TRIB1 rs17321515 gene polymorphism increases the risk of coronary heart disease in general population and non-alcoholic fatty liver disease patients in Chinese Han population

Background Present evidences suggested that TRIB1 rs17321515 polymorphism was tightly associated with the increased risk of NAFLD and CHD. CHD is one of the main complications of NAFLD, whether TRIB1 rs17321515 polymorphism could affect the risk of CHD in general population and NAFLD patients in Chinese Han population was remain unknown. The present study was designed to investigate the association between TRIB1 rs17321515 polymorphism and the risk of CHD in general population and NAFLD patients in Chinese Han population, and investigate the effect of TRIB1 rs17321515 polymorphism on serum lipid levels. Patients and methods TRIB1 rs17321515 gene polymorphism was genotyped using the polymerase chain reaction (PCR) in healthy controls (n = 175), CHD patients (n = 155), NAFLD patients (n = 146), and NAFLD+CHD patients (n = 156). Serum lipid profiles were determined using biochemical methods. Statistical analyses were performed using SPSS 24.0 statistical software. Results The TRIB1 rs17321515 AA+GA genotypes were the significant risk factors for the CHD in general population (OR = 1.788; 95% CI: 1.104–2.897; P = 0.018) and in the NAFLD patients (OR = 1.760; 95% CI: 1.071–2.891; P = 0.026). After adjusted for age, gender, and body mass index, the risk for CHD in general population (OR = 1.857; 95% CI: 1.116–3.089; P = 0.017) and NAFLD patients was still significant (OR = 1.723; 95% CI: 1.033–2.873; P = 0.037). In addition, TRIB1 rs17321515 A carriers possess the higher lipid profiles in the included subjects. Conclusions TRIB1 rs17321515 AA+GA genotypes were significant associated with the risk of CHD in general population and in NAFLD patients in Chinese Han population. The rs17321515 A allele increases the serum lipid profiles in included subjects.


Background
Non-alcoholic fatty liver disease (NAFLD) is defined histologically as when more than 5% of liver cells are found to be suffering from steatosis, which is the manifestation of metabolic syndrome in liver [1]. The disease spectrum of NAFLD includes non-alcoholic fatty liver (NAFL), non-alcoholic steatohepatitis (NASH), fibrosis, liver cirrhosis and even hepatocellular carcinoma [2]. NAFLD has exceeded viral hepatitis and become the most prevalent liver disease in the world, and become the second largest cause of liver transplantation and the third largest cause of liver cancer [3]. Coronary heart disease (CHD) is a form of the metabolic syndrome, more and more epidemiology and clinical studies had been conducted to investigate the tightly association of NAFLD and CHD [4]. Increased evidences suggested that NAFLD is not only a marker of CHD and other structural, functional and arrhythmia complications, but also takes effects in the development and progression of these cardiac complications [5,6]. Therefore, patients with NAFLD will benefited from the more rigorous monitoring and early treatment interventions to reduce the risk of CHD and other cardiac and arrhythmia complications.
NAFLD and CHD possess several the same influence factors such as lipids metabolism disorder, obesity and insulin resistance [7]. In addition, genetic factor such as gene polymorphism was also the significant risk factor for the NAFLD and CHD [8][9][10][11]. Genome-wide association study (GWAS) revealed many gene polymorphisms such as PNPLA3 rs738409 and TM6SF2 rs58542926 which were the significant risk factors for NAFLD [12,13]. Abundant studies revealed that gene polymorphisms such as CD36 rs1761667, CDKN2BAS rs496892, and COX2 -765G > C were tightly associated with the risk of CHD in different countries [14][15][16]. Tribbles 1 (TRIB1) encoding Tribbles protein homolog 1 [17], proteins from the tribbles family include a MEK1-binding domain, an E3 ubiquitin ligase (COP1)-binding domain, and a pseudokinase domain, all of which are significant by interacting with respective partners [18]. GWAS in American identified that lots of novel genomic loci participated in the regulation of plasma triglyceride (TG) levels. One of those loci was the 8q24 locus, with the lead single nucleotide polymorphism (SNP) involving a linkage-disequilibrium block that contains the gene TRIB1 [19]. Although TRIB1 undergoes miRNA regulation, it is quite conserved in parts, containing a long 1.5 Kbp 3′ untranslated region (UTR) [20]. Under normal physiological conditions, TRIB1 can negatively regulate carbohydrate-responsive element-binding protein (ChREBP) via proteasome proteolysis ubiquitination, raising the microsomal triglyceride transfer protein (MTTP) to terminate lipogenesis. In the absence of TRIB1, the process of lipogenesis is aberrant and the serum lipid disorders was occurred [21]. GWAS studies have repeatedly confirmed that TRIB1 gene polymorphisms were associated with dyslipidemia [22][23][24][25], and several studies had shown that TRIB1 gene variants can increase the risk of CHD [26,27]. In addition, previous studies have confirmed that TRIB1 gene variation can also increase the risk of NAFLD [28][29][30].
In consideration of the prevalent complication of NAFLD and CHD, understanding of effect of genetic factors on the patients with NAFLD and CHD were Fig. 1 The flow chart of this study urgent and remains to be explored. The aim of this study was to investigate the association of TRIB1 rs17321515 gene polymorphism with the risk of CHD in NAFLD patients in Chinese Han population, and investigate the effect of TRIB1 rs17321515 on the serum lipid profiles of patients with NAFLD and CHD.

