Genetic association between two Estrogen Receptor 2 (ER2) Gene Polymorphism (rs4986938 and rs1256049) and SYNTAX Score in Patients with Coronary Artery Disease: A Report from Southern Iran

Farzaneh Foroughinia Shiraz University of Medical Sciences School of Pharmacy https://orcid.org/0000-0003-3993-2043 Pooyan Dehghani Tehran University of Medical Sciences School of Medicine Mehdi Dianatpour Shiraz Medical School: Shiraz University of Medical Sciences Arghavan Amiri Shiraz University of Medical Sciences School of Pharmacy Iman Jamhiri Shiraz Medical School: Shiraz University of Medical Sciences Parisa Ghasemiyeh (  pa_ghasemeyeh@sums.ac.ir ) Shiraz University of Medical Sciences https://orcid.org/0000-0002-8640-5724


Background
Cardiovascular disease (CVD) is considered as one of the major causes of death worldwide. Coronary artery disease (CAD) and cerebrovascular diseases are among the most common causes of deaths due to CVD [1]. According to the epidemiologic studies, premenopausal females have less risk for CVD in comparison with males with the same age that may be because of the protective effects of estrogen in preventing CVD. Estrogen can serve its effect by binding to estrogen receptor α (ER1), estrogen receptor β (ER2), and G-protein-coupled estrogen receptor 30 (GPR30). The possible protective mechanism of estrogen against CVD would be related to its role on angiogenesis, brosis, vascular function, and oxidative stress processes [2].
Estrogen could induce its cardioprotective effects through a receptor-based biological mechanism.
Recent researches revealed that genetic variants have an important role in cardio-and cerbro-vascular diseases. For instance, ER1 is an important indicator of atheroprotective effect of estrogen on cardiovascular system. [3]. A study evaluated the association between ER1 single nucleotide polymorphism (SNP) and Intima-media thickness (IMT) in carotid artery, an important predictor of CVD, in female Taiwanese population. This research demonstrated a signi cant association between ER1(NM_001122740.1):c.1782G > A [rs2228480] and ER1(NM_001122740.1):c.*1753C > A [rs3798758] of female sex with the IMT of carotid artery. According to this article, women with c.1782G > A polymorphism had an increased IMT of carotid artery and were more prone to the risk of atherosclerotic disorders such as ischemic stroke [4]. A case-cohort design on evaluation of the relationship between ER1 gene polymorphism and the risk of coronary heart disease (CHD) and stroke in Finnish population, revealed the signi cant association of PvuII NM_000125.3:c.453 − 397 T > C [rs2334693 ] polymorphism with the higher risk of CHD in men. On the other hand, this study failed to con rm any signi cant association between ER1 gene polymorphism and the occurrence of ischemic stroke [5]. In addition, there was a report for a signi cant association between ER1 gene polymorphism and low density lipoprotein (LDL) metabolism in women which is an important predictor of atherosclerosis and CVD [6].
Arterial stiffness enhancement and increased wave re ection are the other risk factors for CVD. It has been hypothesized that there is an association between arterial stiffness and genetic variance of ER1, ER2, and CYP19A1 (aromatase). The results of the American population study showed a strong relation between ER1 and ER2 gene polymorphisms and enhanced wave re ection but it is failed to show any association respected to CYP19A1 [7].
The SYNTAX Score (SS) is an angiographic tool using to predict the coronary vasculature characteristics based on the number of lesions and also their complexity, location, and function. SS would be calculated through a computer program using 12 main questions which are about the dominance, number of lesions, segments involved per lesion, total occlusion, trifurcation, bifurcation, aorto-ostial lesion, severe tortuosity, length > 20 mm, heavy calci cation, thrombus, and diffuse disease/small vessels. Higher SS would be a predictor of higher level of disease complexity, more challenging pharmacotherapy, and worse prognosis [8].
Since most of studies have focused on ER1 and few studies evaluated the role of ER2 in atherosclerotic diseases, in this study, we aimed to evaluate the association between two major SNPs of ER2 gene containing AluI [rs4986938] and RsaI [rs1256049] and the severity of CAD expressed as SS.

Methods
The study protocol was assessed and approved by the Ethics Committee of Shiraz University of Medical Sciences (SUMS, Iran) (No: IR.SUMS.REC.1397.369). Written consent form and permission for DNA analyses was obtained from all participants.

Subjects
Inclusion criteria were age of 18-80 years old and con rmed diagnosis of CAD ( 50% luminal stenosis in at least one major coronary artery in angiography). Exclusion criteria include consumption of oral contraceptives and hormone replacement therapy during one month prior to the study, malignancy, schizophrenia, connective tissue disease, or chronic in ammatory disease.
Patients who ful lled the criteria were included into this project. Demographic and clinical data were documented for each patient according to patients' hospital record.

