.014) (Table 5). 3.4. Functional prediction
The clinical and angiographic characteristics of CAD patients with and without restenosis are shown on Table 2. There were no significant differences between patients with or without restenosis with regard to age, gender, type II diabetes, hypertension, dyslipidemia, unstable angina, statin therapy, stent length, and bifurcation. Nonetheless, patients who underwent coronary bare-metal stent (BSM) implantation develop more restenosis (68%) than those patients who underwent drug-eluting stent (DES) implantation (26%) (P b 0.0001). Also, those lesions treated with stents with a diameter b 2.5 mm presented more restenosis (P = 0.04). When we analyzed the baseline characteristics of the CAD patients and healthy controls included in the study, there were significant differences between patients with CAD and healthy controls with regard to gender, type II diabetes, hypertension, and dyslipidemia, but not with smoking habit and age (Table 3).
3.2. Allele and genotype frequencies Observed and expected frequencies in the AGT and REN gene polymorphisms were in Hardy–Weinberg equilibrium. The allele and genotype distribution of the AGT and REN polymorphisms were similar in patients with and without restenosis (data no shown). The analysis made comparing the whole group of CAD patients (with and without restenosis) and healthy controls, showed similar distribution of AGT polymorphism in both groups (data not shown), whereas, an increased frequency of the C allele of the REN C4280A (rs5705) polymorphism in CAD patients (25.6%) when compared to healthy controls (17%) was
The functional prediction analysis obtained by FastSNP (http:// fastsnp.ibms.sinica.edu.tw) and SNP Function Prediction (http:// snpinfo.niehs.nih.gov/snpfunc.htm) bioinformatics tools showed that the presence of the C allele of the C4280A (rs5705) polymorphism produces a motif binding for the SF2/ASF2 protein. 4. Discussion It has been suggest that the RAS is implicated in the pathogenesis of neointimal hyperplasia, and migration and proliferation of vascular smooth muscle cells in restenotic lesions (Pacurari et al., 2014) and could contribute to initiation and progression of atherosclerosis (van Thiel et al., 2015; Sheppard and Schiffrin, 2013; Pacurari et al., 2014). Polymorphisms in the genes that encode these molecules have been associated with risk of developing inflammatory diseases, such hypertension, atherosclerosis, vascular disease, coronary artery disease, blood pressure, coronary artery bypass graft, preeclampsia, and acute pancreatitis (Mansego et al., 2008; Ragia et al., 2010; Dzielinska et al., 2011; Abd El-Aziz et al., 2012; Al-Najai et al., 2013; Brugts et al., 2011; Wang et al., 2014; Skipworth et al., 2015; Mirsha et al., 2012). In the present work, we studied seven polymorphisms, five located in the AGT gene and two located in the REN gene. None of the five polymorphisms of the AGT gene was associated with restenosis or CAD in our group of patients. The association of AGT gene polymorphisms with several diseases is controversial with positive and negative results. The AGT rs699 polymorphism was not associated with CAD in
Table 4 Distribution of REN polymorphisms in CAD patients and healthy controls. SNP
Genotype frequency n (%)
REN G5795T rs5707 Control (n = 610)
TT 346 (0.570)
TG 218 (0.360)
GG 46 (0.075)
0.254
158 (0.658)
71 (0.296)
11 (0.046)
0.193
AA 421 (0.690)
AC 170 (0.278)
CC 19 (0.031)
0.170
129 (0.537)
99 (0.412)
12 (0.050)
0.256
CAD (n = 240) REN A4280C rs5705 Control (n = 610) CAD (n = 240)
MAF
Model
OR (95%CI)
P
Co-dominant Dominant Recessive Heterozygous Log-additive
0.54 (0.24–1.19) 0.72 (0.50–1.03) 0.59 (0.27–1.29) 0.80 (0.55–1.17) 0.74 (0.55–1.00)
0.14 0.072 0.17 0.25 0.05
Co-dominant Dominant Recessive Heterozygous Log-additive
1.76 (0.74–4.20) 1.81 (1.26–2.60) 1.44 (0.61–3.38) 1.75 (1.21–2.54) 1.59 (1.17–2.15)
0.006 0.001 0.42 0.003 0.003
CAD, coronary artery disease; MAF, minor allele frequency; OR, odds ratio; CI, confidence interval. The P-values were calculated from logistic regression analysis and the ORs were adjusted for gender, age, diabetes, hypertension, dyslipidemia and smoking habit. Bold numbers indicate significant associations.
