Association of AGT rs2493132 Polymorphism and the Risk of Coronary Artery Disease in Patients with Non-alcoholic Fatty Liver Disease in Chinese Han Population


 Background: Previous studies proved that AGT gene polymorphisms were associated with the serum lipids metabolism; however, it is not clear whether AGT rs2493132 increases the risk of NAFLD and CAD. This study was designed to investigate the relationship between AGT rs2493132 gene polymorphism and the developing risk of NAFLD and CAD in Chinese Han population.Methods: A total of 142 NAFLD patients, 109 NAFLD + CAD patients, and 246 health controls were included in this study. PCR method was used to measure AGT genotype. The anthropometric and clinical data were measured in clinical laboratory department of Qingdao Municipal Hospital. Statistical analyzes were conducted using the SPSS statistical software, version 21.0. Results: The AGT rs2493132 CT + TT genotype was an important risk factor for CAD in patients with NAFLD (OR = 1.785; 95%CI: 1.061-3.003; P = 0.029), and the risk of CAD in patients with NAFLD was still marked after adjusted for gender, age, and BMI (OR = 4.718; 95%CI: 1.502-14.824; P = 0.008). The T allele of AGT rs2493132 also significantly increased the risk of CAD in NAFLD patients (OR = 2.436; 95% CI: 1.184-5.011, P = 0.016) after adjusted for age, gender, and BMI. In addition, AGT rs2493132 T allele carriers possess the higher TC and LDL levels compared to the non-carriers in all the participants (Both P < 0.05).Conclusions: AGT rs2493132 CT + TT genotype and T allele significantly increased the developing risk of CAD in the patients with NAFLD in Chinese Han population. AGT rs2493132 T allele associated with the increased serum TC and LDL levels.


Background
Non-alcoholic fatty liver disease (NAFLD) is the most prevalent chronic liver disease, and the prevalence of NAFLD in the Asian was as high as 29.62% [1]. As a hepatic manifestation of metabolic syndrome, NAFLD has been proven to be an independent risk factor for cardiovascular disease (CVD) [2][3][4][5]. The occurrence rate of coronary artery disease (CAD) increased 4.7 times in the adult NAFLD population and the severity of CAD was also signi cantly associated with the presence of NAFLD [6]. Multiple factors contribute to the pathogenesis of NAFLD and CAD [7]. Accumulated evidence suggested that gene polymorphism is an important risk factor not only for the occurrence of NAFLD or CAD in the general population, but also for the occurrence of CAD in the NAFLD patients [3,8]. Simons et al. investigated the effect of three variants in the NAFLD susceptibility genes TM6SF2, MBOAT7, and PNPLA3 on the developing risk of CAD in the NAFLD patients [9]. The results showed that TM6SF2 rs5854292 and PNPLA3 rs738409 possess the protective effect for the development of CAD, while the variant in MBOAT7 rs641738 exerted a neutral effect on CAD risk. Glucokinase regulatory protein gene (GCKR) participates in the de novo lipogenesis, and the case-control studies suggested that the variation of GCKR rs780094 was tightly related to NAFLD [10,11]. A recent meta-analysis (include 274,625 individuals) demonstrated a signi cant association between the GCKR rs780094 and the risk of CAD [12]. The different effects of gene polymorphisms on plasma lipids may be a vital mediator between NAFLD and the risk of CAD [3].
Angiotensinogen (AGT) mainly derives from hepatocytes, and is the precursor of all the angiotensin peptides, which plays an undoubted role in blood pressure regulation [13][14][15]. In addition, AGT has been also found possesses the potential role on metabolic disorder [16]. In the low-density lipoprotein receptor-/-mice, down-regulated the expression of AGT could maintain the lower plasma cholesterol levels and minimize atherosclerotic damage compared to the wild-type mice, whereas up-regulated the expression of AGT could increase the risk of atherosclerosis, diet-induced body weight gain and liver steatosis [14]. In addition, Tao et al. found that hepatocyte-speci c AGT de cient in mice could protect the mice from western diet-induced liver steatosis and resisted to insulin resistance [16]. Recently, several clinical studies have pointed out the relationship between AGT gene polymorphism and the regulation of serum lipid metabolism. AGT rs699 CC genotype was signi cantly associated with high-density lipoprotein (HDL) levels in peripheral arterial diseases patients [17]. Khamlaoui et al. reported that AGT M235T genotype was associated with the serum levels of low-density lipoprotein (LDL) and total cholesterol (TC) (all P < 0.05), and AGT M235T polymorphism increase the risk of overweight-obesity [18]. Ji et al. suggested that AGT rs2493132 was associated with the hypertension in Chinese Han population, which indicated that AGT rs2493132 might be the risk factor for CAD [19].
Although the relationship of AGT gene polymorphisms with the serum lipids metabolism and CAD has been investigated in several studies, but the de nite relationship of AGT gene polymorphism with the developing risk of NAFLD and CAD is remains unclear. This study was designed to investigate the effect of AGT rs2493132 on the developing risk of NAFLD and CAD in the Chinese Han population, and investigate the effect of AGT rs2493132 on the serum lipid pro les.