Coronary angiography
Patient who were candidates for invasive strategy with coronary angiography were enrolled into the study. Patients received Aspirin, clopidogrel, statins, and anticoagulation according to current guidelines prior to coronary angiography. Procedure was performed with local anesthesia and via radial or femoral approaches. Ultravist ® or Visipaque ® was used as contrast agents.
Subjects with 50% stenosis in at least one major epicardial artery greater of 1.5 mm were included into the study. Coronary angiographic data, including vessels' involvement, percentage of stenosis and SS was evaluated by an interventional cardiologist who was blinded to other data. SS was calculated using the o cial calculator from SYNTAX score website [9].
The nal plan of the management for patients were varied from medical therapy (11%) to percutaneous coronary intervention (PCI) (67%) and coronary artery bypass grafting in the remaining (22%).
Severe CAD is de ned as ≥ 70% luminal stenosis in one major epicardial vessel or ≥ 50 % stenosis in the left main coronary artery (LMD) and Multivessel disease (MVD) is de ned as more than one coronary artery involvement with ≥ 70% stenosis [10,11].

Biological samples and genotyping
Blood samples were collected in EDTA vacutainer tubes from all patients in order to further assessment of ER2 gene polymorphism. Whole blood was stored at −20°C freezer until use. Genomic DNA for PCR was extracted from whole blood by DNA extraction kit (Yekta tajhiz, Iran) according to manufacturer protocol.
Selected regions of ER2 gene were ampli ed by polymerase chain reaction (PCR) using speci c primers listed in Table 1. All primers used for ampli cation of RsaI and AluI polymorphisms regions in ER2 gene were designed by the Primer 3 software and were obtained from metabion (metabion international AG, Martinsread, Germany). The reaction program for RsaI and AluI polymorphisms ampli cation was as follows: initial denaturation for 4 min at 94°c, followed by 35 cycles of 94°c for 50 s, 66°c for 50 s, 72°c for 50 s and nal elongation at 72°c for 7 min. The PCR reactions were performed in Veriti Thermal Cycler (Applied Biosystems, Foster City, CA, USA).
For RFLP, the PCR products of RsaI and AluI polymorphisms were digested with RsaI (#ER1121, 5U at 37°C for 16h) and AluI (#ER0011, 5U at 37°C for 16h) (Fermentas), respectively. DNA fragments from RFLP were electrophoresed on 2% agarose gel to determine the RsaI and AluI polymorphic patterns. SD and the differences between male and female were analyzed by independent samples t test.
Categorical variables were showed as absolute number and percentages and were tested by using the chi-square test.
For comparison between ER2 genotypes and allele frequencies and SS or sex, chi-square test and Fischer's exact test were applied. P-value of <0.05 was considered as signi cant.

Results
Totally, 148 patients were enrolled into the study. The mean age of all patients was 59.06 ± 11.42. 110 (74.7%) male with the mean age of 57.62 ± 11.75 and 38 (25.3%) female with the mean age of 63.29 ± 9.28 were participated in our study. The same gender distribution was found in previous investigation studied Iranian patients undergoing elective PCI [12].
Clinical baseline characteristics of patients were described in Table 2, dividing participants in two groups according to the sex. Both groups were similar in all variables with exception of age (p = 0.01), weight (p= 0.001), glomerular ltration rate (GFR) (p= 0.001) and frequency of hypertension (p = 0.02), diabetes mellitus (DM) (p = 0.01), and active smoking (p= 0.001). In addition, no signi cant difference was found between sex and endpoints such as SS, severe CAD, and the number of diseased vessels. All samples were examined for ER2 gene polymorphisms (RsaI and AluI polymorphisms). The gel electrophoresis results after enzymatic digestion are shown in Fig. 1.
Of 148 samples analyzed for RsaI polymorphisms, a statistically signi cant relationship was found between SS and RsaI genotypes of ER2 gene (P = 0.01) but not with RsaI alleles (P = 0.05). Of note, AG genotype was found to be the more prevalent ones in patients with SS ≥ 23. In contrast, there was no association between AluI genotypes and alleles and SS ( Table 3). The same results were reported for the association between sex and RsaI and AluI polymorphisms of ER2 gene.

