{'fraction_non_alphanumeric': 0.03505370709981661, 'fraction_numerical': 0.01828661252292376, 'mean_word_length': 4.810045662100457, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 5, 'https://': 0, 'lorem ipsum': 0, 'www.': 1, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 2, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Is Endothelin Gene Polymorphism Associated with Postoperative Atrial Fibrillation in Patients Undergoing Coronary Artery Bypass Grafting?

Background: The mechanism of development of atrial fibrillation (AF) in patients undergoing coronary artery bypass grafting (CABG) has not been clearly defined, and the involvement of multiple factors such as advanced age, withdrawal of β-blockers, inadequate atrial protection, and electrolyte imbalance, particularly hypomagnesemia has been documented by several authors. Despite all the available pharmacologic prophylaxis, incidence of AF still remains high in this group of patients. This unexplained cause could be genetic inheritance of endothelin-1 (ET-1) gene which is thought to have a pro-arrhythmogenic effect. Aim: This study aims to investigate the relationship between plasma ET-1 concentrations, ET-1 gene polymorphisms in loci -1370 T/G, -134 (3A/4A) Ins/del, Lys198Asn (G/T), and occurrence of AF in patients undergoing CABG. Methodology: Ninety-eight nonrelated, nondiabetic patients over a period of 4 years undergoing routine CABG were selected for the present study. All patients were genotyped for three single nucleotide polymorphisms (SNPs) in loci -1370 T/G, -134 (3A/4A) Ins/del, and Lys198Asn (G/T) in the ET-1 gene by gene sequencing. The plasma ET-1 concentrations were measured using an ET immunoassay. Results: Plasma ET-1 concentrations were higher in AF+ group (P = 0.001) as compared to AF− group. The allele frequencies between AF+ and AF− group were significantly different only with respect to the Lys198Asn (G/T) SNP of the ET-1 gene. Conclusion: The study described the possible correlation of polymorphism of ET gene in CABG population from India. The ET-1 gene might play a disease-modifying role in atrial fibrillation.


Introduction
Atrial fibrillation (AF) is a frequent postoperative complication seen after coronary artery bypass grafting (CABG) with a reported incidence of 20%-50%. [1] The frequency of AF has not decreased despite recent developments in cardiac surgical techniques, anesthetic management, and optimal myocardial protection. [2] Different drugs have been used for the prophylaxis of AF in these patients although conflicting evidence exists. [3,4] Although the mechanism of development of AF in these group of patients has not been clearly defined, the involvement of multiple factors such as advanced age, withdrawal of β-blockers, inadequate atrial protection, and electrolyte imbalance, particularly hypomagnesemia has been documented by several authors. [5] In some patients, the cause is still not known. AF is often associated with structural heart disease, and traditionally, has not been considered to have a genetic association. While familial forms of AF have long been reported, a genetic predisposition for the same in the topic of recent interest. [6,7] The genetics of AF is complex and heterogeneous. More recently, common variant analyses have identified 14 genetic loci associated with AF. [8] Once a condition is found to be heritable, there are several techniques that are commonly employed to identify the genetic basis of a disease, namely, linkage analysis, candidate gene resequencing, and association studies. [9] In recent years, genome-wide association studies (GWASs) have been made possible by advancements in genotyping technology that allows investigators to assay hundreds of thousands of single nucleotide polymorphisms (SNPs) spread over the entire human genome. [9] Such studies have been used successfully in the past year to identify potential novel pathways for diabetes, macular degeneration, and repolarization. [9] Genetic etiology for AF has been attributed in "lone AF" and familial AF. Although there are several causes for AF in adults undergoing CABG and despite all the available pharmacologic prophylaxis, incidence of AF still remains high. This unexplained cause could be genetic inheritance. Several gene loci encoding ion channels, gap junction proteins, interleukins, renin-angiotensin-aldosterone axis, calcium handling, various SNPs, etc., have been identified to be associated with AF. However, the various SNPs associated fail to explain the mechanism for the development of AF.
Endothelins (ETs) are a family of 21 amino acid peptides comprising three isoforms such as ET-1, ET-2, and ET-3. [10] The only isoform thought to be constitutively released from endothelial cells is ET-1, which is believed to play an important role in regulating vascular function. The elevation of ET level in patients suffering from coronary artery disease is described by few authors. [11,12] ET-1 is thought to play an important role in the etiologies of hypertension, atherosclerosis, and cardiovascular disease as well as other vascular events. Apart from its vasoconstrictor properties, inotropic and chronotropic effects, ET-1 is thought to have a pro-arrhythmogenic effect as well. [13] We hypothesized that there exists an association between ET-1 gene polymorphism and postoperative AF in patients undergoing CABG.