Study subjects
This study was approved by the Ethical Committee of Qingdao Municipal Hospital (Qingdao, China), and this study was conducted in accordance with the principles of the Helsinki declaration and its appendices [31]. All the subjects had signed the informed consent before participating in this study.
From June 2018 to November 2018, 146 NAFLD patients (65 females and 81 males, median age 64.00 years) diagnosed by B-type ultrasonography, 155 CHD patients (72 females and 83 males, median age 60.00 years) diagnosed by coronary angiography, 156 patients (80 females and 76 males, median 64.00 years) with both NAFLD and CHD diagnosed by B-type ultrasonography and coronary angiography, and 175 healthy controls (87 females and 88 males, median 62.00 years) that matched for sex and age were enrolled in this study. Clinical data of all the subjects were collected from the department of Gastroenterology  and Cardiology, and the Medical center of Qingdao Municipal Hospital. All the individuals were unrelated Northern Han Chinese origin. NAFLD was diagnosed by a standard clinical evaluation, according to the criteria of the American association for the study of liver diseases (AASLD) [32]. CHD was diagnosed by a percutaneous coronary angiography, with the presence of at least 50% stenosis in at least one of the coronary arteries. Subjects who had other liver diseases, other cardiac disorders or diabetes mellitus were excluded.

Biochemical analyses
The basic clinical information (name, gender, age, body height, and weight) were obtained by a standard study questionnaire. The body mass index (BMI) was calculated using the equation that mass (kg)/height (m) 2 . After a 12-h overnight fasting, blood sample was collected from median vein of each subject, and the blood sample was placed into an ethylene diamine tetraacetic acid (EDTA)-containing tube. The serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyltranspeptidase (GGT), alkaline phosphatase (ALP), triglyceride (TG), total cholesterol (TC), high-density lipoprotein (HDL), low-density lipoprotein (LDL), total bilirubin (TBIL), fasting plasma glucose (FPG) were measured by standard clinical laboratory techniques (IChem-540 automatic biochemical analyzer, Shenzhen, China), respectively. Environmental factors were excluded in the study.

Genomic DNA extraction and genotyping
Blood genomic DNA was isolated with a genomic DNA purification kit (TIANGEN, Beijing, China) according to the manufacturer's instructions and stored at − 20°C until use. The genotyping of TRIB1 rs17321515 was conducted using the polymerase chain reaction (PCR). Primers for PCR were 5′-ACGTTGGATGTAGAAGTCCCCTTCCC TTAG-3′ and 5′-ACGTTGGATGGAACAAGGACTT TCGTCCTC-3′. PCR amplification was performed under the following conditions: an initial denaturation at 94°C for 5 min, followed by 45 cycles of denaturing at 94°C for 20 s, annealing at 56°C for 30 s, and extending at 72°C for 1 min. The genotypes of rs17321515 were detected by direct DNA sequencing using the ABI veriti-384 Prism sequence detection system, and the raw data were analyzed using MassARRAY TYPER4.0 software (Agena, Inc). Genotyping was performed in blinded fashion and the success rates were > 95%.

