7R genetic polymorphisms in coronary heart disease in Chinese Han population

International Immunopharmacology 79 (2020) 106084

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Contribution of IL-7/7R genetic polymorphisms in coronary heart disease in Chinese Han population

T

Yuxiao Suna,b,c,d, Jifeng Yana,b,c, Jiliang Zhanga,b,c, Aifeng Wanga,b,c, Jie Zoua,b,c, ⁎ Chuanyu Gaoa,b,c,d, a

Department of Cardiology, Henan Provincial People's Hospital, Zhengzhou 450003, People's Republic of China Fuwai Central China Cardiovascular Hospital, Zhengzhou 450003, People's Republic of China c People’s Hospital of Zhengzhou University, Zhengzhou 450003, People's Republic of China d Henan Provincial Key Laboratory for Control of Coronary Heart Disease, Zhengzhou 450003, People's Republic of China b

ARTICLE INFO

ABSTRACT

Keywords: Coronary heart disease IL-7/7R Genetic association

Background: Coronary heart disease (CHD) is a common chronic inflammatory disease. Interleukin (IL)-7/IL-7R has been reported to be involved in the development of CHD. However, the relationship between IL-7/7R genetic polymorphisms and CHD among the Han Chinese population remains unclear. Methods: To examine whether IL-7/7R variants contributed to CHD, six single-nucleotide polymorphisms (SNPs) were genotyped by using the Agena MassARRAY platform in 499 CHD patients and 496 controls. Logistic regression was used to calculate the odds ratios (ORs) and 95% confidence intervals (CIs). The linkage disequilibrium was analyzed using Haploview software. The association between clinical parameters and IL-7/7R polymorphisms was determined by a one-way ANOVA. Results: IL-7R rs969129 G (OR = 1.20, 95% CI: 1.00–1.43, p = 0.047) allele and GG (OR = 1.45, 95% CI: 1.01–2.08, p = 0.044) genotype carriers had a higher risk for CHD. IL-7R haplotype “ACAG” (OR = 1.43, 95% CI: 1.09–1.87, p = 0.010) conferred an increased CHD risk. Rs969129, rs6451231, and rs117173992 were related to CHD susceptibility in males and/or the subgroup of individuals aged > 61 years. IL-7R rs969129, rs10053847, rs6451231, and rs118137916 variants were associated with diabetes in patients with CHD. Moreover, rs969129, rs6451231, and rs117173992 were associated with high-density lipoprotein cholesterol (HDL-C) concentrations, whereas rs118137916 and rs10053847 were associated with low-density lipoprotein cholesterol (LDL-C) levels (p < 0.05). Conclusion: IL-7/7R variants were related to the genetic predisposition of CHD in the Chinese Han population. These findings increase our knowledge regarding the effect of IL-7/7R on CHD.

1. Introduction Coronary heart disease (CHD) is a common chronic inflammatory disease, leading to ischemia, hypoxia, or necrosis of cardiac myocytes [1]. It is characterized by remodeling and narrowing of blood vessels (coronary arteries). CHD is the major cause of mortality in the world [2]. In China, the steady rise in prevalence of CHD is likely to continue into the future [3]. CHD is a complex and heterogeneous disease caused by both polygenic and environmental factors [4]. Epidemiological studies have revealed that age, gender, hyperlipidemia, diabetes, hypertension, lack of exercise, and dietary factors (smoking and drinking) are risk factors for CHD [5]. Various gene polymorphisms have been identified as being related to CHD risk, such as CDKN2BAS, PPAP2B,

⁎

TCF21, and SCARB1 [6–8]. In CHD, inflammatory cells promote plaque formation by recruiting secreted inflammatory mediators into the injured vascular system [9]. Interleukin 7 (IL-7) is a member of the type I cytokine subfamily that prompts monocyte and natural killer cell activation, and causes the production and increase of adhesion molecules and pro-inflammatory cytokines [10]. The levels of IL-7 were higher in the plasma of patients with acute coronary syndrome, and are involved in the inflammatory processes of atherosclerosis and acute coronary syndrome by interaction with monocytes, platelets and chemokines [11]. IL-7 recruits monocytes/macrophages to the endothelium via the PI3K/AKT-dependent and -independent activation of NF-κB, playing an active role in atherogenesis [10]. IL-7 receptor (IL-7R) blockade can reduce

Corresponding author at: #7 Weiwu Road, Jinshui District, Zhengzhou 450003, Henan, People's Republic of China. E-mail address: [email protected] (C. Gao).

https://doi.org/10.1016/j.intimp.2019.106084 Received 13 August 2019; Received in revised form 13 November 2019; Accepted 24 November 2019 1567-5769/ © 2019 Elsevier B.V. All rights reserved.

