Polymorphism of IL‐12/IL‐23 axis is associated with coronary heart disease

Abstract IL12B encodes the shared p40 subunit (IL‐12p40) of IL‐12 and IL‐23, which have diverse immune functions and are closely related to the occurrence and development of atherosclerosis (AS). However, the exact role of IL12B in coronary heart disease (CHD) was still unknown. A case–control association analysis was performed between five single nucleotide polymorphisms (SNPs) of IL12B (rs1003199, rs3212219, rs2569254, rs2853694 and rs3212227) and CHD in Chinese Han population (1824 patients with CHD vs. 2784 controls). Logistic regression analyses were used to study the relationships between SNPs and CHD, while multiple linear regression analyses were used to test the association between the SNP and the severity of CHD. In addition, the plasma IL12B concentration of CHD patients were detected by ELISA. We detected a significant association between one of the SNPs, rs2853694−G and CHD (p adj = 2.075 × 10−5, OR, 0.773 [95% CI, 0.686–0.870]). Stratified analysis showed that rs2853694 was associated with CHD in both male and female populations and was significantly associated with both early‐ and late‐onset CHD. In addition, rs2853694 is also related to the different types of CHD including clinical‐CHD and anatomical‐CHD. Moreover, there are significant differences in serum IL12B concentrations between rs2853694−TT carriers and rs2853694−GT carriers in CHD patients (p = 0.010). A common variant of IL12B was found significantly associated with CHD and its subgroups. As a shared subunit of IL‐12 and IL‐23, IL‐12p40 may play a key role in IL‐12/IL‐23 axis mediated AS, which is expected to be an effective therapeutic target for CHD.


| INTRODUC TI ON
Coronary heart disease (CHD) is an ischemic heart disease in which blood vessels narrow due to coronary atherosclerosis (AS), resulting in myocardial infarction (MI), hypoxia and even necrosis.Coronary heart disease, as a high incidence of cardiovascular diseases (CVDs), is one of the highest mortality diseases globally.It affects as many as 11 million people in China, and the annual growth rate of 20%, there are 1 million deaths due to CHD every year.Coronary heart disease has caused serious economic burden and health burden to the world, so it is urgent to seek effective prevention and treatment strategies of CHD.
In recent years, accumulating studies indicated inflammation are closely related to the progression of CHD. 1,2Interleukins (ILs) are inflammatory cytokines involved in pro-inflammatory and antiinflammatory responses through interactions with a variety of receptors, such as Toll-like receptors (TLRs). 3Research has suggested that ILs and TLRs could represent promising targets for the therapeutic intervention of inflammation-associated diseases. 4The pathogenic role of inflammation is well-established, and many animal studies have suggested that anti-inflammatory therapy can inhibit the progression of AS. 5 The CANTOS study found that the use of the IL-1β blocking antibody (canakinumab) significantly reduced cardiovascular events in patients with CHD.It confirmed that IL-1β is an effective target for anti-inflammatory therapy in CHD in the complex inflammatory regulatory network. 6,7Based on JUPITER studies, some scholars have proposed that the therapeutic effect of statins on CHD is partly due to the anti-inflammatory effect. 8However, due to the complex regulatory network of inflammatory response, how to find out the key regulatory factors in this network and use them as targets to effectively regulate inflammation is the key to the success of anti-inflammatory therapy.
Results from epidemiological surveys, twin studies and relative risk studies all show a significant genetic predisposition to CHD. [9][10][11] Relatives of people with CHD have a 2 to 3.9 times increased risk of developing CHD compared with relatives without CHD.Although numerous susceptibility genes for CHD have been discovered through genetic studies, these susceptibility genes can only explain about 28% of the heritability of CHD, and these conclusions are mainly drawn from western European populations. 12Moreover, many of those susceptibility genes for CHD have not yet been elucidated.The full genetic basis of CHD and its molecular mechanism of action in the disease through various research strategies is still needed, so as to provide theoretical basis for precision medicine in the future.
IL12B encodes the p40 subunit (IL-12p40) of IL-12 and IL-23, which have been reported to be closely related to AS.The expressions of both IL-12 and IL-23 are increased in human atherosclerotic plaques, and IL-12 promoted the formation of AS in mice. 13,14rrently, IL-12/IL-23 axis is considered a therapeutic target for autoimmune inflammation, 15 which may also be a new target for treating AS. 16 Studies have found that polymorphism on IL12B is associated with susceptibility to some inflammatory diseases, [17][18][19] and in some disease states, the polymorphism on Il12B regulate IL-12p40 and IL-23 transcription and affects their serum levels. 20,21 addition, recent research has found IL12B polymorphisms are associated with the presence of premature CHD and with cardiovascular risk factors in Mexican population. 22However, genetic relationship between IL12B and CHD in the Chinese population is unclear.Our study explores the role of IL12B in the genetic factors of CHD by studying the association between its polymorphisms and CHD (Figure 1), in order to find the susceptible polymorphism of CHD, and further understand the role of IL-12/IL-23 axis in the occurrence and development of CHD, so as to clarify the pathological process of CHD.