Statistical analysis
Statistical analysis was performed using the statistical package for the social sciences (SPSS) version 24.0 (SPSS Inc. Chicago, IL, USA). The Hardy-Weinberg equilibrium of expected and observed genotype distribution was analyzed by Pearson's χ 2 test. After testing for normality, continuous variables were shown as the mean ± standard deviation or median (interquartile range) for normal and abnormal distributed parameters, respectively. The t-test and Wilcoxon rank sum test were used for comparison of continuous data between groups. The Genotypes and allele frequencies were evaluated using the χ 2 test. The association between SNP and CHD risk in NAFLD patients was estimated by computing odds ratios (ORs) and 95% confidence intervals (CI) from the binary logistic regression analyses. P < 0.05 was considered as statistically significant.

Clinical characteristics of the study participants
The flow chart of this study was shown in the Fig. 1. The clinical characteristics of CHD patients and healthy controls are shown in the Table 1. The two groups were matched for gender and age (all P > 0.05). The CHD patients had higher BMI value and serum levels of ALT, GGT, TG and FPG than healthy controls (all P < 0.05), besides, the serum level of HDL and LDL in CHD patients was significant lower compared to the healthy controls (all P < 0.05). The clinical characteristics of NAFLD+CHD patients and Genotype and allele distributions of TRIB1 rs17321515 The genotype distributions of TRIB1 rs17321515 in the four groups were in accordance with the Hardy-Weinberg equilibrium (all P > 0.05) ( Table 3). As described in the Table 4, there was significant difference in the allele distribution of TRIB1 rs17321515 between CHD patients and controls group (P = 0.031). The TRIB1 rs17321515 GA + AA genotypes were the significant risk factor for the development of CHD (OR = 1.788; 95% CI: 1.104-2.897; P = 0.018), after adjusted for age, gender, and body mass index, the risk of TRIB1 rs17321515 GA + AA genotypes were still marked (OR = 1.857; 95% CI: 1.116-3.089; P = 0.017) ( Table 5). In addition, there was significant difference in the allele distribution of TRIB1 rs17321515 between NAFLD+CHD patients and NAFLD group (P = 0.021) ( Table 4). The TRIB1 rs17321515 GA + AA genotypes were the significant risk factor for the development of CHD in NAFLD patients (OR = 1.760; 95% CI: 1.071-2.891; P = 0.026), after adjusted for age, gender, and body mass index, the risk of TRIB1 rs17321515 GA + AA genotypes were still marked (OR = 1.723; 95% CI: 1.033-2.873; P = 0.037) ( Table 5).

Association of the TRIB1 rs17321515 gene polymorphism with clinical parameters characteristics in each group subjects
We compared the TRIB1 rs17321515 A allele with the clinical characteristics of the four groups to estimate whether the rs17321515 A allele was correlated with clinical parameters. As the results shown in the Table 6, there was no significant difference between the A allele carriers and non-carriers in the overall series and in the NAFLD patients (all P > 0.05) ( Tables 6 and 7). Higher serum level of FPG in the A allele carriers was observed compared to the non-carriers in the CHD group (P = 0.021) ( Table 8). In the NAFLD+CHD patients, the A allele carriers had the higher serum HDL level than non-carriers (P = 0.015) ( Table 9). In the healthy controls, the A allele carriers had the higher serum TC level than non-carriers (P = 0.001) ( Table 10).

