Polymorphisms and Gene-Gene Interaction in AGER/IL6 Pathway are Associated with Diabetic Ischemic Heart Disease

Background: The aim of the present study is to demonstrate the association of AGER and IL6R gene polymorphisms with diabetic ischemic heart disease (IHD), and to investigate the effect of gene-gene interaction on disease risk. Methods: Our study included 204 ischemic heart disease cases who have previously been diagnosed as diabetes before the diagnoses of IHD, and 882 health controls. Polygenic risk score (PRS) was calculated by summing the number of risk alleles of all the candidate single nucleotide polymorphisms (SNPs). Logistic regression was used to nd the association of candidate SNPs and PRS with diabetic ischemic heart disease. Generalized multifactor dimensionality reduction (GMDR) was used to illustrate gene-gene interaction. Haplotypes were identied and analyzed via Haploview and Plink software. Results: The rs184003 and rs2070600 in AGER gene were signicantly associated with the risk of diabetic ischemic heart disease (P additive =0.005; P additive =0.025, respectively). For IL6R rs4845625, CT and TT genotype were associated with lower risk of the disease comparing with CC genotype (OR=0.692, P=0.045; OR=0.503, P=0.003, respectively). After adjustment for covariates, the association of rs4845625 with disease remained statistically signicant. Haplotypes in AGER gene (rs184003-rs1035798-rs2070600-rs1800624) and IL6R gene (rs7529229-rs4845625-rs4129267-rs7514452-rs4072391) were both signicantly associated with diabetic ischemic heart disease (P=0.008; P=0.007). PRS was associated with the disease (OR=1.106, P=0.020) after adjusting for covariates. The GMDR analysis suggested that rs184003 and rs4845625 was the best interaction model after permutation testing (P=0.001) with a cross-validation consistency of 10/10. Conclusions: SNPs and haplotypes in AGER and IL6R gene and the interaction of rs184003 in AGER with rs4845625 in IL6R were signicantly associated with diabetic ischemic heart disease. ethnicity, subjects with rs2070600 risk allele were at higher risk of coronary arterial disease (CAD) in the Chinese population, rather than non-Chinese population. However, our study found the association between rs2070600 and diabetic ischemic heart disease was null. Another research also found rs2070600 was associated with the circulating levels of esRAGE but not with CAD in Chinese patients with T2DM [18] . These results might indicate that the association between rs2070600 and CAD may also be different in general population and T2DM patients.


Introduction
Cardiovascular disease (CVD) is the leading cause of mortality in people with type 2 diabetes mellitus (T2DM). About 68% of deaths in type 2 diabetic patients are caused by cardiac complications [1] . However, it is not clear how diabetes promotes cardiac dysfunction. The widely acceptable hypothesis is that many signaling cascades, ultimately resulting in pro-in ammatory reaction, oxidative stress or thrombotic pathways, and subsequently leading to vascular in ammation [2] . It has been demonstrated that advanced glycation end products (AGER) / interleukin-6 (IL-6) pathway plays an important role in the physiological mechanism of diabetic cardiovascular complication. High glucose level can trigger neutrophil to release S100 calcium-binding protein A8/A9 (S100A8/A9), which binds to AGER on Kupffer cells, and leads to IL-6 secretion [3] . IL-6 / IL-6R complex activate JAK2 / STAT3 pathway, which can mediate pro-in ammatory response and increase platelet thrombopoiesis by inducing thrombopoietin (TPO) production [4,5] .
Several single nucleotide polymorphisms (SNPs) in AGER gene have been reported to be associated with diabetes or its complications. The Atherosclerosis Risk in Communities Study showed an association between rs2070600 and an approximate 50% reduction in soluble AGER levels [6] . A meta-analysis has highlighted a signi cant association of rs2070600 with the risk of diabetic nephropathy development [7] .
However, the association between rs2070600 and diabetic cardiovascular disease are under-reported. On IL6R gene, the rs1800624 and rs1800625 are in absolute or strong linkage disequilibrium, and were reported to be protective factors for cardiovascular disease [8,9] . However, the effect of them on the vascular complications in T2DM remains inconsistent [10,11] , In addition, the gene-gene interactions on the increased risk of the disease requires further clari cation .
The current study aimed at illustrating the association of AGER and IL6R gene polymorphisms with the risk of diabetic cardiovascular disease, and assess the modulatory effect of gene-gene interaction between these variants on disease risk. The result would provide evidence on the precise prevention of ischemic heart disease in diabetes.