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populations could help define the true role of this polymorphism as risk factor for developing CAD.
Table 5 Frequencies (%) of REN haplotypes in patients with CAD and healthy controls.
Haplotype H1 (TA) H2 (GA) H3 (TC) H4 (GC)
CAD (n = 240)
Controls (n = 610)
Hf 0.554 0.190 0.252 0.004
Hf 0.582 0.248 0.164 0.006
OR
95%CI
131
P
Author disclosure statement NS NS 1.53 NS
No competing financial interests exist. 1.11–2.10
0.014
Acknowledgments
Abbreviations: Hf = haplotype frequency, CAD = coronary artery disease, P = P value, OR = odds ratio, 95%CI = confidential interval. The order of the polymorphisms in the haplotypes is according to the positions in the chromosome (rs5705 and rs5707). The OR was adjusted for gender, age, diabetes, hypertension, dyslipidemia and smoking habit. The significant P value is in bold.
This work was supported in part by grants from the Consejo Nacional de Ciencia y Tecnología (Project number 182962), Mexico City, Mexico. The authors are grateful to the study participants. References
Korean population (Ryu et al., 2002). Similar data were obtained by Renner et al., who reported no association of two AGT polymorphisms (rs699 and rs4762) with blood pressure and CAD in German population (Renner et al., 2005). On the other hand, two AGT polymorphisms studied by Ragia et al., were not associated with CAD and coronary artery bypass grafting in Greek population (Ragia et al., 2010). In contrast to these results, Al-Najai et al., reported that the rs5051, and rs699 polymorphisms were associated with risk of developing myocardial infarction in native Saudi population (Al-Najai et al., 2013). Similar data were obtained by Mehri et al., who reported the association of the AGT rs699 polymorphism with CAD and acute myocardial infarction in a Tunisia population (Mehri et al., 2011). On the other hand, in our study, the REN rs5707 (G5795T) polymorphism was not associated with risk of developing restenosis or CAD. Similar data were obtained by Wang et al., who studied this polymorphism and reported no association with hypertension in Han Chinese population (Wang et al., 2014). In contrast with these data, the rs5707 polymorphism has been associated with increased risk of developing hypertension and acute pancreatitis in European populations (Mansego et al., 2008; Skipworth et al., 2015). In our study, the REN rs5705 (C4280A) polymorphism was associated with increased risk of developing CAD. Association studies of this polymorphism with some pathologies are very scarce and controversial. Sun et al., reported association of this polymorphism with hypertension in a Caucasian population (Sun et al., 2011), whereas Vefring et al., did not detect association with preeclampsia in mother–father–child triads of preeclampsia pregnancies in a Norway population (Vefring et al., 2010). The two REN polymorphisms were in high linkage disequilibrium and one haplotype (TC) was associated with increased risk of developing CAD. Sun et al., studied two [rs5705 (C4280A) and rs10900555 in the intron 1] REN gene polymorphisms and reported the association of the “AA” haplotype with risk of hypertension (Sun et al., 2011). Unfortunately, in the literature there is no evidence of functional studies of the REN polymorphisms. Nonetheless, we determined the potential effect of the polymorphism associated with CAD (rs5705) using bioinformatics tools, as FastSNP and SNP function prediction database. This analysis showed that the presence of the C allele of the C4280A (rs5705) polymorphism produces a motif binding for the SF2/ASF2 protein. These proteins belong to family of SR proteins that regulate alternative splicing (Sureau et al., 2001). Individuals with this allele would have increased binding of the SF2/ASF2 proteins and, in consequence, would have increased expression of the renin isoforms. However, the predictions of functional consequences of this polymorphism need further experimental testing. In summary, our data suggest that the REN C4280A (rs5705) polymorphism plays an important role in the risk of developing CAD with the highest risk for C allele, but do not support its role as a risk factor for developing restenosis after coronary stenting. In our study, it was possible to identify one risk haplotype (TC) for CAD. Our data is still preliminary due to the study sample size and additional studies in other
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