Association between MIF gene promoter rs755622 and susceptibility to coronary artery disease and inflammatory cytokines in the Chinese Han population

Macrophage migration inhibitory factor (MIF) is an essential mediator of atherosclerotic plaque progression and instability leading to intracoronary thrombosis, therefore contributing to coronary artery disease (CAD). In this study, we investigated the relationship between MIF gene polymorphism and CAD in Chinese Han population. Three single nucleotide polymorphisms (SNP, rs755622, rs1007888 and rs2096525) of MIF gene were genotyped by TaqMan genotyping assay in 1120 control participants and 1176 CAD patients. Coronary angiography was performed in all CAD patients and Gensini score was used to assess the severity of coronary artery lesions. The plasma levels of MIF and other inflammatory mediators were measured by ELISA. The CAD patients had a higher frequency of CC genotype and C allele of rs755622 compared with that in control subjects (CC genotype: 6.5% vs. 3.9%, P = 0.008, C allele: 24.0% vs. 20.6%, P = 0.005). The rs755622 CC genotype was associated with an increased risk of CAD (OR: 1.804, 95%CI: 1.221–2.664, P = 0.003). CAD patients with a variation of rs755622 CC genotype had significantly higher Gensini score compared with patients with GG or CG genotype (all P < 0.05). In addition, the circulating MIF level was highest in CAD patients carrying rs755622 CC genotype (40.7 ± 4.2 ng/mL) and then followed by GC (37.9 ± 3.4 ng/mL) or GG genotype (36.9 ± 3.7 ng/mL, all P < 0.01). Our study showed an essential relationship between the MIF gene rs755622 variation and CAD in Chinese Han population. Individuals who carrying MIF gene rs755622 CC genotype were more susceptible to CAD and had more severe coronary artery lesion. This variation also had a potential influence in circulating MIF levels.


Results
Clinical characteristics of the study population. We recruited 1176 CAD patients and 1120 controls in our study. The Clinical characteristics were shown in Table 1. We found that the CAD patients had higher levels of LDL-C, TG, TC and glucose and lower HDL-C level versus control participants (all P < 0.05). CAD patients were older and had a higher prevalence of hypertension and diabetes than control group (all P < 0.05), while gender ratio, smoking, BMI were comparable between the two groups.
CC genotype of MIF gene rs755622 was the susceptible factor for CAD. The distributions of the three SNPs (rs755622, rs10078888 and rs2096525) in MIF gene were according with Hardy-Weinberg equilibrium both in CAD and control groups. The detected frequency of rs755622 CC genotype was higher in CAD patients than in controls (6.5% vs. 3.9%, P = 0.008). We also found that the C allele was more common in CAD patients versus controls (24.0% vs. 20.6%, P = 0.005, Table 2). For the other two SNPs, rs1007888 and rs2096525, we did not observe prominent difference about the distribution of genotype or allele between the two groups Table 1. Clinical characteristics of including control participants and CAD patients. Continuous variables are expressed as mean ± SD. Categorical variables are expressed as percentages. CAD coronary artery disease, BMI body mass index, TG triglyceride, TC total cholesterol, LDL-C Low density lipoprotein-cholesterol, HDL-C high-density lipoprotein-cholesterol.   www.nature.com/scientificreports/ CC genotype of MIF gene rs755622 was associated with elevated MIF levels. We also investigated whether variation of MIF gene affect the expression of inflammatory cytokines such as MIF, MMP-9, IL-6 and IL-8 in randomly selected 123 controls and 283 CAD patients. The circulating MIF level in CAD patients was highest in rs755622 CC genotype carriers (40.7 ± 4.2 ng/mL) and then followed by GC (37.9 ± 3.4 ng/mL) or GG genotype carriers (36.9 ± 3.7 ng/mL, both P < 0.01 vs. CC genotype, Fig. 2A). These differences of MIF levels in CAD patients remained after correcting by age, lipids, hypertension and diabetes via general liner model analysis (CC vs. GG genotype P < 0.0001, CC vs. GC genotype P = 0.034). Moreover, MIF levels in CAD group were all higher than that in the counterparts of control group (all P < 0.05). For control participants, the circulating levels of MIF were comparable among the three genotypes of rs755622. The levels of MMP-9, IL-6 and IL-8 were comparable among the three genotypes of rs755622 in both control and CAD groups except that CAD patients carrying rs755622 GC genotype had higher IL-6 level compared with controls carrying the same genotype (P < 0.05, Fig. 2B-D). In addition, the levels of circulating MIF, MMP-9, IL-6 and IL-8 were comparable among CAD patients and controls carrying different genotypes of rs1007888 and rs2096525 polymorphisms (all P > 0.05, data were not shown).