| Study objects
This study collected a total of 1824 patients with CHD and 2784 controls in the Wuhan Union Hospital (Wuhan, China) from January 2013 and November 2015.Of these, 768 CHDs and 768 controls were in the Phase I discovery study, and the remaining samples were used for further extended sample validation of single nucleotide polymorphism (SNP) that showed significant result in Phase I study (Figure 2).All of our samples have been obtained with DNA and relevant clinical information including: age, gender, height, weight, the history of smoking, drinking, hypertension and diabetes, blood lipid and fasting blood glucose, as well as other biochemical detection indicators information. 23In addition, there are ECG, CT, heart colour ultrasound, coronary angiography and other imaging data of patients with CHD.The inclusion and exclusion criteria for CHD and control were as described in our previous public article 24,25 (Figure 1).According to the different diagnostic criteria, we divided was multiplied with a specific factor judged as the vessel importance and size (ranging from 5.0 to 0.5) and the total sum weights of the segments is the Gensini index for each patient. 26Our study meets the requirements of the World Medical Association Declaration of Helsinki and has been approved by the local ethics committee.

| Tag SNPs selection of IL12B
A Linkage disequilibrium (LD) Block of CHB + JPT (China and Japan) population was established in the Haploview software (V4.2 version) covering the entire IL12B gene and its upstream and downstream 5 kbp region (Figure 3).The Tag SNPs selection principles were as follows 24 : (1) Minimum allele frequency (MAF) >0.05, r 2 > 0.8.(2)   SNPs reported to be associated with inflammation in the literature is preferentially selected.(3) SNP located in promoter and exon region is preferentially selected.According to the selection principle above, a total of five SNPs were included in our study, namely rs1003199, rs3212219, rs2569254, rs2853694 and rs3212227 (Figure 2).For detailed SNP information, see Table S1.

| Association analysis
In this study, age, body mass index (BMI), total cholesterol (Tch),  were corrected as covariables using logistic regression analysis, and multiple linear regression was carried out to analysis the effects of rs2853694 on Gensini scores (SPSS, v.16.0).Allelic and genotypic association analysis were carried out by 2 × 2 and/or 2 × 3 pearson chi-squared contingence tables respectively. 27Mann-Whitney Utests evaluated the relationship between different genotypes of rs2853694 and LN of Gensini scores. 24As five SNPs were covered in this association analysis, the statistically significant p value was ≤0.01 (0.05/5) after Bonferroni correction.

| Plasma level of IL12B in CHD patients
A total of 56 CHD patients were selected from the combined population for analysis of the association between rs2853694 genotypes and the plasma level of IL12B.Blood samples were collected in tubes containing EDTA as an anticoagulant.After centrifuging blood samples for 15 min at 1000 g at 4°C within 30 min of collection, supernatant was obtained.100 μL plasma of each sample was aspirated for

| Characteristics of study objects
In the Phase I discovery study, we compared the characteristics of those groups (768 CHDs vs. 768 controls) and found that age and BMI of the CHD group were higher than those of the control group, and the proportion of males, smoking, hypertension and diabetes in the CHD group were also higher compared with the control group.
In addition, the Tch, TG and LDL-C concentrations were significantly higher compared with the control group, while the HDL-C concentration was lower compared with the control group.For the subsequent study, 1824 CHDs and 2784 controls were included, and the characteristics of those samples were similar to those of phase I. See Table 1 for details.