Study design and population
A total of 204 diabetic ischemic heart disease cases and 882 health controls were enrolled from communities in Beijing. All subjects gave written informed consent. This study was approved by the Ethics Committee of Capital Medical University (No:2016SY24).
Inclusion criteria for the cases were as follows: (1) T2DM patients diagnosed according to American Diabetes Association Criteria [12] , or receiving pharmaceutical treatment on T2DM; (2) Ischemic heart disease de ned by clinical history, including acute myocardium infarction, angina pectoris and/or ischemic electrocardiographic alterations; (3) T2DM was diagnosed earlier than ischemic heart disease. (4) The medical records or copies should be provided to verify the diagnose of diseases.
Inclusion criteria for the controls were as follows: (1) Subjects had not been diagnosed as T2DM before, and fasting blood glucose was less than 5.6 mmol/L in the current survey. (2) Subjects did not have cardiovascular disease, which included ischemic heart disease, ischemic stroke, or cerebral hemorrhage.
(3) Subjects did not have chronic kidney disease. (4) Subjects were not in the acute phase of infection.

Measurements
Life style risk factors were obtained from structured questionnaire. Smoking status was categorized as: "currently smoking" and "past / never smoking". Current smoking was de ned as at least 1 cigarette per day, lasting for more than 1 year. Those who have never smoked before or have not smoked for at least 3 months were de ned as past / never smoking. Alcohol drinking was categorized as "currently alcohol drinking" and "past / never alcohol drinking". Currently drinking was de ned as at least drink once per week and still drank at that frequency in the previous month. Those who never drink alcohol or have not drank alcohol for at least one month were de ned as never / past alcohol drinking.
Blood pressure (BP) was measured in the morning before participants use anti-hypertensive medication.
Participants were asked to rest for at least 30 minutes before BP measurement if they had just smoked or had caffeinated products. BP (mmHg) was measured for three times at sitting positions by mercury sphygmomanometer. The average of the last two measurements was used for data analysis.

Serum markers
After an overnight fasting, all participants underwent fasting blood sampling. Fasting blood samples are collected and restored in 2% EDTA vacutainer for each participant. After centrifuging, plasma and blood cell samples are separated into two cryovials. Fasting plasma glucose (FPG), total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDLC), low-density lipoprotein cholesterol (LDLC) were tested using the Beckman coulter chemistry analyzer AU5800 in the clinical laboratory of Beijing Hepingli Hospital.
Venous blood samples were obtained and stored in 4 o C refrigerator. All the hematological analysis was done within 8 hours. Serum glucose and biochemical determinations were measured by an enzymatic method using a chemistry analyzer (Beckman LX20, Beckman, Brea, CA, USA) at the central laboratory of the hospital.

Genotyping
Important functional SNPs and previously reported susceptible SNPs were selected as candidate SNPs.
Genomic DNA was extracted from 1 ml of peripheral blood cell using TIANGEN DNA kit (TIANGEN Biotech, China, DP319-01) according to the manufacturer's protocol. Primers were designed by the AssayDesigner3.1 software, and they were synthesized by Thermo Fisher Scienti c Co., Ltd. Detailed information of the primers were shown in supplementary table S1. A Sequenom MassARRAY® matrixassisted laser desorption/ionization-time of ight mass spectrometry (MALDI-TOF MS) platform (Sequenom Inc., San Diego, CA, USA) were used to genotype SNPs.
De nition of diseases and recommend level of their risk factors T2DM was de ned as FPG ≥ 7.0 mmol/L or self-reported physician-diagnosed diabetes and/or on use of antidiabetes agents, according to American Diabetes Association Criteria [12] . Ischemic heart disease (IHD) was de ned as non-fatal ischemic heart disease, including acute myocardial infarction and angina pectoris. The incident of ischemic heart disease in T2DM patients were de ned as diabetic ischemic heart disease. Hypertension was de ned as systolic blood pressure (SBP) ≥ 140 mmHg and/or diastolic blood pressure (DBP) ≥ 90 mmHg and/or on current antihypertensive medication. Participants with TG ≥ 2.3 mmol/L, or TC ≥ 6.2 mmol/L, or LDLC ≥ 4.1 mmol/L, or HDLC ≤ 1.0 mmol/L were de ned as dyslipidemia according to the criteria of the 2016 Chinese guidelines for the management of dyslipidemia in adults [13] .