International Immunopharmacology 79 (2020) 106084

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atherosclerotic plaque inflammation induced by post-myocardial infarction by inhibiting binding of IL-7 to IL-7R [12]. These studies suggest that IL-7/IL-7R have a significant role in the development of CHD, which led us to formulate the hypothesis that IL-7/7R polymorphisms could be of importance in CHD susceptibility. However, the association between IL-7/7R genetic variants and CHD has not been reported. Here, we aimed to evaluate the relationship between IL-7/7R polymorphisms and CHD risk among the Chinese Han population, and to focus on the effect of age, gender, and CHD with diabetes and hypertension on the association.

Table 1 Characteristics of patients with CHD and controls.

2. Materials and methods 2.1. Study participants A cohort of 499 CHD patients and 496 age-and sex-matched health controls were enrolled into the study from the Affiliated Hospital of Yan′an University. All recruited individuals were unrelated Chinese Han people. All patients were identified as diagnosed by two-experienced interventional cardiologists. CHD was defined by at least one of the three main coronary arteries or their major branches having severe coronary stenosis (≥50%) via a coronary angiography [13]. Patients with congenital heart disease, concomitant cardiomyopathy, valvar heart disease, inflammatory disease or other disease were excluded. Healthy controls were enrolled at their annual health examination at the hospital. These controls did not have any chest symptoms or electrocardiogram abnormalities suggesting CHD, without any history of CHD and were free from cardiovascular and cerebrovascular diseases, as well as immunological diseases. Demographic and clinical information was gathered via medical records and questionnaires. This study was performed with the approval of the Institutional Ethics Committee of the Affiliated Hospital of Yan′an University and was in compliance with the principles of the Declaration of Helsinki. Written informed consent was obtained from all participants before the experiment.

Variable

Cases (n = 499)

Controls (n = 496)

p

Age, year > 61 ≤61

61.34 (11.70) 249 (49.9%) 250 (50.1%)

61.29 (8.94) 273 (55.0%) 223 (45.0%)

0.939

Gender Male Female

319 (63.9%) 180 (36.1%)

320 (64.5%) 176 (35.5%)

Smoking Yes No Missing

180 (36.1%) 319 (63.9%)

121 (24.4%) 169 (34.1%) 206 (41.5%)

Total cholesterol (mmol/L) HDL-C (mmol/L) LDL-C (mmol/L) Total protein (g/L) Total bilirubin (μmol/L) Urea (mmol/L) Triglyceride (mmol/L) Hemoglobin (g/L) Leukocyte (109/L) RBC (109/L) Platelet (109/L)

4.09 (1.14) 1.09 (0.28) 1.92 (0.88) 66.39 (6.36) 13.11 (4.5) 5.42 (1.74) 1.46 (0.63) 130.58 (29.82) 7.52 (3.03) 4.72 (0.71) 182.97 (59.56)

3.61 (0.95) 1.08 (0.27) 1.51 (0.78) 71.05 (3.75) 15.51 (4.23) 5.28 (1.31) 1.52 (0.68) 145.53 (13.24) 5.83 (1.51) 4.86 (0.55) 210.33 (53.31)

Concomitant disease With hypertension Without hypertension With diabetes Without diabetes

295 204 101 398

0.795

< 0.001 0.571 < 0.001 < 0.001 < 0.001 0.025 0.257 < 0.001 < 0.001 0.002 < 0.001

(59.1%) (40.9%) (20.2%) (79.8%)

CHD, coronary heart disease; HDL-C, high-density lipoprotein cholesterol; LDLC, low-density lipoprotein cholesterol; RBC, red blood cell. Bold indicate that p < 0.05 indicates statistical significance.

clinical parameters under different genotypes of IL-7/7R polymorphisms was tested by one-way analysis of variance (ANOVA). A p < 0.05 was statistically significant.

2.2. Genotyping

3. Results

Blood samples were collected from all subjects in EDTA-coated tubes. The GoldMag DNA Purification Kit (GoldMag Co. Ltd, Xi′an, China) was used for DNA extraction according to the manufacturer’s protocol. Six SNPs (rs118137916, rs10053847, rs10213865, rs969129, and rs6451231 in IL-7R and rs117173992 in IL-7) were selected. The screening criteria were minor allele frequencies (MAFs) > 0.05 in the 1000 Genomes Project data (http://www.internationalgenome.org/), and a pairwise tagging r2 ≥ 0.8. SNPs genotyping was performed on Agena MassARRAY platform (Agena, San Diego, CA, U.S.A.) in double blind. PCR and single base extension primers were displayed in Supplementary Table 1. Randomly, 5% of samples underwent re-genotyping for quality control, and concordance rates were 100% in duplicate samples.

