Association of single Nucleotide Missence Polymorphism Val109Asp of Omentin-1 gene and coronary artery disease in Pakistani population: Multicenter study

Background & Objective: Coronary artery disease (CAD) is a most important cause of morbidity and mortality worldwide as well as in Pakistan. Recent studies have shown that the combination of obesity, insulin resistance and fluctuation in circulating adipocytokines levels is associated with the pathogenesis of coronary artery disease. Omentin-1 is recently found adipocytokine that is highly expressed in visceral adipose tissue. It has anti- inflammatory properties and is negatively correlated with ischemic heart disease. Therefore, this study was designed to investigate the relationship between omentin-1 Val109Asp polymorphism and CAD in Pakistani population. Methods: A total of 350 subjects were included in the study. Two hundred fifty were diagnosed with coronary artery disease while 100 served as healthy controls. PCR-RFLP was performed at Dr. A Q. Khan Institute of Biotechnology (KIBGE) to analyze Val109Asp polymorphism. In this, case control study SPSS software version 16 (Chicago, IL, USA) was used for data analysis. Continuous variables and categorical variables were presented as mean±SD or in percentage. Independent sample test and chi-square test was performed to compare the differences in means between cases and controls. Genotype distribution was analyzed by chi-square test and results were presented as percentage and frequency. Multivarible regression analysis indicated that Val109Asp SNP might be an independent risk factor for CAD susceptibility after adjustment for some well- known CAD risk factors including age, gender, body mass index, smoking, hypertension, diabetes mellitus and lipid abnormalities. There was estimation of odd ratios (OR) and 95% confidence intervals (CIs) to determine the correlation between genotypes and the risk of CAD. (p> 0.05). Genotype frequencies were compared by Chi-square test. Results: There was prevalence of Omentin-1 Val109Asp polymorphism in both case and control groups. However, Val/Asp (heterozygous mutant) genotype was detected more frequently in patients with CAD, OR(95%)=1.921; CI=1.173-3.1469 in comparison of Asp/Asp and Val/Val genotypes. Conclusion: Individuals having Val/Asp heterozygous gemotype of omentin-1 gene polymorphism are at more risk of developing CAD in Pakistani population, further studies are required in different populations and ethnicities to confirm our findings.


INTRODUCTION
Coronary artery disease (CAD) is emerging as the principal killer of the 21st century in both developed and developing countries. Globally 17.4 million deaths, that is29% of all deaths occur due to CAD, per year. 1 The prevalence of CAD in Pakistan is about 6%. 2,3 Atherosclerosis is the single most significant cause of CAD. It is characterized by chronic inflammation of arteries with development of fatty plaque in arterial wall, leads to ischemia of heart, brain and extremities. 4 Inflammation is caused by several hormones like proteins called adipokines, released from adipose tissues, which now, considered as main endocrine tissue of the body. 5 Omentin-1 is a recently identified, visceral adipose tissue derived adipokine consists of 295 amino acids, coded by gene located at chromosome 1q 21-23. 6,7 Omentin-1 is reported to be highly expressed in epicardial adipose tissue (EAT) around the heart and coronary arteries. 8 Omentin-1 is considered as an anti-inflammatory adiopokine. 9 Recent studies have shown that Serum levels of omentin-1 significantly reduced in coronary heart disease and associates negatively with markers of atherosclerosis including: body mass index (BMI), waist circumference, HDL and hsC-reactive protein. So, omentin-1 may serve as a novel biomarker for early detection of CAD. 10,11 Single gene missence polymorphism in exon 4 of omentin-1 gene was identified by Scaffler in 2007 by random sequencing and made known that nucleotide +326 is polymorphic A/T. However; significance of missense polymorphism Val109Asp in the human Omentin-1 gene in connection with CAD has not been studied yet in Pakistan.
This study aimed to establish local perspective as there is paucity of local data and was designed to determine whether omentin-1 gene polymorphism is a predisposing factor in development of CAD in Pakistani population because coronary artery disease and its management is emerging as a great challenge to deal with, hence, data collected from this study will help in establishing the role of omentin-1 in pathophysiology and therapy of CAD.

