The Correlation Of FTO And LEP Gene Polymorphisms With Coronary Artery Disease In Chinese Han Nationality

BackgroundAt present, coronary artery disease is a polygenic complex diseases and obesity is one of the risk factors of coronary artery disease. The pathological basis of coronary artery disease is coronary atherosclerosis, the occurrence and development of CAD are closely related with genetic and environmental factors. FTO (fat mass and obesity associated, FTO) gene is by far the strongest obesity susceptibility gene. In2007, Frayling first reported the FTO gene. Genome-wide association studies have identified FTO gene common variants that are robustly associated with obesity, body mass index (BMI) and the risk of diabetes, hypertension, the metabolic syndrome, atherosclerosis and C-reactive protein. Since these cardiovascular risk factors play an important role in the etiology of coronary artery disease(CAD), obesity, diabetes, inflammation are involved in the onset and severity of CAD. Obesity is related to chronic subclinical inflammation which plays an essential role in the development of atherosclerosis. In a certain sense, diabetes is equal to the coronary artery disease. Therefore, We assume that the coronary artery disease and obesity, diabetes may have a common genetic basis, FTO gene polymorphisms may be associated with the risk of coronary artery disease. According to the literature retrieval, studies on obesity related gene polymorphisms and CAD are very little, only a few scholars in the study of type2diabetes mellitus. Otherwise subgroup analysis of the relationship between diabetes with coronary heart disease and FTO gene polymorphisms were taken. In2010Hubacek first reported that there were a significant association between the FTO gene rs17817449polymorphism and risk of acute coronary syndrome (ACS) in Caucasian males. But research on Africa Indian showed that FTO variant was not related with metabolic syndrome with acute myocardial infarction. The association of FTO gene single nucleotide polymorphism (SNP) and CAD is still uncertainty. Up to now, there is no direct study on the association between FTO gene polymorphism and CAD in China. Different races have genetic heterogeneity, susceptibility gene of CAD found by genome-wide association study (GWAS) need to be duplicated in a large sample of different races. Researches in the last ten years had shown that leptin was associated with obesity, dyslipidemia, insulin resistance, hypertension and atherosclerosis, it was one of the risk factors of CAD.The LEP gene encoding leptin was one of obesity-associated gene. Lep gene and FTO gene play an important part mainly by hypothalamus, inhibiting the food intake to keep energy balance in the same way. Researches also confirmed the existence of correlation of leptin and FTO gene, the specific mechanism is still not clear. Obesity related SNP in FTO gene are mainly located in the first intron and it was confirmed in the highly conserved gene sequence.The most well-studied locus rs9939609were in strong linkage disequilibrium with rs17817449and other loci, and its genotyping was high success rate. A large number of studies have reported a significant association of the intronic SNP rs9939609of FTO gene with obesity and with a number of obesity-related features. Therefore our experiment chooses the typical sites of FTO rs9939609, rs17817449 and LEP rs10954174as tagging SNP. We evaluated the relationships between the3SNPs and CAD.This was a case-control study, It was the first time that we studied the relationship between the3SNPs polymorphisms and risk of CAD in Chinese Han populations. It is well-known that DNA sequencing is the highest accuracy method for detecting gene polymorphisms. So we took direct sequencing method to determine the genotypes of FTO and LEP gene and analyze the correlation between gene variant and CAD-related subclinical phenotype (hypertension, high blood lipid, high blood sugar and high homocysteine, etc.). We aim to investigate the association between FTO, LEP gene polymorphisms and CAD in Chinese Han populations.Objective①To assay the allele and genotypes of fat mass and obesity-associated(FTO) gene rs9939609(A/T)、rs17817449(G/T) and LEP gene rs10954174(A/G) in Chinese Han CAD and Control patients.②To explore the correlation between FTO rs9939609(A/T)、rs17817449(G/T) and LEP rs10954174(A/G) polymorphisms and coronary artery disease(CAD).③To evaluate the relationship between the polymorphisms of FTO rs9939609(A/T)、rs17817449(G/T) and LEP rs10954174(A/G) and a number of CAD-related clinical parameters.Methods1. Subjects A total of245subjects,147CAD and98matched non-CAD(Control) patients, were randomly enrolled in this case-control study. All participants were from the department of cardiology, Shenzhen Longgang Central Hospital. The cases group included127male and20female, The control group included88male and10female. They were all Chinese Han populations by self-report. Informed written consent was obtained from all subjects before participation. The studies were approved by the regional ethical committees and were in accordance with the principles of the Declaration of Helsinki.2. Biochemical and anthropometrical measurements Anthropometrics including weight, height, waist circumference were measured at the baseline examination by trained persons with standardized methods. and Cardiometabolic risk factors, like blood pressure, fasting plasma glucose, total cholesterol, LDL-cholesterol, HDL-cholesterol, triglycerides, homocysteine and uric acid were measured by routine laboratory methods. Risk factors of CAD questionnaires were administered in both groups. Medical history were obtained from medical records of the subjects. Information about smoking, personal and family history of cardiovascular disease, and history of hypertension, diabetes