We recruited 499 cases (319 males and 180 females; 61.34 ± 11.70 years) and 496 controls (320 males and 176 females; 61.29 ± 8.94 years) for this study. Features of CHD patients and health controls were presented in Table 1. There were no statistical differences with regard to the frequency distribution of age, sex, HDL-C and triglycerides (p > 0.05) between cases and controls. The concentration of total cholesterol, LDL-C, total protein, total bilirubin, urea, hemoglobin, leukocytes, red blood cells (RBCs) and platelets in two groups had significant differences (p < 0.05). Six SNPs in IL-7/7R were identified and genotyped in the cases and controls. The MAF of all SNPs was higher than 5% of the study population. All SNPs were in HWE in the controls (Table 2). For SNP rs969129 in IL-7R, the frequency distribution of G allele was high among CHD patients (49.0%) compared to controls (44.6%), and showed a 1.2-fold increased CHD risk (p = 0.047). We also found that the frequency of GG genotype was high among CHD patients (23.2%) compared to controls (19.0%, Table 3). IL-7R rs969129 conferred an increased CHD risk in the homozygote (OR = 1.45, 95% CI: 1.01–2.08, p = 0.044) and additive (OR = 1.20, 95% CI: 1.01–1.44, p = 0.044) models. However, the allele and genotype distributions of other SNPs were not significantly different (p > 0.05, Table 2 and Supplementary Table 2). The results of haplotype analysis revealed that rs10213865, rs969129, rs118137916 and rs10053847 in IL-7R were in linkage disequilibrium, as shown in Fig. 1. Further analyses of association between IL-7R haplotypes and CHD risk found that “ACAG” haplotype distribution frequency was significantly higher in the CHD group than the control group (Table 4), and conferred an increased risk of CHD after

2.3. Data analysis SPSS version 18.0 (SPSS Inc., Chicago, IL, USA) and PLINK software were used for statistical analyses. Demographic and clinical data between patients and controls were compared by student's t-test or χ2 test. Continuous variables were expressed as means (SD), while categorical variables were presented as absolute numbers (percentage value). A goodness-of-fit χ2 test was used to test Hardy–Weinberg equilibrium (HWE) among controls. Logistic regression analysis was carried out to estimate the possible effect of the IL-7/7R genotypes on CHD by odds ratios (ORs) and 95% confidence intervals (CIs) adjusted by age and sex. Linkage disequilibrium (LD) analysis was measured by Haploview software (version 4.2) and PLINK software. Multifactor dimensionality reduction (MDR) method was used to assess the association between IL7/7R SNPs and the occurrence risk of CHD [14]. The distribution of 2

International Immunopharmacology 79 (2020) 106084

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Table 2 The information about IL-7/7R SNPs and associations with the risk of CHD in allele model. Genes

IL-7R IL-7R IL-7R IL-7R IL-7R IL-7

SNPs ID

Chr: Position

rs10213865 rs969129 rs118137916 rs10053847 rs6451231 rs117173992

5:35857748 5:35861166 5:35863436 5:35878038 5:35878825 8:78779168

Alleles (minor/major)

Frequency (MAF) Case

Control

C/A G/T G/A A/G C/T G/A

0.188 0.490 0.080 0.154 0.416 0.085

0.178 0.446 0.085 0.161 0.382 0.075

O (HET)

E (HET)

p-value for HWE

OR (95% CI)

p

0.300 0.512 0.157 0.278 0.481 0.129

0.293 0.494 0.155 0.271 0.472 0.138

0.649 0.467 1.000 0.620 0.775 0.178

1.07 1.20 0.94 0.94 1.15 1.16

0.566 0.047 0.714 0.639 0.128 0.384

(0.85–1.34) (1.00–1.43) (0.68–1.30) (0.74–1.20) (0.96–1.38) (0.83–1.60)

SNP, single nucleotide polymorphism; CHD, coronary heart disease; MAF: minor allele frequency; HWE, Hardy-Weinberg equilibrium. Bold indicate that p < 0.05 means the data is statistically significant.