Discussion
CAD as a complex and multifactorial disorder is characterized by the interaction between traditionally environmental factors and genetic changes. Many genetic factors involve in the development and progression of the CAD. Recently, Schunkert and colleagues analyzed previously published GWAS and reported that 164 chromosomal loci have been identified to affect the risk of CAD. These chromosomal loci were involved in modulating traditional risk factors such as immune system and inflammation (CXCL12, HDAC9, IL5, etc.), lipid metabolism (LDLR, PCSK9, APOE, etc.), blood pressure (ARHGAP42, LPL, NOS3, etc.) and glycometabolism (IRS1, GIP, SLC22A4) 32 . Hypercholesterolemia is a primary trigger for the initiation of atherosclerosis. It leads to the accumulation of lipoproteins in the intima and also results in the formation of oxidized lipoproteins and release of inflammatory mediators 33 . Inflammation can enhance atherosclerotic plaque instability presented as plaque fissuring, erosion and rupture which provides the substrate for the thrombotic response that eventually causes myocardial ischemia or infarction 34 . The high blood pressure and elevated glucose levels are key risk factors for CAD and both can expedite the progression of atherosclerosis by impairing endothelial function 35 . MIF plays an  www.nature.com/scientificreports/ important role in cell recruitment and arrest through binding to the chemokine receptors CXCR2 and CXCR4 36 . However, it cannot be classified into one of the four typical chemokine classes, such as CXCL12, CCL2, due to its absence of a characteristic cysteine motif in its N-terminus, and is therefore called a chemokine-like function chemokine 37 . Although the effectively clinical treatments and lifestyle modification have decreased its incidence and mortality largely, CAD was still considered the leading cause of adults mortality worldwide 38,39 . Therefore, detecting the association between susceptible gene and CAD has been considered as a valuable approach for identifying risk and improving management of CAD. Our present study documented that the frequency of rs755622 CC genotype and C allele were more common in CAD patients than that in controls and individuals carrying CC genotype had an increased susceptibility of CAD. These findings were supported by others. It has been reported that MIF rs755622 C allele polymorphism was associated with CAD and the C allele might be a risk factor for CAD in other Chinese population 31,40 . The MONICA/KORA Augsburg study suggested that rs755622 C allele and rs2070766 G allele were related to the higher risk for CAD in German females after 10 years follow-up 10 . Another case-control study showed that MIF gene rs755622 C allele was also associated with increased risk of ACS in a Chinese Han population 41 . Our previous study which conducted in a Kazakh population, a Chinese minority ethnic, found that the frequencies of MIF rs755622 CC genotype and C allele were significantly higher in CAD patients than that in control subjects 42 . Some different results were also reported. Tereshchenko et al. investigated the distribution of MIF rs755622 G/C genotypes, alleles or carriage rates in Czech or Russian population, no significant difference was found between patients with MI and control subjects. However, they observed that the GG genotype of the MIF rs1007888 was associated with MI in Czech female patients 43 . Except the findings of rs755622 and rs1007888, another MIF gene SNP, rs5844572, contains a 4-nucleotide microsatellite positioned at -794 in the MIF promoter (CATT [5][6][7][8] ). The CATT repeat number regulates the activity of the MIF gene promoter and higher CATT repeat numbers lead to stronger activity of the promoter 44 . It was reported that MIF − 794 CATT 5-8 was also associated with susceptibility and severity of many in immune-related disease and CAD. A Mexican population-based case-control study documented that MIF -794 CATT 6/7 genotype was associated with susceptibility to ACS 45 . Qian et al. showed that among 70 CAD patients in China, MIF -794 CATT [5][6][7][8] was not associated with CAD, but carriers with -794 CATT 5 allele showed lower plasm MIF levels than those carriers with -794 CATT 7 allele 46 . Taken together, these findings clearly indicated the MIF gene rs755622 and -794 CATT 5-8 variations were more likely to be universal genetic variants associated with increased risk of CAD.