Study subjects
This study was approved by the ethics committee of Qingdao Municipal Hospital and was conducted according to the principles of the Helsinki declaration and its appendices [20]. All the participants lled out the informed consent form before this study. From July 2017 to December 2018, a total of 109 NAFLD patients with CAD (NAFLD + CAD), 142 NAFLD patients without CAD, and 246 healthy controls were recruited in Qingdao Municipal Hospital. The diagnosis of NAFLD was based on the guidelines of the American association for the study of liver disease (AASLD) [21]. CAD was diagnosed by a percutaneous coronary angiography, with at least 50% stenosis in at least one of the coronary arteries. Subjects with other liver diseases, such as autoimmune hepatitis, drug-induced hepatitis, viral hepatitis, hepatocellular carcinoma, were excluded from this study.

Biochemical Analyses
Demographic characteristics (age and gender), anthropometric (height and weight), and blood pressure were recorded by the questionnaire. The body mass index (BMI) was calculated as weight (kg) / height (m) 2 . Blood samples were collected from each subject's median vein for biochemical analysis following 12 hours of fasting. Serum aspartic aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), gamma-glutamine transpeptidase (GGT), triglyceride (TG), TC, HDL, LDL, and fasting blood glucose (FPG) were detected in clinical laboratory department of our hospital.

Genomic Dna Extraction And Genotyping
Genomic DNA was extracted from EDTA anticoagulated whole blood using TIANamp Blood DNA Kit (Tiangen Biochemical Technology, Beijing, China) and stored at -20 °C until use. AGT rs2493132 was genotyped by polymerase chain reaction with the corresponding primers 5′-ACGTTGGATGGGATTTGACATGAGAGTGGC-3′ and 5′-ACGTTGGATGATTTACCTAGCCTCCGGATG − 3′. ABI veriti-384 Prism sequence detection system was used to detect the genotype of rs2493132 by direct DNA sequencing, and MassARRAY TYPER4.0 software (Agena, Inc) was used to analyze the original data.

Statistical analysis
All analysis was performed using SPSS software (version 21.0; SPSS, Chicago, IL, USA). The Pearson's χ 2 test was used to analyzing the Hardy-Weinberg equilibrium in the distributions of genotype among subjects. Continuous variables were compared by unpaired Student's t-test, and expressed as mean ± standard deviation (SD) when normally distributed. Categorical variables were compared using the Chisquare test. The association between AGT rs2493132 and the risk of NAFLD and NAFLD + CAD was calculated by computing the crude and adjusted odds ratios (ORs) and 95% con dence intervals (CI) from the logistic regression analyses. Statistical signi cance was considered as P < 0.05.