| Allelic and genotypic association analysis of SNPs in IL12B gene with CHD in discovery population
In the phase I discovery study, only rs2853694 of the five SNPs    3).

| Allelic and genotypic association analysis of rs2853694 in IL12B gene with CHD in combined population
Through the analysis of relatively small samples in the phase I discovery stage, we found that a SNP in IL12B gene, rs2853694, was significantly correlated with CHD.Therefore, we expanded the sample size to further explore the relationship between rs2853694 and CHD.
Finally, we found for allele analysis, rs2853694 −G were significantly associated with CHD in more than 4500 samples (1824 CHDs vs.  4).

| Association of rs2853694 in IL12B gene with CHD in subgroup populations
Considering the influence of gender and age on CHD, we conducted  associated with late-onset CHD in the ADD (GG/GT/TT) and REC (GG/GT + TT) modes (p adj = 0.007 for ADD model; p adj = 0.005 for REC model) (Table 5).
Second, we divided the population into male and female according to sex, and the results showed that in allele analysis, rs2853694 −G was significantly correlated with CHD in both male and female groups (p adj = 0.004 for male; p adj = 4.311 × 10 −4 for female).
Further genotype analysis showed that rs2853694 was also correlated with CHD in ADD (GG/GT/TT) and DOM (GG + GT/TT) mod- for DOM model in female group) (Table 6).7).

| Associations between rs2853694 in IL12B and the severity of CHD
It is also possible that rs2853694 is associated with the severity of CHD.We evaluated the severity of CHD using Gensini scoring system for each patient with CHD.Since Gensini score data in the sample did not conform to normal distribution, logarithmic conversion (LN) of Gensini score data was performed before analysis.First, we use linear regression model to conduct quantitative trait association.
Our analysis results showed that rs2853694 was not related to the severity of CHD either in allele mode or genotype mode (p > 0.05) (Table 8).Then we divide the LN of Gensini scores into quartiles, 4th (highest) and 1st (lowest) quartiles of the LN of Gensini scores were choose to conduct case-control association analysis.Results showed that rs2853694 was not related to the severity of CHD either in allele mode or genotype mode (p > 0.05) (Table 8).In addition, we evaluated the relationship between different genotypes of rs2853694 and LN of Gensini scores through Mann-Whitney U-tests.Results also showed no difference between LN of Gensini TA B L E 6 Allelic and genotypic association analysis of rs2853694 with CHD in gender subgroups.scores and rs2853694 genotypes (p > 0.05) (Figure S1).These results suggest that there is no correlation between rs2853694 and the degree of CHD.

| Association of rs2853694 genotypes with plasma IL12B concentration in CHD patient
Among the 56 CHD patients, the detection rate of plasma were significant differences in plasma IL12B concentration between rs2853694 −TT carriers and rs2853694 −GT carriers (p = 0.010) (Figure 4).