mellitus, gout, hyperlipidemia,myocardial infarction, stroke and physical activity was recorded3. Genotyping Genomic DNA was isolated from peripheral whole blood cells by standard methods according to the manufacturer’s instructions and stored at-20℃. Genotyping of FTO gene (rs9939609, rsl7817449) and LEP rs10954174polymorphisms for all subjects were carried out using sequences retrieval method through the ABI3730sequencing equipment (Applied Biosystem) and genotypes were read using automated software. Reactions were run in5μL volumes using an amplification protocol of95℃for2minutes, followed by25cycles of95℃for10seconds,51℃for10seconds, then60℃for190seconds.4. Statistical analysis Statistical analysis was performed by SPSS version17.0statistical software. Data were presented as means±standard deviation (SD) or as percentages for discrete variables. Differences in quantitative traits between the two groups were analyzed by the independent sample T Test, the three groups by one-way ANOVA analysis. Differences in discrete variables were analyzed using χ2analysis and odds ratios (OR) were calculated using the "case-control" method. Frequency distribution of genotypes and alleles in the3SNPs were consistent with the Hardy-Weinberg equilibrium evaluating with χ2tests. Association between3SNPs and CAD and CAD-related phenotypes(BMI, WC, FPG and TC, TG, LDL-C, HDL-C, et al.) were analyzed by using the logistic regression analysis. The3SNPs were included as a dichotomous variable in dominant models. Statistical significance was taken at a P value<0.05for all comparisons.Results1. Basic characteristics of the participants The basic characteristics of the two groups under study were summarized. Gender, BMI, Systemic blood pressure and Diastolic blood pressure, TG, LDL-C, and UA were similar to the both groups (P>0.05). History of diabetes mellitus (DM) and gout were no significance in the two groups (P>0.05). The CAD patients have higher prevalence of smoking, hypertension and hyperlipidemia (P<0.05). Age, BMI, WC, FPG,TC and HCY were higher in CAD group than in Control. And HDL-C lower than that of the control group, the difference was statistically significant (P<0.05).2. In both analyzed groups, The genotype frequencies of FTO rs17817449were in accordance with the Hardy-Weinberg equilibrium. Distributions of FTO rs9939609and LEP rs10954174genotypes did not deviate from the Hardy-Weinberg equilibrium (P>0.05).3. The genotype frequencies of AT+AA and TT were26.53%and73.47%, and the A allele frequency of rs9939609was13.27%in CAD group and13.30%in control groups (P>0.05). 4. G allele frequency at rs17817449was16.67%in CAD and8.2%in Control group. The frequency of the GG genotype and G allele was significantly higher in CAD group than in control(4.8%vs.1.0%,16.67%vs8.2%, P<0.05). Carriers of rs17817449GG+GT genotype had the increased risk of CAD,4.85times higher than that of homozygous TT genotype (P=0.038, OR=4.85,95%CI:0.587～40.053). rs17817449at least one G allele carriers had a higher risk of CAD than non-G allele carriers (P=0.007, OR=2.250,95%CI:1.239～4.084).5. The LEP rs10954174A allele frequency was3.4%and4.08%, respectively in CAD and Control group (P>0.05).The frequency of the GA+AA genotype was lower in CAD group than in control(6.8%vs.8.2%, P>0.05).6. The2SNPs rs9939609and rs17817449in FTO gene were strongly in linkage disequilibrium (D=3.4%, r2=0.97>0.33).7. There was a significant association between rs9939609(A/T) variation and BMI, WC, SBP, DBP, FPG, TC, LDL-C in both CAD and control group (P<0.05). There was a higher prevalence of hypertension and hyperlipidemia history in AA+AT genotypes than that of TT genotypes (P<0.05). There was no significant association between FTO rs9939609(A/T)) polymorphisms and TG, HDL-C, UA, HCY and medical history of smoking, diabetes and gout in both groups (P>0.05).8. There was no significant association between FTO rs17817449(G/T) polymorphisms and DBP, UA,FPG, TG, TC, HDL-C and HCY in CAD and Control group (P>0.05). The history of diabetes, gout and hyperlipidemia were similar in the both groups(P>0.05). BMI,WC, SBP, LDL-C were higher in FTO rs17817449GG homozygotes than that of TT genotype. Difference were significant in the two kinds of genotypes(P<0.05).9. All of the CAD-related parameters were no any significant differences in LEP rs10954174GG genotype and GA+AA genotypes in the both groups (P>0.05).10. The results of Logistic regression analysis showed, The risk factors of CAD were body mass index (BMI), fasting blood glucose (FPG), triglyceride (TG), HDL-C, hyperlipidemia history and rs17817449G allele. The rs17817449G allele was significantly associated with coronary artery disease risk (P<0.05, OR=25.81,95%CI:2.348～285). The association was independent of BMI, WC, smoking, history of hypertension, history of diabetes and serum total cholesterol, low density lipoprotein cholesterol, uric acid, homocysteine.Conclusions1. Association of FTO gene rs9939609(A/T) polymorphisms with coronary artery disease(CAD) in Chinese Han nationality was not observed. the allele A may be a risk factor of CAD in Chinese Han populations.2. The single nucleotide polymorphism (SNP) of rs17817449(G/T) in FTO gene was associated with CAD, GG genotypes carriers have higher risk of CAD than TT carriers in Chinese Han populations.3. No any association was found between LEP rs10954174polymorphisms and CAD-related quantitative traits and obesity-associated parameters.4. These results replicate the association of FTO rs9939609(A/T) and rs17817449(G/T) variants with BMI in Chinese Han populations. And FTO rs9939609and rs17817449were strongly in linkage disequilibrium.5. Our data provide preliminary evidence that FTO and LEP polymorphisms may lead to increased risk of CAD in Chinese Han populations. The influence of these genotypes on CAD risk warrants further investigation.