Clinical characteristics of the study population
The clinical characteristics of 142 patients with NAFLD (mean age 40.95 ± 5.96 years), 109 patients with NAFLD + CAD patients (mean age 59.05 ± 7.95 years), and 246 health controls (mean age 47.40 ± 11.52 years) were summarized in the Table 1. As the results shown, patients with NAFLD had the higher BMI, serum ALT, AST, ALP, TG levels, SBP and DBP than the health controls (all P < 0.05). There were the high levels of BMI, serum ALT, ALP, TG levels, and SBP, and the low serum levels of TC, HDL, LDL in the patients with NAFLD + CAD compared to the patients with NAFLD (all P < 0.05). In addition, patients with NAFLD + CAD had the higher BMI, serum levels of GGT, TC, HDL, and LDL, and the lower serum ALP level and SBP than patient with NAFLD (all P < 0.05).

Genotypes And Allele Distribution Of Agt Rs2493132
The genotype distributions of AGT rs2493132 in health controls group, NAFLD group and NAFLD + CAD group were con rmed with the Hardy-Weinberg equilibrium (P = 0.921, 0.298, and 0.579; respectively) ( Table 2). There was no signi cant difference of the genotype distributions of AGT rs2493132 between the NAFLD group and health controls group, and between the NAFLD + CAD group and NAFLD group (Both P > 0.05). In addition, the distribution of AGT rs2493132 allele between the NAFLD group and health controls group, and between the NAFLD + CAD group and NAFLD group were also not signi cant different (Both P > 0.05) ( Table 3). The results of logistic regression analysis suggested that AGT rs2493132 CT + TT genotype was the important risk factor for the CAD in the NAFLD patients (OR =    Association of the AGT rs2493132 polymorphism with the clinical characteristics in the overall series To investigate the effect of AGT rs2493132 polymorphism on the clinical parameters in the enrolled subjects, we compared the difference of clinical parameters of the AGT rs2493132 T allele carriers and non-carriers in the overall series. As the results shown in the Table 5, the serum levels of TC and LDL in the T allele carriers were higher than that of non-carriers (P = 0.045 and 0.044). There was no signi cant difference in other parameters such as ALT, AST, GGT, ALP, TG and HDL between the T allele carriers and non-carriers (all P > 0.05). The above results suggested that AGT rs2493132 T allele variant may increase the serum TC and LDL levels in the general population. Discussion NAFLD is a multisystem disease and tightly associated with the risk of CVD [22]. Previous studies proven that NAFLD is an independent risk factor for CAD, and CAD causes the majority death among NAFLD patients [22][23][24]. In a study conducted in Korea, Lee et al. showed that NAFLD was a independent predictor of CAD, and coronary artery stenosis was tightly correlated with fatty liver at the gradedependent manner (P = 0.025) [25]. Accumulated studies had demonstrated that gene polymorphisms were the signi cant risk factor for the NAFLD and CAD. In the Chinese Han population, Liu et al. reported that genetic variants of TRIB1 rs17321515 could increase the risk of CAD in patients with NAFLD [26]. Cheng et al. showed that the TNF-α-238 gene polymorphism also increases the developing risk of CAD in NAFLD patients in Chinese Han population [27]. Moreover, variants in PNPLA3 and TM6SF2 predispose to NAFLD conferred a mild protective effect for CAD, and both TM6SF2 and PNPLA3 gene polymorphisms were associated with lower plasma lipid levels [3,28]. These results suggested that gene polymorphisms play an important role in the development of CAD in patients with NAFLD, gene polymorphisms may exert the protective or risk dangerous role for the CAD in patients with NAFLD.
Previous study showed that the AGT M35T gene polymorphism was strongly associated with the increased risk of heart failure [29]. A study in Saudi Arabia reported that AGT variants (rs5051C and rs7079G) were related to CAD [30]. In the current study, we investigated the effect of AGT rs2493132 polymorphism on the risk of NAFLD in general population and the risk of CAD in patients with NAFLD for the rst time. We found that the AGT rs2493132 CT + TT genotype was tightly associated with the developing risk of CAD in the NAFLD patients, but not the AGT rs2493132 T allele. After adjusted for the age, gender, and BMI, the association of AGT rs2493132 CT + TT genotype with the risk of CAD was remains exist. In addition, the AGT rs2493132 T allele also signi cant increases the developing risk of CAD in the patients with NAFLD after adjusted for gender, age, and BMI.
Dyslipidemia is a common feature of both NAFLD and CAD patients which characterized by the elevated plasma triacylglycerol, circulating LDL particles and the reduced HDL [31][32][33][34]. Brouwers et al. suggested that plasma lipids act as the major mediators in the relationship between NAFLD and CVD [3]. The characteristics of subjects in this study showed that NAFLD patients possess the higher levers of BMI, AST, ALT, TG, LDL, GGT, and decreased level of HDL compare to health controls, that is consistent with the changes of serum lipids pro les in patients with NAFLD in previous studies [32,[35][36][37]. In the overall series in this study, we found that AGT rs2493132 T allele carriers were likely to possess the higher serum TC and LDL levels compared to the non-carriers. Previous study suggested that the decreased LDL levels could protect the NAFLD patients from the cardiovascular disease, and the NAFLD susceptibility genes confer to the strong relationship between plasma lipids and the risk of CAD [38,39]. These data suggested that AGT rs2493132 gene polymorphism may increase the risk of CAD in the NAFLD patients by affect the concentrations of serum TC and LDL levels.
There were several limitations in this study. Firstly, the diagnosis of NAFLD was conducted by ultrasonography rather than the liver biopsy due to the limited acceptability of liver biopsy in the clinical examination. Secondly, the CAD is usually occurred in the older peoples; therefore, the mean age in patients with NAFLD + CAD is higher than the NAFLD patients and the health controls. Thirdly, all the study subjects in the study were Han Chinese in Qingdao. Therefore, the role of AGT rs2493132 in the risk of NAFLD and CAD should be studied in other countries and ethnicity.