adjusted by age and gender (OR = 1.43, 95% CI: 1.09–1.87, p = 0.010). Moreover, we analyzed whether the genotypic effects on CHD risk were dependent on gender and age (Table 5). Stratified by gender, we found that IL-7R rs969129, IL-7R rs6451231 and IL-7 rs117173992 polymorphisms were related to increased CHD risk in males. In the male subgroup, IL-7R rs969129 polymorphism increased CHD risk in allele (OR = 1.33, p = 0.012), homozygote (OR = 1.84, p = 0.009), recessive (OR = 1.63, p = 0.014) and additive (OR = 1.34, p = 0.011) models. For rs6451231 in IL-7R, individuals carrying CC genotype had 1.57 times increased risk of CHD, and showed a marginal p value (p = 0.048) in males. In addition, IL-7 rs117173992 conferred high CHD risk in allele (OR = 1.57, p = 0.034), heterozygote (OR = 1.70, p = 0.030), dominant (OR = 1.66, p = 0.030) and additive (OR = 1.50, p = 0.047) models among males. Stratified by age, rs969129 and rs6451231 in IL-7R increased susceptibility to CHD in the subgroup of individuals aged > 61 years. The G allele (G vs T: OR = 1.32, p = 0.025; additive: OR = 1.35, p = 0.025) and GG genotype (GG vs TT: OR = 1.88, p = 0.020; GG vs GT-TT: OR = 1.71, p = 0.020) of rs969129 contributed to increased CHD susceptibility among the population with age > 61 years. The C allele of rs6451231 had 1.30 times greater risk of CHD compared to T allele under allele (p = 0.037) and additive (p = 0.047) models at age > 61 years. To evaluate the combined effect of CHD and diabetes or hypertension, CHD patients were divided into two groups according to the presence or absence of diabetes or hypertension (Table 6 and Supplementary Table 3). Rs969129, rs10053847, rs6451231 and rs118137916 variants in IL-7R were found associated with diabetes in patients with CHD under multiple genetic models (p < 0.05). The results were as follows: IL-7R rs969129 (allele: OR = 1.45, p = 0.018; homozygote: OR = 2.24, p = 0.016; dominant: OR = 1.85, p = 0.034; additive: OR = 1.48, p = 0.016), rs10053847 (allele: OR = 1.75, p = 0.005; homozygote: OR = 4.61, p = 0.010; dominant: OR = 1.70, p = 0.024; recessive: OR = 4.08, p = 0.017; and additive: OR = 1.74, p = 0.006) rs6451231 (allele: OR = 1.66, p = 0.001; homozygote;

Fig. 1. Haplotype block map for five SNPs in IL-7R. The block includes rs10213865, rs969129, rs118137916 and rs10053847. The LD between two SNPs is standardized D′.

OR = 2.73, p = 0.002; heterozygote: OR = 2.01, p = 0.011; dominant: OR = 2.20 , p = 0.003; recessive: OR = 1.76, p = 0.036; and additive: OR = 1.66 , p = 0.002) and rs118137916 (allele: OR = 0.48,

Table 3 Relationships between IL-7R rs969129 variant and CHD risk. SNP ID

Model

Genotype

Control

Case

Crude analysis OR (95% CI)

rs969129

Codominant Dominant Recessive Log-additive

TT TG GG TT TG-GG TT-TG GG

126 257 116 126 373 383 116

148 254 94 148 348 402 94

1 1.19 1.45 1 1.26 1 1.30 1.20

Adjusted by age and gender p-value

(0.89–1.60) (1.01–2.08)

0.250 0.044

(0.95–1.66)

0.106

(0.95–1.76) (1.01–1.44)

0.097 0.044

SNP, single nucleotide polymorphism; CHD, coronary heart disease; OR, odds ratio; 95% CI, 95% confidence interval. p values were calculated by logistic regression analysis with and without adjustments for age and gender. Bold indicate that p < 0.05 means the data is statistically significant. 3

OR (95% CI) 1 1.19 1.45 1 1.26 1 1.30 1.20

p-value

(0.88–1.59) (1.01–2.08)

0.252 0.044

(0.95–1.66)

0.106

(0.95–1.76) (1.01–1.44)

0.097 0.044

International Immunopharmacology 79 (2020) 106084

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Table 4 Haplotype frequencies in IL-7R and their associations with CHD risk. SNP

Haplotype

rs10213865|rs969129|rs118137916|rs10053847 rs10213865|rs969129|rs118137916|rs10053847 rs10213865|rs969129|rs118137916|rs10053847 rs10213865|rs969129|rs118137916|rs10053847 rs10213865|rs969129|rs118137916|rs10053847

AGAA ATGG CGAG AGAG ATAG

Frequency

Crude analysis

Adjusted by age and gender

Case

Control

OR (95% CI)

p

OR (95% CI)

p

0.154 0.080 0.189 0.147 0.431

0.159 0.085 0.177 0.108 0.469

0.96 0.94 1.08 1.43 0.86

0.727 0.721 0.507 0.010 0.088

0.96 0.94 1.08 1.43 0.86

0.731 0.720 0.510 0.010 0.088

(0.75–1.22) (0.68–1.30) (0.86–1.36) (1.09–1.87) (0.72–1.02)

(0.75–1.22) (0.68–1.30) (0.86–1.36) (1.09–1.87) (0.72–1.02)