| DISCUSS ION
Our study examined the relationship between SNPs of IL12B and CHD in a Chinese Han population.We detected a significant association between one of the SNPs, rs2853694 and CHD in a small sample.Further significant correlation between rs2853694 and CHD remained in the expanded sample.Stratified analysis showed that rs2853694 was associated with CHD in both male and female populations and was significantly associated with both early-and late-onset CHD.In addition, rs2853694 is also related to the different types of CHD including clinical-CHD and anatomical-CHD.However, no association was found between rs2853694 and the severity of CHD.In addition, there are significant differences in serum IL12B concentrations between rs2853694 −TT carriers and rs2853694 −GT carriers in CHD patients.
IL12B gene located on chromosome 5q31, a region where we have previously identified multiple CHD susceptibility SNPs. 23,26,2812B was related to lower risk of insulin resistance.However, Morahan et al. 31 found rs2853694 was not susceptible to type 1 diabetes.
Moreover, IL12B or IL-12 were not related to insulin sensitivity. 32 addition, Chung et al. 33 found rs2853694 was associated with coronary AS in patients with rheumatoid arthritis (RA) with a relatively small sample (140 RA patients for Caucasian).In our study, we found rs2853694 was associated with CHD including both early-and late-onset CHD, moreover, our results also found that rs2853694 were associated with different disease states of CHD.
Another SNP of IL12B, rs3212227 was found to be no associated with MI in British Caucasian population 34 and not related to both presence or severity of CHD in Japanese population. 35Similarly, no correlation was found between rs3212227 and CHD in our study.
In addition, our results found that there were significant differences in plasma IL12B concentration between rs2853694  blocking IL-12 significantly reduced this effect. 41,42All of these studies suggest that IL-12 have pro-atherogenic effects, indicated that it may be a prospective therapeutic target.Although there is a lack of evidence that IL-23 acts directly on AS, increasing studies show that IL-23 also has the effect of promoting AS.For CHD patients undergoing percutaneous coronary intervention drug-eluting stents, peripheral blood mononuclear cell circulating IL-23 concentration were higher in patients with in-stent restenosis. 43 IL-12p40 in asymptomatic carotid plaques were significantly higher than the symptomatic samples. 51Another study found compared to APOE −/− mice, IL-12p40 −/− APOE −/− mice showed reduction of lesion because of reduced aggregation of macrophages in the aortic root. 52cently study found a significant difference between the percentage methylation of promoter DNA of IL12B in CHD patients and control subjects. 53The author holds that maybe the M1/M2 macrophage polarization marker.The balance of M1/M2 macrophages is closely related to the occurrence and development.Zhou et al. 54 suggest that curcumin can be a therapeutic for AS, as curcumin can inhibit M1 macrophage polarization and the production of TNFα, IL-

| CON CLUS ION
Our study investigated the relationship between IL12B gene and CHD from a genetic perspective in a Chinese Han population.A common variant of IL12B was found significantly associated with CHD and its subgroups.As a shared subunit of IL-12 and IL-23, IL-12p40 may play a key role in IL-12/IL-23 axis mediated AS, which is expected to be an effective therapeutic target for CHD.

| 3 of 12 DONG
the CHD patients into two subgroups.The anatomical-CHD group was defined as severe coronary stenosis (≥50% coronary stenosis in any one of the main vessels [right coronary artery, left anterior descending, left main and left circumflex artery]) which was diagnosed by coronary angiography and the clinical-CHD group is MI or revascularization which is by clinical diagnosis.The severity of CHD was assessed using Gensini score performed according to coronary angiography.The coronary segment's score was judged as 32, 16, 8, 4, 2 and 1 for its stenosis of 100%, 99-91%, 90-76%, 75-51%, 50-26% and 25-0%, respectively.And the coronary segment's score K E Y W O R D S coronary heart disease, genetic, IL12B, polymorphism, rs2853694 et al.
triglyceride (TG), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) levels were tested by independent sample t-test to test whether there were differences in the mean values of these clinical indicators between the two groups and their respective populations.Traditional risk factors for CHD F I G U R E 1 Study design flow chart.
ELISA test.According to the manufacturer's protocol (R&D Systems), standard recombined human IL12B provided by this kit was diluted to eight different concentrations to generate standard curves.After incubation, colour development was measured at 420 nm by means of a spectrophotometer and OD value was gained for each sample.Based on the standard curve drew by the different concentrations of standard recombined human IL12B, we got the corresponding IL12B concentration of each sample.The correlations among the rs2853694 genotypes and IL12B plasma concentrations were studied by non-parametric test.