Aim of the study
The aim of the study was to elaborate the relationship between ET-1 gene polymorphism and postoperative AF in patients undergoing elective CABG.

Study design
The study design was a prospective clinical observational study.

Number of cases
Based on a retrospective data from our institute patients, the incidence of perioperative AF in CABG patients was found to be 25%. Considering a precision of 9%, the estimated sample size was 89 patients. We analyzed 98 patients within the period of study.

Selection of the subjects
Ninety-eight nonrelated, nondiabetic patients over a period of 4 years undergoing routine CABG in Cardiothoracic Sciences Centre, All India Institute of Medical Sciences were included in the study. The study was performed after Hospital Ethical Committee clearance and written informed consent from the patients. The patients with family history of AF or any other arrhythmia, associated comorbidities such as diabetes, chronic obstructive pulmonary disease, hyperthyroidism, psychiatric illness, cardiomyopathy, patients on ventilator, those with severe left ventricular dysfunction, or any drug or alcohol abuse were excluded from the study.

Sample collection and plasma separation
Ten milliliters of venous blood sample was collected before surgery in prechilled tubes containing ethylenediaminetetraacetic acid. Plasma was separated by centrifugation at the rate of 4000 rpm for 15 min at room temperature. The plasma was stored in aliquots of 1 ml each at −70°C until the time of assay. An aliquot of the plasma was lyophilized and reconstituted in 250 ml of double-distilled water before the assay. This was done to ensure that the plasma levels of ET levels being in the range of 1-3 pg/ml. The concentration factor was taken into account at the calculation of results.

Endothelin immunoassay
Plasma levels of ET were determined by acetylcholine esterase-based immunoassay -ACETM (Cayman Chemical Co., USA#583151) which is a double-antibody "sandwitch" ELISA technique where each well of microtiter plate was precoated with a microclonal antibody specific for ET. A purified recombinant human ET-1 served as standard. The concentration of standard ET-1 used was 80, 70, 60, 50, 40, 30, 20, and 10 g/ml. The standard curve of ET was plotted by taking concentration (pg/ml) on X-axis and corresponding absorbance on Y-axis. The concentration of ET in unknown samples was calculated from the standard curve.
The peripheral blood mononuclear cells and red blood cells left after the separation of blood plasma was taken as the starting material for isolation of genomic DNA.

Primer design for endothelin gene and its receptor
The primers were designed using bioinformatics tools and the software gene runner for primer design. The genomic sequel(s) of the contig(s) spanning region of interest in the present study were obtained from Reference Sequence database available at the National Center for Biotechnology Information site (http://www.ncbi.nlm.nih.gov). The designed primer sequences are mentioned in Table 1 and primers were custom synthesized commercially.

Polymerase chain reaction
The polymerase chain reaction (PCR) reactions were carried out in the Eppendorf Masterycler personal system. All sequencing reactions were carried out using ABI PRISM BigDye Terminator Ready Reaction Kit version 3.1 (PE Applied Biosystems, CA, USA) and cycle sequencing was carried out on the thermal cycler (Eppendorf Mastercycler personal). According to the objectives of work plan, it was proposed to genotype DNA extracted from peripheral blood of all study participants to look for five SNPs in ET gene and its receptors [ Table 2].