IL-7R block comprises the four closely linked SNPs rs10213865, rs969129, rs118137916 and rs10053847. SNP, single nucleotide polymorphism; CHD, coronary heart disease; OR: odds ratio; 95% CI: 95% confidence interval. p values were calculated using logistic regression analysis with and without adjustment by gender and age; p < 0.05 indicates statistical significance. Table 5 Relationships between IL-7/7R polymorphisms and CHD risk according to the stratification by gender and age. SNP ID

Model

Male

Female

> 61 years

≤61 years

OR (95%CI)

p

OR (95%CI)

p

OR (95%CI)

p

OR (95%CI)

p

IL-7R rs969129

Allele Homozygote Heterozygote Dominant Recessive Additive

1.33 1.84 1.21 1.36 1.63 1.34

(1.07–1.66) (1.17–2.90) (0.84–1.74) (0.96–1.93) (1.10–2.40) (1.07–1.68)

0.012 0.009 0.317 0.083 0.014 0.011

0.99 0.97 1.16 1.10 0.88 0.99

(0.74–1.33) (0.53–1.76) (0.70–1.90) (0.69–1.75) (0.53–1.46) (0.74–1.34)

0.953 0.909 0.568 0.705 0.616 0.955

1.32 1.88 1.15 1.32 1.71 1.35

(1.04–1.69) (1.11–3.19) (0.75–1.76) (0.88–1.97) (1.09–2.70) (1.04–1.76)

0.025 0.020 0.513 0.178 0.020 0.025

1.06 1.13 1.13 1.13 1.04 1.06

(0.82–1.37) (0.67–1.89) (0.73–1.74) (0.75–1.70) (0.68–1.61) (0.82–1.38)

0.636 0.651 0.593 0.569 0.846 0.635

IL-7R rs6451231

Allele Homozygote Heterozygote Dominant Recessive Additive

1.20 1.59 1.03 1.14 1.57 1.20

(0.96–1.50) (0.98–2.59) (0.73–1.45) (0.83–1.57) (1.00–2.45) (0.96–1.51)

0.115 0.060 0.860 0.424 0.048 0.112

1.07 1.10 1.16 1.14 1.01 1.07

(0.79–1.44) (0.60–2.01) (0.73–1.85) (0.74–1.77) (0.59–1.73) (0.79–1.43)

0.667 0.756 0.528 0.546 0.974 0.672

1.30 1.71 1.29 1.38 1.48 1.30

(1.02–1.67) (0.99–2.96) (0.87–1.91) (0.95–2.00) (0.90–2.44) (1.00–1.69)

0.037 0.056 0.204 0.091 0.126 0.047

1.00 1.04 0.81 0.86 1.17 0.98

(0.77–1.29) (0.60–1.78) (0.54–1.21) (0.59–1.27) (0.72–1.91) (0.75–1.27)

0.980 0.894 0.302 0.451 0.523 0.865

IL-7

Allele Homozygote Heterozygote Dominant Recessive Additive

1.57 1.34 1.70 1.66 1.26 1.50

(1.03–2.39) (0.36–5.06) (1.05–2.75) (1.05–2.62) (0.33–4.73) (1.01–2.24)

0.034 0.662 0.030 0.030 0.734 0.047

0.71 / 0.73 0.71 / 0.69

(0.41–1.20)

0.195 / 0.266 0.222 / 0.184

1.23 2.65 1.12 1.20 2.62 1.24

(0.78–1.92) (0.45–15.52) (0.67–1.88) (0.73–1.97) (0.45–15.28) (0.79–1.93)

0.371 0.279 0.663 0.482 0.286 0.357

1.08 0.28 1.34 1.23 0.27 1.11

(0.67–1.74) (0.03–2.78) (0.79–2.28) (0.74–2.06) (0.03–2.68) (0.69–1.79)

0.743 0.278 0.277 0.428 0.264 0.654

rs117173992

(0.41–1.28) (0.40–1.23) (0.40–1.19)

SNP, single nucleotide polymorphism; CHD, coronary heart disease; OR, odds ratio; 95% CI, 95% confidence interval. p values were calculated by logistic regression analysis with and without adjustments for age and gender. Bold indicate that p < 0.05 means the data is statistically significant.