METHODS
This study was approved by the ethical review board committees of concerned institutions. A total of 250 patients were enrolled in this study between the period of January 2016 to April 2016 in Karachi Institute of Heart Disease (KIHD), Civil Hospital Karachi (CHK) and Darulsehat Hospital Karachi. All patients had undergone coronary angiography. CAD was defined as more than 50% obstruction of one or more coronary arteries. Severe chest pain, ST segment elevation and T wave inversions in more than two leads in ECG, elevated Troponin-I >0.01ng/ml) and positive stress test for ischemia were indications of angiography. The acute infections, malignancy, valvular heart disease, liver disease (ALT >59 units/L) and renal disorders (Creatinine >1.2 mg/dl) were excluded from the study. History of risk factors such as diet, lack of exercise, smoking, family history of cardiac disease, hypertension, diabetes mellitus, and hypercholesterolemia was recorded. Age and gender matched 100 controls were included in this study. Controls were defined as individulas had negative exercise tolerance test or less than 50% stenosis of coronary arteries on angiography, moreover, those control subjects who reported arthritis, diabetes, renal disorders and history of recent infections were excluded from the study. All participants were informed about the methods and benfits of this medical research and their informed consents were taken. Clinical and laboratory measurements: The history of disease was recorded in detail, Body mass index of all participants were calculated as kg/m 2 . Waist circumference was measured between upper margins of iliac crests and lower border of rib cage. Morning blood samples were collected in EDTA tubes after 12 hour overnight fasting. Routine lab tests including serum cholesterol, triglycerides (TC), high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C), fasting blood sugar (FBS), serum creatinine were analyzed by an automated analyzer (Olympus Irish branch) in hospital laboratory. DNA Extraction: Genomic DNA was extracted from whole blood via salting out extraction method (Miller et al.,1988). Extracted DNA was quantified by Nano drop (Thermo scientific USA). The integrity of DNA was checked by resolving 3ul of genomic DNA in 0.8% agarose gel in horizontal gel electrophoresis. Statistical analysis: In this study SPSS software version 16 (Chicago, IL, USA) was used for data analysis. Continuous variables and categorical variables were presented as mean ± SD or in percentage. Independent sample t-test and chi-square test was performed to compare the differences in means between cases and controls. Genotype distribution was analyzed by chisquare test and results were presented as percentage and frequency. Multivariate logistic regression analysis was performed with adjustment for age, BMI,waist circumference and the prevalence of smoking, hypertension, diabete and family history of CAD. The association of AT genotype and CAD was estimated using dominant model (defined as AT vs. TT + AA) and recessive model (defined as TT + AT vs. AA), respectively.
Odds ratios (OR) with their 95% confidence intervals (CI) were reported. A multivariate logistic regression analysis was used for association analyses with adjustments for age, gender, BMI, smoking, hypertension, and diabetes mellitus. P-value < 0.05 was considered statistically significant. There was estimation of odd ratios (OR) and 95% confidence intervals (CIs) to determine the correlation between genotypes and the risk of CAD (p> 0.05).

RESULTS
In current study, 250 patients diagnosed with CAD (92 females and 158 males with a mean age 51.39 ± 6.3 years) and 100 control subjects with normal blood vessels (21 females and 79males with mean age 49.78 ± 6.4) were recruited. The demographic, clinical and anthropometric parameters of the both case and control groups are shown in Table-I. There is no significant difference between two groups in Shazia Nazar et al.  (Table-III). Since CAD patients had higher frequency of abnormality in serum levels of HDL-C and blood glucose and were found with strong history of cardiac disease in family and hypertension as compared to controls, so, multivariate logistic regression analysis was used to exclude the effect of confounding factors on genetic association. AT genotype still demonstrated the strong association with CAD in our samples after adjustment for those well-known CAD risk factors including age, gender, BMI, smoking, hypertension, diabetes mellitus, lipid abnormalities suggesting that AT genotype might be an independent risk factor for CAD susceptibility (Table-IV).