Statistical analysis
Continuous variables with normal distribution are expressed as means ± standard deviations (SDs). Categorical variables were expressed as number (percentage). Student's t test was used to compare the difference of each continuous variables. Polygenic risk score (PRS) was calculated by summing the number of risk alleles of all the eleven candidate SNPs. Logistic regression was used to evaluate the association of the diabetic ischemic heart disease with candidate SNPs and PRS. SPSS25.0 software (SPSS Inc., Chicago, IL, USA) was used for all abovementioned statistical analysis. Generalized multifactor dimensionality reduction (GMDR) method was used to estimate the gene-gene interaction. For the adjustment for multiple testing, a permutation test with 1000 replications was performed. Haplotypes were identi ed and visualized by Haploview software. The association between haplotypes and diabetic cardiovascular disease were demonstrated by using Plink software. A two-sided P ≤ 0.05 was considered statistically signi cant.

Result
General characteristics of the studied participants A total of 882 health controls and 204 diabetic cardiovascular disease cases were included in the current study. DBP, TG and FPG were signi cantly higher in cardiovascular disease cases than controls (P < 0.001). SBP, TC, LDLC and HDLC were signi cantly higher in controls compared with cases (P < 0.001). According to recommendation of "2017 Guidelines for the prevention and treatment of type 2 diabetes in China", the percentage of SBP, DBP, HDLC, LDLC, TG and TC in ideal range were signi cantly higher in control group compared with cases (P < 0.001). In people with diabetic cardiovascular disease, the proportion of current smoker or alcohol drinker was signi cantly lower than controls (P < 0.001). Details were show in Table1. BMI body mass index, SBP systolic blood pressure, DBP diastolic blood pressure, FPG fasting plasma glucose, TG triglyceride, TC total cholesterol, LDL-C low density lipoprotein cholesterol, HDL-C high density lipoprotein cholesterol, AGEs advanced glycation end products, IL-6 interleukin 6. **P < 0.01.
Association of AGER, IL6R polymorphisms with diabetic cardiovascular disease All polymorphisms were in Hardy-Weinberg equilibrium (all P-values were more than 0.05). For AGER rs184003, participants with CA and AA genotype have signi cantly higher risk of diabetic cardiovascular disease compared with CC genotype (OR = 1.435, P = 0.039; OR = 2.525, P = 0.030, respectively). The A allele is associated with an increased risk of diabetic cardiovascular disease by 50% in additive and dominant models (P = 0.005; P = 0.013, respectively). For AGER rs2070600, the T allele is associated with a lower risk of diabetic cardiovascular disease by 30% in additive and dominant models (P = 0.025; P = 0.030, respectively). However, after adjusting for potential confounders, the association between the above two SNPs and disease were null.
For IL6R rs4845625, participants with CT and TT genotype have signi cantly lower risk of diabetic cardiovascular disease compared with CC genotype (OR = 0.692, P = 0.045; OR = 0.503, P = 0.003, respectively). The T allele can signi cantly decrease the risk of diabetic cardiovascular disease in additive and dominant models (OR = 0.707, P = 0.005; OR = 0.632, P = 0.013, respectively). The association between rs4845625 and disease was still signi cant after adjusting for potential confounders. Details were shown in Table 2. Polygenic risk score is also associated with an increased risk of diabetic cardiovascular disease by 10% (OR = 1.098, 95%CI: 1.041 ~ 1.160, P = 0.001). After adjusting for dyslipidemia, hypertension, smoking, and drinking status, PRS was consistently associated with the disease (OR = 1.106, 95%CI: 1.016 ~ 1.205, P = 0.020).

Association between haplotypes and diabetic cardiovascular disease
Four out of ve SNPs in AGER gene (Block1: rs184003-rs1035798-rs2070600-rs1800624) and ve out of seven SNPs in IL6R gene (Block2: rs7529229-rs4845625-rs4129267-rs7514452-rs4072391) showed linkage disequilibrium, see Fig. 1. These two blocks were both signi cantly associated with diabetic cardiovascular disease (Block1: P = 0.008; Block2: P = 0.007). Four haplotypes were constructed in block 1, and two of them associated with diabetic ischemic heart disease (CGTA: P = 0.018; AGCA: P = 0.004). Four haplotypes were constructed in block 2, and two of them associated with diabetic cardiovascular disease (TCCTC: P = 0.033; TTCTC: P = 0.001). Details of haplotype analysis were shown in Table3. The effect of gene-gene interaction on diabetic cardiovascular disease GMDR analysis were performed to assess the gene-gene interaction on diabetic cardiovascular disease risk, after adjustment for dyslipidemia, hypertension, smoking, and drinking. The GMDR analysis suggested that rs184003 in AGER gene and rs4845625 in IL-6R gene was the best model in terms of statistical signi cance after permutation testing (P = 0.001). The two-locus models had a cross-validation consistency of 10/10, and had a testing accuracy of 0.597. Logistic regression was subsequently used to obtain the odds ratios (ORs) and 95% con dence intervals (CI) for the interaction between rs184003 and rs4845625. In additive model, the joint effect of rs184003 and rs4845625 is associated with an increased risk of diabetic cardiovascular disease by 38% (OR = 1.38, 95%CI: 1.13-1.69, P = 0.002).