GG genotype carriers have a higher CHD risk in the Chinese Han population. The haplotype “ACAG” (rs10213865, rs969129, rs118137916 and rs10053847) of IL-7R conferred an increased CHD risk. IL-7R rs969129 and rs6451231 were related to an increased risk of CHD among males and the subgroup of individuals aged > 61 years, and IL-7 rs117173992 contributed to the susceptibility of CHD in males. IL-7R rs969129, rs10053847, rs6451231 and rs118137916 variants were associated with diabetes in patients with CHD. We also found that rs969129, rs6451231 and rs117173992 were associated with HDL-C concentrations, while rs118137916 and rs10053847 were associated with LDL-C levels. This study was the first to find a relationship between IL-7/7R variants and CHD susceptibility among the Chinese Han population. IL-7, encoded by IL-7 (located at 8q21.13), is a pleiotropic cytokine promoted the production of inflammatory cytokines by regulated the proliferation of T lymphocyte and monocytes activation, thus, affect the pathogenesis of CHD [15]. IL-7R, located at 5p13.2, encodes IL-7 receptor protein that participate in the development of immune cells [16]. Mihailovica et al found the IL-7/7R pathway might be a potential intervention target to alleviate the inflammatory cascade in atherosclerotic plaques [12]. Previously, IL-7/7R variants may be involved in various diseases, including osteoarthritis and multiple sclerosis [17–19], but not CHD. Here, we firstly revealed that IL-7R rs969129 G allele conferred to the risk of developing CHD in a Chinese Han population. Previously, very few studies have analyzed the SNP rs969129 to now, only one study reported that

p = 0.037; dominant: OR = 0.48, p = 0.047; and additive: OR = 0.47, p = 0.043). However, no significant correlation was found in CHD patients with hypertension (Supplementary Table 3). MDR was used to assess the accumulated effect of all risk factors of IL-7 rs117173992, IL-7R rs6451231 and IL-7R rs969129. The SNP-SNP interaction analysis revealed risk effect among markers IL-7 rs117173992, IL-7R rs6451231 and IL-7R rs969129, which conferred higher risk towards the occurrence of CHD (OR = 1.46; 95% CI: 1.10–1.95, p = 0.0095), as shown in Supplementary Table 4. We also assessed the association of selected SNPs and clinical variables in patients (Table 7). Significant association was observed between rs10213865 genotypes and levels of total bilirubin (p = 0.023) and urea (p = 0.032). We also found that the rs969129 genotype was related to HDL-C (p = 0.021) and total bilirubin (p = 0.036) concentrations. We observed a significant difference in LDL-C concentrations among rs118137916 (p = 0.014) and rs10053847 (p = 0.023) genotypes. A significant association of rs6451231 polymorphism with HDL-C (p = 0.031) and leukocytes (p = 0.008) was identified. Subjects carrying the rs117173992 “GG” genotype had higher HDL-C (p = 0.001) and total bilirubin (p = 0.036), while lower hemoglobin (p = 0.010) than those carrying “GA” and “AA” genotypes. 4. Discussion In this study, our results revealed that IL-7R rs969129 G allele and 4

5

AA AC CC

TT TG GG

AA AG GG

GG GA AA

TT TC CC

AA AG GG

IL-7R rs10213865

IL-7R rs969129

IL-7R rs118137916

IL-7R rs10053847

IL-7R rs6451231

IL-7

87 14 0

22 54 25

63 32 6

92 9 0

17 54 30

64 34 3

With diabetes

332 61 5

151 183 64

294 97 6

329 67 2

109 203 86

263 122 13

Without diabetes

1 0.87 (0.47–1.64) /

1 2.01 (1.17–3.46) 2.73 (1.43–5.20)

1 1.52 (0.94–2.47) 4.61 (1.44–14.79)

1 0.49 (0.23–1.02) /

1 1.68 (0.93–3.05) 2.24 (1.16–4.34)

1 1.13 (0.71–1.81) 0.99 (0.27–3.58)

0.671 /

0.011 0.002

0.089 0.010

0.056 /

0.086 0.016

0.613 0.984

0.80 (0.43–1.50)

2.20 (1.31–3.67)

1.70 (1.07–2.70)

0.48 (0.23–0.99)

1.85 (1.05–3.26)

1.12 (0.71–1.76)

OR (95% CI)

OR (95% CI)

p-value

Dominant

Genotype

SNP, single nucleotide polymorphism; CHD, coronary heart disease; OR, odds ratio; 95% CI, 95% confidence interval. p values were calculated by logistic regression analysis with and without adjustments for age and gender. Bold indicate that p < 0.05 means the data is statistically significant.

rs117173992

Genotype

SNP_ID

Table 6 Association of IL-7/7R polymorphisms in CHD patients with and without diabetes.