Gensini score was a well-recognized method to evaluate the degree of coronary artery stenosis 47 . In our present study, we observed that CAD patients carrying rs755622 CC genotype had higher Gensini scores than other genotype carriers. This finding was similar to our previous study, which also indicated that CAD patients with rs755622 C allele (CC or CG genotype) had higher Gensini score compared to non C allele carriers in Kazakh population 42 . MIF is an evolutionarily ancient and highly conserved cytokine and plays as a key factor in pathogenesis of cardiovascular disease 36,48 . MIF has been identified as a major regulator of atherogenesis 49 through the www.nature.com/scientificreports/ following mechanisms (1) inducing recruitment of monocytes and T cells to atherosclerotic lesions by various pro-atherogenic stimuli e.g., oxidized LDL or angiotensin II 50 ; (2) regulating smooth muscle cell migration and proliferation, which may promote lesion growth and (3) increasing foam-cell transformation of macrophages and enhancing degradation of extracellular matrix proteins therefore contributing to plaque instability 51 . These results indicate that MIF gene rs755622 may a potential genetic marker for predicting risk and the severity of CAD. As a pleiotropic cytokine, MIF could regulate the release of other pro-inflammatory cytokines through controls the inflammatory 'set point' 52 . In our present study we randomly measured circulating levels of MIF, MMP-9, IL-6 and IL-8 in CAD patients and control subjects. MMP-9 is capable of degrading components of the extracellular matrix which plays an essential role in the progression and instability of atherosclerotic lesions 53 . IL-6 is a central mediator of inflammatory process 54 . Many inflammatory diseases were accompanied with elevated circulating IL-6 levels especially, in atherosclerosis, CAD, acute MI and metabolic syndromes 55 . IL-8 is also a proinflammatory cytokine that is expressed in macrophage-rich areas of atherosclerotic lesions 56 and the elevated levels of IL-8 were associated with an increased risk of CAD 57,58 . In the present study, we observed that plasma MIF level was significantly elevated in CAD patients carrying of MIF gene rs755622 CC genotype. This finding was similar to our previous study, which showed MIF levels in ACS patients with CC genotype was significantly higher than ACS patients carrying GG genotype of MIF gene rs755622 41 . Ji et al. 40 also reported that the plasma MIF level was higher in MIF rs755622 C allele carriers than in G allele carriers. Moreover, Qian et al. reported that MIF level was higher in MIF -794 CATT 7 allele carriers than that of CATT 5 allele carriers in CAD patients 46 . However, we did not observe significant difference in plasma levels of MMP-9, IL-6 and IL-8 between the 3 genotypes of rs755622. Except for CAD, an association of MIF rs755622 CC genotype with higher levels of MIF was also reported in other inflammatory diseases such as juvenile idiopathic arthritis 59 and rheumatoid arthritis 27 . These results suggested that MIF gene rs755622 and -794 CATT 5-8 variations had a potential impact on the expression of MIF.
There are several limitations in our study. First, we did not investigate the other variations of the MIF gene such as -794 CATT5-8 which had been reported positively related with CAD. Second, as blood samples were randomly selected, MIF and other inflammatory cytokine levels were not relevant to the severity of coronary artery lesion, therefore, we did not observe a correlation between MIF levels and Gensini score in CAD patients. Third, MIF levels were somewhat high in our control participants. This phenomenon may be related to other concomitant "unhealthy conditions" that might influence their baseline levels of MIF, in other words, they were not really healthy individuals. Moreover, although these control participants had no history and no signs of CAD, they might be exposed to same risk factors of CAD. At last, the small sample size for measuring plasma levels of MIF and other inflammatory factors limited the power of MIF genetic variation influencing expression and production of these inflammatory mediators.