Anesthetic management
All the patients were managed by a single anesthesia, surgical, and perfusion team. The anesthesia management protocol was similar for all the patients. All the patients were treated with 2 g magnesium chloride postanesthesia induction. Patients underwent CABG on standard cardiopulmonary bypass (CPB) technique. The hemoglobin was maintained between 9 and 10 g/dl throughout CPB and >10 g/dl postoperatively. Postoperative pain management was done with intravenous fentanyl till extubation and oral tramadol or ketorolac thereafter. Weaning from mechanical ventilation and catecholamine infusion was guided by institute protocol. Patients were discharged from the Intensive Care Unit (ICU) to the nursing ward as soon as their hemodynamics, and respiratory condition was stable. Continuous monitoring of vital parameters and 5-lead electrocardiogram (ECG) was done using Siemens 7000 Bedside monitor (Siemens, Danvers, USA) during their ICU stay and thereafter continuously during the hospital stay along with a standard 12-lead ECG examination was carried out twice daily till the time of discharge. All the patients were monitored for any new onset of AF. AF was considered to be significant if it is persistent for >15 min and/or requires treatment because of hemodynamic compromise. Throughout the study duration, the serum potassium was maintained at a level of 4-4.5 meq/L.

Statistical analysis
It was carried out using Stata version 9.0 (College Station, Texas, USA). Data were presented as number% or mean ± standard deviation as appropriate. The univariate association between qualitative variable was evaluated using Chi-square test and Student's t-test was used for continuous variables. Bivariate, univariate, and logistic regression was carried out to find predictors of AF. Comparison of plasma level of ET along with genotype distributions together with their probability of occurrence and their association with plasma ET levels were determined by applying nonparametric Kruskal-Wallis and Mann-Whitney rank-sum test, and distribution of genotype was determined by Chi-square analysis. The association of SNP and plasma ET level was determined by Spearman's rank coefficient of correlation. A result was considered to be significant at P ≤ 0.05.

Results
A total of 98 patients were evaluated. The primary intention of this study was to target the drug therapy at the molecular genesis of AF in this subset of patients. The first part of the study was dedicated to identify ET-1 gene polymorphism and record the incidence of the development of AF at any given point of time from the beginning of the case till the time of discharge of the patient, whereas the second part of the study examined the predictors of AF and if there is any association between ET-1 gene polymorphism and AF. The summary statistics of demographics and preoperative profile are presented in Table 3.
Before the detailed analysis, the patients were further divided into two groups depending on the presence (AF+) or absence of AF (AF−). A comparative study of the clinical profile and perioperative variables of the groups is presented in Table 4. Bivariate analysis shown that the incidence of AF is more in males (71.42% vs. 28.5%, P = 0.05) and those with endarterectomy (26.035 vs. 45.71%, P = 0.02).   The occurrence of -1370 T/G All the three possible combinations of alleles, i.e., homozygous T/T, heterozygous T/G, and homozygous G/G were observed at varying frequencies in the AF+ and AF− groups and it is not significant as indicated by their "P" values [P = 0.05 in each case, Table 5].

The -134 (3A/4A) Ins/del
ET-1 gene site as reported in literature for hypertensive patients located in 5' flanking noncoding region of ET-1 gene. This site was amplified by PCR for all the participants and does not differ between the groups [P > 0.05 in each case, Table 6].

Lys198Asn (G/T) single nucleotide polymorphism of endothelin-1 gene
Three possible combinations of alleles, i.e., homozygous G/G, heterozygous G/T, and homozygous T/T were observed at varying frequencies in both the groups. There is a significant difference between probability of occurrence of T/T and G/T alleles with a higher number of both in AF+ group patients [40.62% vs. 21.21%, P = 0.04 and 25% vs. 45.05%, P = 0.05, respectively, Table 7].

His 323 His (C/T) single nucleotide polymorphism of endothelin receptor type A gene
Site in located in +69 bps from the begging of exon 6 of endothelin receptor type A gene. There is no difference between the occurrence of all the three alleles, i.e., C, C/T and T/T in both the groups [P > 0.05 in each, Table 8].

Leu 277 Leu (G/A) single nucleotide polymorphism of endothelin receptor type B gene
The site of this gene is located in +30 positions from the beginning of exon 4 of endothelin receptor type A gene. All the three possible combinations of alleles, i.e., homozygous G/G, heterozygous G/A, and homozygous A/A were observed at varying frequency in both the groups. The probability of occurrence for G/G, G/A, and A/A between both the groups as shown in Table 9 is comparable (P > 0.05 in each).