Conclusions
In summary, this study investigated the effect of AGT rs2493132 polymorphism on the risk of NAFLD and CAD in Chinese Han population. The results showed that AGT rs2493132 CT + TT genotype was tightly associated with the developing risk of CAD in the NAFLD patients. After adjusted for the age, gender, and BMI, AGT rs2493132 CT + TT genotype and T allele remain signi cantly increased the risk of CAD in patients with NAFLD. In addition, AGT rs2493132 T allele carriers possess the higher LDL and TC levels compared to the non-carriers in all the subjects. This is the rst time to investigate the effect of AGT rs2493132 polymorphism on the developing risk of NAFLD and CAD, the role of AGT rs2493132 in the risk of NAFLD and CAD should be studied in other countries and ethnicity in the further studies.
Abbreviations AASLD, American association for the study of liver disease; AGT, Angiotensinogen; ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartic aminotransferase; CI, con dence intervals; CAD, coronary artery disease; CVD, cardiovascular disease; FPG, fasting blood glucose; GCKR, Glucokinase regulatory protein gene; GGT, gamma-glutamine transpeptidase; HDL, high-density lipoprotein; LDL, low-density lipoprotein; NAFLD, Non-alcoholic fatty liver disease; OR, odds ratio; SD, standard deviation; TC, total cholesterol; TG, triglyceride Declarations Availability of data and materials All data generated or analyzed in this study are available from the corresponding author for the reasonable request.
Authors' contributions DMZ and ZJ, MXF, and LSS acquisition of data, analysis and interpretation of data, drafting the article, and nal approval; DMZ, ZJ, MXF, WMK, and WYF, acquisition of data, analysis and interpretation of data. XYN and LSS design this study and revision of this article. All authors have read and approved the manuscript.

Consent for publication
All the authors consent for this publication.
Ethics approval and consent to participate Not applicable.

Competing interests
The authors do not have any disclosures to report.

Funding
This study was supported by Grants of National Natural Science Foundation of China (31770837).