DISCUSSION
The present study has established that omentin-1 gene Val109Asp (326 +A/T) missense polymorphism is frequently present in both groups of current Shazia Nazar et al.   (Table-II). To the best of our knowledge there is a single study in literature by Yörük et al. who investigated the relatioship of omentin-1 gene Val109Asp polymorphism and CAD in Turkish population but found no significant association reason might be recruitment of small number of CAD patients, however, it was observed that Val/Val heterozygous genotype was more frequent in CAD patients. 12 We analyzed omentin-1 gene Val109Asp polymorphism in CAD patients in Pakistani population and found the significant difference between cases and controls regarding this SNP in terms of genotype distribution, although we could not find any significant association between Val109Asp polymorphism and standard clinical, and metabolic factors. Subjects reported with strong family history of CAD had been identified with more heterozygous genotype Val/Asp. Male predominance with this SNP had also been observed in current study (Table-III). By multivariable regression analysis, our data showed that AT genotype still remained the strong association with HU after adjustment for some well-known CAD risk factors included age, gender, BMI,waist circumference, smoking, blood pressure, blood glucose, cholesterol and diet, suggesting Val109Asp might be an independent risk factor for CAD susceptibility (Table-IV). It has been documented by various resent studies that the adipose tissue is not just a fat deposit but it also acts as an endocrine tissue and produces numerous hormone like proteins called adipokines including: leptin, adiponectin, vaspin, TNFa, IL 6, IL 10, resistin, visfatin and omentin. 13 Visceral adipose tissue is dynamically involve in inflammation and its hypertrophy is associated with pathogenesis of various disorders like diabetes, metabolic syndrome, arthritis and CAD. 14 Omentin-1 is newly identified protein secreted by omental visceral fat. It has metabolic effects on myocytes, hepatocytes and adipocytes. It acts as an anti-inflammatory protein via nuclear factor Kappa b(NFkb) pathway. 15 The location of omentin-1 gene is q22-q23 of chromosome 1, this location has found to be linked with Type-2 diabetes mellitus in some populations. In-vitro experiments have been shown that omentin-1 recombinant therapy enhanced glucose uptake by cells via Akt signaling pathway 16,17 , moreover, plasma concentration of omentin-1 and its mRNA expression in VAT is found to be reduced in obesity and diabetes. 18 Some studies have made known the correlation between obesity, insulin resistance adipokines and pathogenesis of CAD. 19,20 Yamawaki et al. had demonstrated that omentin-1 may directly induced vascular relaxation via inhibition of endothelial nitric oxide synthase (eNOS). 21 As omentin-1is highly expressed by epicardial fats, so it might has protective role in coronary artery atherosclerosis and obesity related cardiac events. 22 After considering these outcomes that serum omentin-1 circulating levels are significantly associated with coronary artery atherosclerosis, it seems reasonable to investigate omentin-1 gene polymorphism and its correlation with of CAD in pakistani population. Although, several disease like psoriasis 23 , diabetes 14 , rheumatoid arthritis 24 had been investigatd in association with Val109Asp SNP but found no significant correlation.
In current study CAD patients were recruited from three different centers of Karachi, belong to different ethinic groups. The common factor that observed was central obesity even with normal BMI. As omentin-1 expressed from visceral fats so it seem reasonable to investigate the omentin-1 polymor-  phism and coronary artery disease. So the data from this study will help the researchers to investigate the pathophysiology behind the association between polymorphism and CAD, which is still unknown. Current study made evident that there is a significant association between omentin-1 polymorphism and risk of developing coronary artery disease.

CONCLUSION
This is perhaps the first study in Pakistan that has established a significant association between omentin-1 Val109Asp polymorphism and coronary artery disease. It also revealed that heterozygous Val/ Asp genotype of omentin-1 gene is more associated with risk of developing CAD in Pakistani population while healthy subjects were found with more Asp/ Asp genotype, however studies with different ethnicities are required to confirm our findings.

Rationale of study:
Our study, to best of our knowledge, is the first study in Pakistan that established the association of omentin-1 gene with coronary artery disease. This study not only revealed the genetic aspect of coronary artery disease but also provide a local data which help in future studies.