0.487

0.003

0.024

0.047

0.034

0.638

p-value

/

1.76 (1.04–2.98)

4.08 (1.28–12.96)

/

1.55 (0.95–2.54)

0.95 (0.26–3.41)

OR (95% CI)

Recessive

/

0.036

0.017

/

0.079

0.935

p-value

0.76 (0.42–1.36)

1.66 (1.21–2.27)

1.74 (1.17–2.58)

0.47 (0.23–0.98)

1.48 (1.07–2.04)

1.08 (0.73–1.61)

OR (95% CI)

Additive

0.350

0.002

0.006

0.043

0.016

0.701

p-value

0.76 (0.42–1.38)

1.66 (1.21–2.26)

1.75 (1.18–2.59)

0.48 (0.23–0.97)

1.45 (1.07–1.99)

1.08 (0.73–1.60)

OR (95% CI)

Allele

0.366

0.001

0.005

0.037

0.018

0.695

p-value

Y. Sun, et al.

International Immunopharmacology 79 (2020) 106084

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Table 7 Comparisons of clinical characteristics among T2D patients with different genotypes of SNPs in IL-7/7R. Characteristics

rs10213865

rs969129

AA

CA

CC

p

TT

GT

GG

p

Total cholesterol (mmol/L) HDL-C (mmol/L) LDL-C (mmol/L) Total protein (g/L) Total bilirubin (μmol/L) Urea (mmol/L) Triglyceride (mmol/L) Hemoglobin (g/L) Leukocyte (109/L) RBC (109/L) Platelet (109/L)

4.07 (1.16) 1.09 (0.28) 1.92 (0.92) 66.34 (6.17) 12.81 (4.30) 5.29 (1.73) 1.47 (0.65) 128.53 (31.66) 7.34 (2.85) 4.75 (0.68) 180.67 (61.22)

4.12 (1.12) 1.11 (0.29) 1.93 (0.81) 66.43 (6.85) 13.9 (4.78) 5.71 (1.78) 1.46 (0.58) 134.01 (26.65) 7.89 (3.35) 4.65 (0.75) 185.73 (56.91)

4.23 (1.07) 1.03 (0.10) 1.78 (0.70) 67.12 (5.77) 11.56 (4.88) 5.04 (0.97) 1.45 (0.50) 137.92 (12.07) 7.38 (3.00) 4.85 (0.66) 199.63 (51.25)

0.804 0.585 0.796 0.888 0.023 0.032 0.990 0.122 0.195 0.280 0.369

3.93 (1.10) 1.12 (0.31) 1.80 (0.76) 66.13 (6.42) 12.37 (4.37) 5.23 (1.53) 1.37 (0.64) 129.36 (30.43) 7.97 (3.12) 4.73 (0.74) 181.89 (66.66)

4.12 (1.20) 1.11 (0.30) 1.96 (0.98) 66.22 (6.51) 13.61 (4.54) 5.50 (1.85) 1.51 (0.63) 130.51 (30.36) 7.26 (2.89) 4.71 (0.64) 180.27 (58.62)

4.19 (1.05) 1.03 (0.15) 1.95 (0.75) 67.04 (5.97) 12.79 (4.45) 5.43 (1.67) 1.47 (0.59) 132.08 (28.04) 7.61 (3.19) 4.74 (0.82) 190.16 (53.11)

0.200 0.021 0.247 0.460 0.036 0.398 0.166 0.795 0.109 0.915 0.360

Characteristics

rs118137916

rs10053847

AA

GA

GG

p

AA

AG

GG

p

Total cholesterol (mmol/L) HDL-C (mmol/L) LDL-C (mmol/L) Total protein (g/L) Total bilirubin (μmol/L) Urea (mmol/L) Triglyceride (mmol/L) Hemoglobin (g/L) Leukocyte (109/L) RBC (109/L) Platelet (109/L)

4.11 (1.13) 1.09 (0.27) 1.93 (0.89) 66.51 (6.38) 13.19 (4.52) 5.43 (1.75) 1.47 (0.62) 130.16 (30.11) 7.44 (3.07) 4.71 (0.71) 183.19 (57.62)

3.95 (1.20) 1.11 (0.31) 1.77 (0.71) 65.78 (6.35) 12.78 (4.42) 5.31 (1.68) 1.43 (0.69) 132.87 (28.35) 7.85 (2.71) 4.77 (0.71) 181.69 (69.19)

5.41 (1.99) 1.13 (0.18) 3.52 (2.14) 65.50 (6.36) 9.15 (0.21) 5.85 (0.21) 1.54 (0.65) 142.00 (29.63) 10.6 (3.67) 5.15 (0.21) 183 (120.21)

0.157 0.925 0.014 0.657 0.358 0.793 0.885 0.732 0.209 0.573 0.982

4.29 (1.43) 0.99 (0.03) 2.50 (0.81) 67.32 (6.52) 12.46 (5.35) 5.67 (2.35) 1.70 (0.74) 136.67 (12.06) 8.70 (4.36) 4.73 (0.62) 190.25 (60.11)

4.13 (1.12) 1.06 (0.22) 2.01 (1.09) 66.32 (6.12) 13.43 (4.07) 5.44 (1.85) 1.54 (0.65) 129.68 (30.80) 7.03 (2.69) 4.70 (0.77) 180.74 (54.98)