| 5 of 12 DONG
et al.In genotypic association analysis, rs1003199 was related to CHD in ADD model before correcting for risk factors (p obs = 0.046), rs2853694 was significantly associated with CHD in all three genotype modes (p obs = 8.358 × 10 −5 for ADD model; p obs = 2.305 × 10 −4 for DOM model; p obs = 0.001 for REC model).However, after adjusting for risk factors, rs1003199 was no longer F I G U R E 3 (A) Haploview linkage disequilibrium (LD) block of IL12B region based on the HapMap CHB and JPT data Sets.Each diamond represents the LD degree between the SNPs.The number within each diamond represents the r 2 value.The colour indicates the D′: regions of high LD are showed in dark red; regions of low LD are showed in white.The selected Tag SNPs are showed in black box.(B) The location of the selected SNPs on the IL12B region.associated with CHD (p adj = 0.354), while the correlation between rs2853694 and CHD still exists (p adj = 2.192 × 10 −5 for ADD model; p adj = 1.561 × 10 −4 for DOM model; p adj = 0.005 for REC model).In addition, after correcting for risk factors, rs3212227 was associated with CHD in a DOM model (p adj = 0.046), but did not pass multiple correction tests (Table 2784 controls) (p obs = 0.004), remained significant after adjusting for risk factors (p adj = 2.075 × 10 −5 , OR, 0.773 [95% CI, 0.686-0.870]).In genotype analysis, rs2853694 was significantly associated with CHD in the ADD (GG/GT/TT) and REC (GG/GT + TT) models (p obs = 0.008 for ADD model; p obs = 0.007 for REC model), the DOM (GG + GT/ TT) model is also associated with CHD (p obs = 0.024).The association between rs2853694 and CHD remained after correction for risk factors (p adj = 1.424 × 10 −5 for ADD model; p adj = 3.981 × 10 −5 for DOM model; p adj = 0.008 for REC model) (Table different stratified analyses according to gender and age of onset of CHD.First of all, we divided the population of CHD into early-onset CHD and late-onset CHD based on the age of onset (early-onset CHD: the first onset age ≤55 years for male and ≤65 years for female; late-onset CHD: the first onset age >55 years for male and >65 years for female).Allele analysis showed that rs2853694 −G was significantly associated with both types of CHD (p adj = 3.549 × 10 −4 for early-onset CHD; p adj = 0.009 for late-onset CHD).Genotype analysis showed that rs2853694 was associated with early-onset CHD in ADD (GG/GT/TT) and DOM (GG + GT/TT) models (p adj = 2.716 × 10 −4 for ADD model; p adj = 7.135 × 10 −5 for DOM model), rs2853694 was TA B L E 1 Characteristics of the study population.
els (p adj = 0.003 for ADD model and p adj = 0.010 for DOM model in male group; p adj = 3.691 × 10 −4 for ADD model and p adj = 1.793 × 10 −4 Coronary heart disease has different clinical manifestations due to different pathological anatomy and pathophysiology.To explore the influence of rs2853694 on different types of CHD, we divided CHD population into clinical-CHD and anatomical-CHD.The anatomical-CHD group was defined as severe coronary stenosis and the clinical-CHD group is MI or revascularization.Our results found that in allele analysis, rs2853694 −G was associated with both types of CHD (p adj = 2.750 × 10 −5 for clinical-CHD; p adj = 0.001 for anatomical-CHD).Genotype analysis showed that rs2853694 was also associated with two types of CHD under three genotype patterns (p adj = 2.013 × 10 −5 for ADD model; p adj = 3.384 × 10 −5 for DOM model; p adj = 0.016 for REC model in clinical-CHD; p adj = 0.001 for ADD model; p adj = 0.001 for DOM model; p adj = 0.041 for REC model in anatomical-CHD) (Table belongs to the IL-12 family, consisting of IL-12, IL-23, IL-27 and IL-35.Our previous research found that other genes in the IL-12 family are related to CHD. 25,29 9p21.3 is the first CHD susceptibility site identified by genome-wide association studies (GWASs).Zollbrecht et al. 30 found 9p21.3 haplotype can affect the expression of IL12B, which is higher up-regulated in 9p21.3 risk haplotype carrier.Recently, researcher 22 found rs1363670 of IL12B was associated with a low risk of premature CHD (pCHD) in Mexican population and in pCAD patients, the rs2853694 of IL12B
Subramanian et al.44 found granulocyte-macrophage colony stimulating factor increases the expression of IL-23, which further promotes the differentiation of macrophages and the development of AS.Although both IL-12 and IL-23 belong to the IL-12 family of cytokines, they have different immune functions, particularly in determining the nature of the immune response to be undertaken.IL-12 is a key factor promoting Th1 differentiation,45 while IL-23 is the key factor promoting Th17 differentiation.46Th1 and Th17 cell-mediated immune inflammation paly key role in the occurrence and development of AS.[47][48][49]In clinical studies, our previous research found that the proportion of Th17 cells, Th17 characteristic transcription factor RORγt and Th17-related cytokines (IL-17, IL-23, IL-6) in peripheral blood of patients with acute coronary syndrome (ACS) of CHD were significantly increased, suggesting that Th17 cells mediated the instability of AS plaques.50Th1/Th2 dysfunction exists in AS animals and patients with CHD, and inhibition of Th1 function can delay the progression of AS.IL-12 and IL-23 share the IL-12p40 subunit.Similarly, IL-12 and IL-23 receptors share the IL-12Rβ1 subunit.Therefore, previous interventions targeting IL-12 are likely to actually act on IL-23 by acting on IL-12p40 or IL-12Rβ1 subunits, that is, interventions targeting Th1 actually act on Th17 cells.This made scholars puzzled about the pathogenesis of diseases mediated by Th1 cells.RNA levels of