In conclusion, our study demonstrated an association between the MIF gene variation and risk of CAD in Chinese Han population. Individuals who carried MIF gene rs755622 CC genotype were more susceptible to CAD and likely to have more severe coronary artery lesion. This variation also had a potential influence in circulating MIF levels.

Materials and methods
Study participants. Control participants were randomly selected from the Cardiovascular Risk Survey (CRS) study, which was performed in the multiethnic population in Xinjiang, northwestern part of China from October 2007 to March 2010 60,61 . Individuals with no history of cardiovascular disease and no signs of CAD according to their physical and laboratory examination, electrocardiograph and echocardiography were defined as control participants. But these control participants were not the real healthy individuals. They may have hypertension, dyslipidemia and diabetes, which means these controls exposed to same risk factors of CAD. This study recruited CAD patient during May 2009 and December 2014. CAD patients were recruited from inpatients who received treatment in the First Affiliated Hospital of Xinjiang Medical University. CAD was considered as the presence of coronary artery stenosis (> 50% lumen narrowing) in one or more major coronary arteries according to coronary angiography 42 . Angiographic evaluations were reviewed by two independent interventional cardiologists blinded to the study information. Gensini score was applied to evaluate the severity of CAD as we described previously 42 . We also counted the number of diseased arteries. Patients with more than one major coronary artery stenosis were defined as multi-vessel disease. Exclusion criteria for both CAD and control participants were those with concomitant valvular heart disease, congenital heart disease, non-ischemic cardiomyopathy and acute and chronic inflammatory diseases. As valvular heart disease, congenital heart disease and non-ischemic cardiomyopathy were also involved in genetic disorders, which may be confounding factors interfering with the association between MIF gene variation and susceptibility of CAD.
Definition of cardiovascular risk factors. Body mass index (BMI) was calculated by dividing body weight (in kilograms) with the height in meters squared. Individuals who smoked regularly in the past six months were regarded as current tobacco users. A person whose systolic blood pressure exceeded 140 mmHg and/or diastolic blood pressure was more than 90 mmHg that occurred twice in different occasions was defined as a hypertension 62  www.nature.com/scientificreports/ included blood glucose and lipids including total cholesterol (TC), low density lipoprotein-cholesterol (LDL-C), high density lipoprotein-cholesterol (HDL-C), and triglycerides (TG). All of the plasma samples were measured by standard enzymatic methods in the Central Laboratory of the First Affiliated Hospital of Xinjiang Medical University.
Enzyme linked immunosorbent assay (ELISA). We measured plasma levels of MIF, IL-6, IL-8, and matrix metalloproteinase-9 (MMP-9) in randomly selected control subjects and CAD patients to investigate the potential functional influence of MIF gene variation by ELISA kits (Wuhan USCN Business Co., Ltd, China). The randomization of participant selection for ELISA was performed by following the computer generated sequential numbers (blocked randomization) and the participants' allocations were kept in sealed envelopes according to our previously established method 64 . Statistical analysis. Analyses were carried out by SPSS version 17.0 (SPSS Inc., Chicago, IL). Continuous variables were presented as mean ± standard deviation (SD) and the differences between CAD and controls was detected by Student unpaired t test between. Categorical variables were showed as proportions and their differences between the two groups were analyzed by chi-square test. Chi-square test was also used in the analysis of Hardy-Weinberg equilibrium. The association between MIF gene variation and CAD was detected by multiple logistic regression analysis. The difference in the levels of MIF, MMP-9, IL-6 and IL-8 and Gensini score was analyzed by two-way ANOVA among different genotypes. General linear model analysis was performed to test the associations among different genotypes, plasma levels and Gensini score. P < 0.05 was considered as statistical significance.

DNA extraction and
Ethical approval. Written informed consent was obtained from all of study participants. This study was approved by the Ethics Committee of the First Affiliated Hospital of Xinjiang Medical University (Ethic Approval No. 20070726-8) and conducted according to the standards of the Declaration of Helsinki.