Discussion
Individuals with T2DM are with an increased risk of CVD which cannot be fully explained by elevated glucose [14] . Genetic risk factors contribute a lot to the pathogenesis of diabetic macrovascular complications, but its role has not been fully illustrated yet. In the present community-based case-control study, rs4845625 in IL-6R gene, and the interaction of rs184003 in AGER gene and rs4845625 in IL-6R were signi cantly associated with diabetic ischemic heart disease. Polygenic risk score calculated by summing the number of risk alleles of the SNPs located in the above two genes were also associated with the elevated risk of diabetic ischemic heart disease.
AGER is a multiligand cell surface receptor. Advanced glycation end products (AGEs) which is produced after high glucose exposure can bind to AGER. Their interaction has been implicated in the pathogenesis of atherosclerosis. In addition, HMGB1 (high-mobility group protein 1) and neutrophil-derived S100 calcium-binding family members (S100A8/A9/A11/A12, and S100B) were also ligands of AGER. After ligand binding, proin ammatory and procoagulant pathways will be activated. The rs2070600 was found to be signi cantly associated with diabetic ischemic heart disease in the current study. But after adjustments for covariates, the associations became null. The rs2070600 is located in ligand-binding V domain of the AGER gene, often referred to as Gly82Ser [15] . Genome-wide association studies (GWAS) showed that rs2070600 were strongly and dose-dependently correlated with sRAGE level in whites and blacks from Atherosclerosis Risk in Communities Study and Chinese population [6,16] . Interestingly, although soluble-RAGE levels were found to be associated with diabetic complications in many researches, the association between rs2070600 and cardiovascular disease or other diabetic complications were not consistent. In Atherosclerosis Risk in Communities Study, the rs2070600 was not signi cantly associated with incident coronary heart disease or diabetes in both whites and blacks with a median follow-up of 20 years [6] . Gao et al. has found a signi cant association between rs2070600 and coronary arterial disease in 175 cases and 170 controls [17] . Meta-analysis found that the discrepancy may be attributable to ethnicity, subjects with rs2070600 risk allele were at higher risk of coronary arterial disease (CAD) in the Chinese population, rather than non-Chinese population. However, our study found the association between rs2070600 and diabetic ischemic heart disease was null. Another research also found rs2070600 was associated with the circulating levels of esRAGE but not with CAD in Chinese patients with T2DM [18] . These results might indicate that the association between rs2070600 and CAD may also be different in general population and T2DM patients.
Only few studies had demonstrated the association between rs184003 and cardiovascular disease. A hospital-based case-control study found rs184003 can signi cantly increase the risk of coronary artery disease (OR = 1.23, P = 0.008), and haplotypes C-T-G-G and T-A-G-T in AGER gene (rs1800625-rs1800624-rs2070600-rs184003) were associated with signi cant increases in risk for CAD [19] . In the current study, we also haplotypes C-G-T-A and A-G-C-A in AGER gene (rs184003-rs1035798-rs2070600-rs1800624) were signi cantly associated with diabetic ischemic heart disease. Although the rs184003 was signi cantly associated with diabetic ischemic heart disease in the current study, the associations became null after adjustments for covariates. To our knowledge, few studies illustrated the relationship between rs184003 and diabetic macrovascular complications. More researches are still need to validate our results. Given the fact sRAGE level were found to be signi cantly associated with CAD [20,21] in many researches, the null association between AGER polymorphisms and diabetic ischemic heart disease in the current study indicated that sRAGE level could be served a marker of CAD, but not the a potential intervention targeting of reducing the burden of CAD.
Mendelian randomization analysis illustrated that IL6R signaling might have a causal role in development of coronary heart disease [22] . Previous meta-analysis demonstrated that rs2228145 and rs7529229 in IL6R could signi cantly reduce the risk of coronary heart disease [22,23] . Although the metaanalysis constituting a large sample size, the data from Asian is insu cient. Chen et al. did not nd an association of rs2228145 with coronary stenosis or acute myocardial infarction in the Chinese Han population [24] . Likewise, our current study, showed no association between rs2228145 and diabetic ischemic heart disease in Chinese population. The haplotype T-T-C-T-C (rs7529229-rs4845625-rs4129267-rs7514452-rs4072391) in IL6R gene and the rs4845625 was associated with diabetic cardiovascular disease in our study, and the association held after adjusting for potential confounders. The rs4845625 was found to be signi cantly associated with hypertriglyceridemia in Japanese population [25] , and the T allele was associated with lower serum concentration of creatinine and increased eGFR [26] . Hypertriglyceridemia and chronic kidney disease (CKD) have common pathway leading to metabolic cardiovascular disease, like endothelial dysfunction, dyslipidemia, and in ammation [27] . Although there was seldom any study focus on the association between rs4845625 and diabetic heart disease, its association with triglyceride and kidney function might indicate the potential mechanisms of rs4645625 on diabetic ischemic heart disease.