6 ,
and IL-12p40.Nikolajuk et al. 32 found IL-12p40 was positive associated with fat mass and TG, while negative associated with HDL-C in obese women.As a shared subunit of IL-12 and IL-23, IL-12p40 may pivotal in IL-12 and IL-23 mediated AS.Currently, there are monoclonal antibodies targeting IL-12/IL-23 axis, which have shown good efficacy in the treatment of other inflammatory diseases by blocking the IL-12/IL-23 pathway. 55Considering the important role of IL-12p40 in IL-12/IL-23 axis, it is expected to be a key target for the treatment of AS.This study is the first to investigate the relationship between IL12B gene and CHD from a genetic perspective in a Chinese Han population.The results showed that a SNP of IL12B is related to CHD, which provides a new target for targeted therapy of CHD and provides a theoretical basis for individualized therapy.But limitations remained in this subject: people in different regions may have some differences in genetic background and disease susceptibility due to the inconsistency of geographical environment and living environment; the majority of the samples in this study are from the Han population in central China, and the samples of people in northern China or other areas can be collected for verification in subsequent studies; lacking the laboratory data regarding the inflammatory state of the patients and the sample size for detecting IL12B concentration was small.In addition, this study only found the susceptibility sites and possible pathogenesis of CHD from the perspective of genetics through statistical methods.Further functional experiments are needed to verify the effects of rs2853694 on IL-12p40 and the IL-12/IL-23 axis.

Table 2
Flow chart of the association analysis between IL12B and CHD.
).F I G U R E 2 Note:The data are shown as the mean ± SD.Abbreviations: BMI, body mass index; CHD, coronary heart disease; DM, diabetes mellitus; HDL-C, high-density lipoprotein cholesterol; LDL-C, lowdensity lipoprotein cholesterol; Tch, total cholesterol; TG, triglyceride.Allelic association analysis between IL12B and CHD in discovery population.Genotypic association analysis between IL12B and CHD in discovery population.Association analysis between rs2853694 and CHD in the onset age subgroups.
adj , p value adjusted by the covariates (age, BMI, hypertension, diabetes mellitus, smoking history, Tch, TG, HDL-C and LDL-C); REC, recessive model, rs2853694_GG/GT + TT.Association analysis of rs2853694 with CHD in the disease status subgroups.
Association analysis of rs2853694 with the LN of Gensini scores.
−TT carriers TA B L E 8