Discussion
NAFLD has become a worldwide public health problem. Clinical experience and epidemiological evidences showed  that NAFLD-related mortality not only referred to the liver itself, but also companied with the complication such as the increased risk of CHD [33,34]. NAFLD could aggravate insulin resistance in the body and liver that cause atherosclerotic dyslipidemia, and release a variety of pro-inflammatory, pro-coagulant, and pro-fibrotic mediators, which may play an important role in the pathophysiology of cardiac and arrhythmia complications [33,35]. Single nucleotide polymorphism (SNP) as a significant genetic factor, plays an important role in the development of NAFLD and CHD [36,37]. Multiple studies have confirmed that TRIB1 gene polymorphisms can increase the risk of NAFLD and the risk of CHD [28,29,38]. In this study, we investigated the relationship of TRIB1 rs17321515 gene polymorphism with the risk of CHD, and the risk of CHD in NAFLD patients in Chinese Han population for the first time. The results showed that TRIB1 rs17321515 GA + AA genotypes were significantly associated with the increased risk of CHD in healthy controls and NAFLD patients. Tribbles-1 (TRIB1) is one of the member of tribbles family which were first identified in Drosophila and possess the function to regulate the cell division and migration [39]. Previous studies confirmed that TRIB1 encodes a human homologue of the Drosophila tribbles protein, and the relationship of TRIB1 variant with the serum lipids metabolism was discovered by GWAS in the European population [40,41]. To further explore the role of TRIB1 in lipoprotein metabolism, Burkhardt et al. found the serum levels of TG and  cholesterol were inversely associated with the expression of hepatic TRIB1 in mice [17]. Several studies had reported the effect of TRIB1 rs17321515 on the serum lipids metabolism. In a Chinese Han cohort study, TRIB1 rs17321515 AA genotype carriers had higher TG level than GG genotype carriers [42]. Katalin et al. also found that TRIB1 rs17321515 AA genotype carriers had higher serum TG and TC levels than GG + GA genotypes carriers [43]. In consideration of the higher mortality rate of NAFLD-related CHD than the single liver disease, it is meaningful to investigate the effect of TRIB1 rs17321515 gene polymorphism on the risk of CHD in general population and NAFLD patients, and the effect on the serum lipids levels [4,44]. In this study, we observed that TRIB1 rs17321515 AA+GA genotypes were significantly associated with the risk of CHD in general Chinese Han population, Besides, TRIB1 rs17321515 AA+GA genotypes carriers in NAFLD patients had a higher CHD risk than GG genotype carriers.
Our results showed that TRIB1 rs17321515 polymorphism was a significant risk factor for CHD in the general population and could increase the CHD risk in NAFLD patients in Chinese Han population. Moreover, TRIB1 rs17321515 A allele could affect the serum lipids levels such as FPG, HDL, and TC in each group. Our conclusion is consistent with the previous studies which were conducted in other countries [26,43,45]. Some limitations of our study must be acknowledged. Firstly, all the included subjects in this study are Chinese Han nationality, our conclusion may not be applicable to other nationality absolutely. Secondly, lacking of liver biopsy is the main limitation of our study, however, liver biopsy is invasive, and may lead to a small probability of  serious morbidity. Thereby, we used abdominal ultrasound to diagnose NAFLD in the present study. Thirdly, some of our subjects were long-term users of lipid-lowering drugs, which will affect the accuracy of the results to some extent. Lastly, nutraceuticals can influence the lipids levels [46], but we did not consider this factor in this study.

Conclusion
In summary, we investigated the relationship of TRIB1 rs17321515 with the risk of CHD in general population and NAFLD patients in Chinese Han population for the first time. Our results showed that TRIB1 rs17321515 AA+GA genotypes are associated with an increased risk of CHD in general population and NAFLD patients, and the rs17321515 A allele affects the serum lipids levels of multiple lipid profiles in included subjects. Diverse ethnic groups and larger sample sizes are needed to investigate in further study to confirm the present data.

Funding
This study was supported by Grants of National Natural Science Foundation of China (31770837) and the Qingdao People's Livelihood Science and technology plan (18-6-1-68-nsh).

Availability of data and materials
None.

Ethics approval and consent to participate
The study protocol was approved by the Ethics Committee of Qingdao Municipal Hospital before participation (Approval NO.2017-20). All the subjects have signed written informed consent.

Consent for publication
Not applicable.