Association of atrial fibrillation and plasma endothelin level
A wide variation in plasma ET levels was observed in both the groups as a whole with minimum value obtained being 2 pg/ml and maximum 10 pg/ml. When observed plasma ET levels were compared in both the groups for each genotype, it was found that the mean ET levels in AF+ group were significantly higher than that in AF− group (9.56 ± 3.2 vs. 4.84 ± 2.2, P = 0.001). The association of plasma ET level with SNP of all genotypes of -1370 T/G, -134 3A/4A, Lys198Asn (G/T), His 323 His, and Leu 277 Leu (G/A) gene of both the groups is presented in Table 10. A strong association between higher plasma levels with -134 4A/4A, Lys198Asn G/G, and Leu 277 Leu G/A SNP was observed in patients with AF.

Discussion
The incidence of AF after CABG varies between 10% and 40% and it remains as a drain on hospital resources. [1,2] Incidence of AF in our study was 32.65% even with the use of magnesium and it is comparable with few reports in literature. [14,15] The incidence of AF still varies in spite of so many proven agents being used as prophylaxis. This is because of the fact that AF is multifactorial in origin.    stress and reperfusion injury have been extensively studied. The role of genetics in genesis of AF is the topic of recent interest. Some genetic mutations have been identified in familial AF. [16,17] While doing candidate gene studies, Otway et al. identified a single mutation in KCNQ1 in one family (arginine-to-cysteine change at amino acid position 14), that is associated with AF. [18] In one of the association studies, the frequency of a genetic marker (SNP) was compared between patients with or without AF. However, these studies have typically tested a small number of variants and have been directed at candidate genes previously believed to be involved in AF, i.e., genes encoding products involved in renin-angiotensin axis, calcium handling, ion channels, and interleukins. [19][20][21][22][23] With the advancement of genotype technology, investigators can assess hundreds of SNPs spread over the entire genome. Such studies have been used successfully in the past several years to identify potential novel pathways for diabetes, obesity, etc. Unfortunately, these studies have been limited by a lower probability of any polymorphism truly associated with AF. These studies are further limited by small sample size and a lack of replication in a distinct population and phenotype.

Most of the clinical factors and factors such as oxidative
We studied the SNPs that lies on the upstream of the ET gene (the gene that is associated with vasculature and myocardium) assuming a possible association. A higher ET level in AF patients was again an indirect clue to further study the association of AF with SNPs of ET gene. Lys198 gene is the most extensively studied ET gene polymorphism. T-allele of Lys198Asn has been found to be associated with higher ET-1 levels, increased receptor responsiveness to ET-1, increased risk of developing coronary artery disease, heart failure, ischemic stroke, variant angina, and induced hypertension. [13] The novel finding in our study is that it is the first study showing the association of Lys198Asn with another commonly prevalent disorder that is AF.
In general, for the chosen SNPs, there was no difference between the different allele types between the groups. However, there was a difference in the groups with a particular genotype. The strong association between higher plasma levels with -134 4A/4A, Lys198Asn G/G, and Leu 277 Leu G/A SNP in patients with AF. This may have an important implication on the expression levels of ET during the genesis of AF.

Limitations
While GWAS has the potential to identify new pathways for disease, they also have a number of limitations. In particular, with hundreds of thousands of individual associations being tested, these studies have a high likelihood of producing a false-positive result. There is still discussion within the field of what the threshold P value should be for genome-wide significance. False-positive results can also emerge from population stratification or the failure to properly control for ethnicity, thus resulting in over or under representation of spurious ethnic specific markers. While there have been proposed variations in study design in an effort to eliminate false associations, ultimately, replication of the results in other populations may be the best test of whether a result is a true positive. [9]

Conclusion
The study described the possible correlation of polymorphism of ET gene in CABG population from India. Polymorphisms of this gene may not have a direct effect on the initiation of AF, but it may have a modifying effect on the sustainability of AF. This study may provide a theoretical basis to further study regarding the genesis of AF and develop a targeted therapy for the refractory cases, the first steps toward a more individualized treatment of the arrhythmia. Ultimately, a greater understanding of the genetics of AF should yield insights into novel pathways, therapeutic targets, and diagnostic testing for this common arrhythmia in CABG patients. [9]