It has been found that, in response to hyperglycemia, AGER will be activated by S100A8/A9 on hepatic Kupffer cells, leading to the secretion of IL-6. IL-6 would subsequently bind to its receptor (IL6R) on hepatocytes to enhance the production of thrombopoietin, thereby regulating platelet production and resulting diabetes-induced thrombocytosis [28] . In the current study, we found that gene-gene interaction between AGER and IL6R would increase the risk of diabetic ischemic heart disease. We subsequently used GeneMANIA to construct gene network and predict gene function. IL6R and AGER have physical interactions with each other, and several pathways including NF-kB /RelA and JAK/STAT are involved in these interactions. Details were shown in Supplementary Figure S1. These interactions illustrated that the interaction of SNPs in IL6R and AGER was not only a statistical interaction, but also a biological interaction. To our knowledge, this is the rst study aimed to identify interaction of AGER and IL6R gene, and our results provided a genetic evidence on the physiological mechanism of diabetic macrovascular complications. Whether the main effect and gene-gene interaction in these two genes could be used to predict the risk of diabetic macrovascular complications are still need to be validated by cohort study in the future. Although we found the signi cant interaction of AGER gene and IL6R gene, the association between circulating IL-6 and diabetic ischemic heart disease was null. This result indicates that the role of circulating IL-6 in the pathogenesis and development of T2DM cardiovascular complications is complex. The most common hypothesis is that local IL-6 production and dynamics of sIL-6R which indicated the activation of IL-6 trans-signaling pathway were more likely to affect the TPO production and macrovascular complications [28,29] .
In the current study, SBP, TC, LDLC level and the proportion of people with smoking and drinking habits were signi cantly lower in cases than in controls, which is not consistent with other researches.
According to "2017 Guidelines for the prevention and treatment of type 2 diabetes in China", diabetes patients have more stringent standards on blood pressure (BP) and blood lipid compared with health population, and diabetes patients with ischemic heart disease should quit smoking and drinking [30] .
Diabetes patients might change their lifestyles and medication to maintain their BP or blood lipid at a lower level. Due to the case-control study design of the current study, we were not able to collect the lifestyle risk factors and blood sample before the incident of diabetic ischemic heart disease. However, the percentage of SBP, DBP, HDLC, LDLC, TG and TC in ideal range were signi cantly higher in control group compared with cases (P < 0.001, supplementary table S2). Due to the above limitation of our study, more longitudinal researches are still needed to demonstrate whether genetic variants will increase the incident of diabetic macrovascular complications. What is more, medication information was not included in the investigation. Given the fact that some antidiabetic medication, like SGLT-2 inhibitor [31] , will reduce the risk of cardiovascular disease in diabetes patients, future researches considering antidiabetic medication are still needed to validate the genetic effect on diabetic macrovascular complications.
List of abbreviations Ethical Approval and consent to participate: This study was approved by the Ethics Committee of Capital Medical University (No:2016SY24). All participants enrolled in this study have signed informed consent.

Consent for publication: Not applicable
Availability of data and materials: The datasets generated and/or analyzed during the current study are not publicly available due to the regulations of the people's Republic of China on the administration of human genetic resources, but part of the dataset is available from the corresponding author on reasonable request.
Competing interests: The authors declare that they have no competing interests.
Funding: This study was supported by grants from National Science Foundation of China (81602908), and National key research and development program of China (2016YFC0900600/2016YFC0900603).
The funding sources had no involvement in the study design, data collection, analysis and interpretation of data, writing of the manuscript, and the decision to submit the article for publication.
Author contributions: LK designed the study and wrote the manuscript, XY analyzed data and visualized the interaction diagram. ZQ provided the statistical plan and helped to revise the manuscript. PW contributed to the veri cation of diabetic ischemic heart diseases in case group. GC and ZJ contributed to the management of blood sample and DNA extraction. ZL contributed to the collection of controls and participated in the study design.
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