Discussion
In this study the association between ER2 gene polymorphisms, RsaI and AluI, and SS was evaluated in 148 Iranian patients with CAD. According to our results, CVD risk factors such as hypertension and active smoking were more prevalent in males compared to females. Higher frequency for history of smoking in males is completely predictable since smoking is not a common habit among Iranian women due to cultural issues. Unfortunately, it is in a growing manner in recent years. In contrast, older age, more cases with diseases such as diabetic mellitus and chronic kidney disease (CKD) were reported to be more prevalent in studied females than males. Older ages in studied women is congruent with the fact that women experienced CVD at higher ages than men. In addition, more rates of CKD involvement in female gender may be assumed due to higher rate of DM among women than men in this study. Although there was signi cant differences regarding some cardiovascular risk factors between genders, it dose not interpreted to difference in SS among these two groups.
Results of polymorphisms analysis revealed that there was neither signi cant association between AluI gene polymorphism nor their alleles with SS, on the other hand; a signi cant association between RsaI genotypes and SS was found. Even though several researches evaluated the association between ER gene polymorphism and CVD prognosis, most of them worked on ER1 gene polymorphisms [13][14][15][16] and only few evidences investigated the association between ER2 gene polymorphisms and CVD progress [17].
A recently published case-control study performed on Chinese Han women population evaluated the association of SNPs of ER2 genes, RsaI and AluI, with CAD. Results of this study revealed that there was no signi cant association between the genotypes and/or haplotypes of these two SNPs and risk of CAD. Further subdivided age-based analysis in this study revealed signi cantly lower risk of CAD in AG genotypes of AluI in comparison to homozygotes GG carriers in patients younger than 40 years of age. In contrast, homozygotes AA carriers had higher risk of CAD. They concluded that A allele of AluI SNP would be an important predictor of CAD risk in Chinese women younger than 40 years of age. These correlations were not found in RsaI SNP [18]. Another case-control study on Brazilian population with premature CAD revealed a statistically signi cant association between AluI SNP and CAD and it was introduced as one of the most important independent risk factors for CAD as well as dyslipidemia, elevated levels of triglycerides and apolipoprotein B and low levels of HDL in this research. The homozygote AA genotype was more prevalent in case group in comparison to control group [19]. Results of these studies are not similar to ours since no signi cant association was found between AluI genotypes and SS in our study. In contrast, AG genotype of RsaI polymorphism was associated to more severe coronary artery stenosis (SS ≥ 23).
There are some other researches worked on other SNPs of ER2 gene, rs1271572. A nested case-control study on Spanish population which was assessed the relationship between three SNPs of ER2 and myocardial infarction (MI) revealed that there was a signi cant association between rs1271572 SNP and enhanced risk of MI occurrence. This nding was limited to the male sex, therefore; this study suggested the potential role of gender in genetic variance of ER2 gene polymorphisms in MI patients [20]. Another study performed on American population con rmed a signi cant association between T allele of rs1271572 SNP and higher risk of MI and CVD, however; this association was just seen in female gender. Because of these controversial results on the association of ER2 polymorphisms and risk of CVD occurrence and severity, further studies with larger sample sizes and assessments of patients with different ethnics and genetics would be necessary [17].
The exact mechanism by which ER signaling and ER gene polymorphism play a role on CAD pathogenesis is uncertain. Recent studies revealed that estrogen could induce anti-brotic effects in the heart that would be occurred through the ER2 [2]. Results of an in vivo animal study revealed that activation of ER2 could stop the effects of angiotensin II (Ang-II) and endothelin-1 (ET-1) pro-brotic signaling thus prevent further cardiac brosis. Ang-II and ET-1 could induce cardiac brosis through the inversion of broblasts to myo broblasts and also through the induction of transforming growth factor-β1 (TGFβ1) which is a known cardiac brosis inducer. Estrogen and ER2 agonists could stop TGFβ1 action via cAMP and protein kinase A pathway [2,21]. Also previous studies revealed that estrogen administration could enhance ER2 transcript expression and therefore induce its anti-brotic effects [22]. It has been reported that ER2 gene overexpression would be a predictive factor of heart function improvement and survival in patients who had a recent history of MI both in males and females [23].
Results of another study on the role of ER1 and ER2 gene expression on the occurrence of neovascularization after ischemic heart disease revealed that both ER1 and ER2 (with the dominance of ER1) might have a pivotal role in estrogen-induced endothelial progenitor cells (EPCs) mobilization and further protection of cardiac function following a recent MI [24]. Another suggestive cardioprotective mechanism of ER2 gene would be through the induction of eNOS expression and further vasodilation in cases of ischemia/reperfusion (I/R) injury, especially in female gender [25]. These researches emphasizes the fact that in addition to the estrogen plasma level and the extent of ER1 and ER2 gene expression, evaluation of ER1 and ER2 genetic variance and polymorphisms would be helpful as predictive measure for identi cation of high risk patients for CVD and also their prognosis.
Because of the controversies around the role of ER2 SNPs in CVD in different populations, further larger investigations would be necessary in order to con rm our observed association between RsaI polymorphism and SS.

Study Limitations
The possible limitations of our study were small sample size and unequal number of participants according to their gender (males and females). Also, it is suggested to evaluate the plasma estrogen levels of patients in combination with investigation of ER2 gene polymorphisms in future studies.

Conclusions
Besides to estrogen level, the genetic variation of its receptors, ER1 and ER2, might play an important role in the pathogensis, severity and complexity of CAD. According to our results, RsaI polymorphism of ER2 gene may assert a pivotal role in the severity of CAD; however this assumption needs to be proved in higher population studies.