4.07 (1.14) 1.11 (0.30) 1.86 (0.79) 66.38 (6.47) 13.03 (4.63) 5.40 (1.68) 1.43 (0.61) 130.96 (29.57) 7.65 (3.07) 4.73 (0.69) 183.48 (61.2)

0.727 0.111 0.023 0.874 0.620 0.865 0.129 0.721 0.064 0.949 0.834

Characteristics

rs6451231

Total cholesterol (mmol/L) HDL-C (mmol/L) LDL-C (mmol/L) Total protein (g/L) Total bilirubin (μmol/L) Urea (mmol/L) Triglyceride (mmol/L) Hemoglobin (g/L) Leukocyte (109/L) RBC (109/L) Platelet (109/L)

rs117173992

TT

CT

CC

p

AA

GA

GG

p

3.94 (1.12) 1.13 (0.33) 1.85 (0.83) 66.16 (6.46) 12.98 (4.65) 5.32 (1.61) 1.38 (0.62) 130.00 (30.45) 8.08 (3.05) 4.75 (0.69) 182.32 (65.89)

4.14 (1.19) 1.09 (0.27) 1.93 (0.96) 66.3 (6.40) 13.52 (4.51) 5.49 (1.81) 1.49 (0.64) 131.52 (29.17) 7.10 (2.94) 4.71 (0.66) 179.8 (56.25)

4.24 (1.04) 1.04 (0.17) 2.01 (0.76) 67.09 (6.10) 12.24 (4.08) 5.4 (1.78) 1.54 (0.62) 129.18 (30.57) 7.56 (3.09) 4.69 (0.85) 192.99 (54.61)

0.105 0.031 0.413 0.511 0.077 0.609 0.138 0.794 0.008 0.775 0.239

4.10 (1.12) 1.09 (0.27) 1.91 (0.79) 66.46 (6.34) 12.93 (4.42) 5.39 (1.72) 1.48 (0.64) 130.7 (29.83) 7.50 (3.00) 4.73 (0.73) 181.14 (56.45)

3.99 (1.25) 1.09 (0.26) 1.92 (1.29) 65.67 (6.51) 13.82 (4.68) 5.57 (1.81) 1.39 (0.56) 132.16 (26.46) 7.58 (3.16) 4.68 (0.61) 191.31 (73.59)

4.95 (1.18) 1.54 (0.51) 2.46 (0.96) 71.46 (3.41) 17.75 (6.34) 5.52 (1.69) 1.34 (0.48) 79.00 (63.24) 8.75 (4.69) 5.00 (0.01) 226.67 (86.64)

0.181 0.001 0.379 0.126 0.036 0.709 0.516 0.010 0.763 0.718 0.192

CHD, coronary heart disease; SNP, single nucleotide polymorphism; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; RBC, red blood cell. Bold indicate that p < 0.05 indicates statistical significance.

rs969129 was associated with rheumatoid arthritis [20]. Rs969129 polymorphism, located in the intron region, might be related to promoter and enhancer histone marks, DNase, motifs changed and selected eQTL hits from HaploReg v4.1 database (https://pubs.broadinstitute.org/mammals/ haploreg/haploreg.php). Several studies reported that intronic SNPs confer susceptibilities by affecting gene expression [21–23]. These study hinted us that IL-7R rs969129 contributed to CHD risk might be associated with its function. However, further study is necessary to explore the function of the SNP. Moreover, we also found ACAG haplotype consisting of rs10213865-rs969129-rs118137916-rs10053847 was the susceptibility haplotype of CHD. CHD is a sex-specific disease, and the incidence of coronary events is higher in male than female [24]. A further stratification analysis by gender, we noticed that IL-7R rs969129, IL-7R rs6451231 and IL-7 rs117173992 were related to an increased CHD risk in males but not in males, which indicates that the effect of these polymorphisms on CHD risk presented sex difference. Aging is a known risk factor of CHD [25]. Our age-stratified correlation analysis revealed that rs969129 and rs6451231 in IL-7R increased the susceptibility to CHD in the subgroup of individuals aged > 61 years, suggesting that the risk association of the polymorphisms might be age-dependent. Diabetes and hypertension

are also the risk factor for CHD [26,27]. Our results also showed IL-7R variants (rs969129, rs10053847, rs6451231 and rs118137916) were associated with diabetes in patients with CHD. However, these SNPs were not significantly associated with hypertension in patients with CHD. Further study is required to validate these findings. Some studies showed that IL-7R rs10053847 variant was related to a decreased lung cancer risk and rs6451231 in IL-7R were associated with an increased risk of rheumatoid arthritis [20,28]. However, this study exported the relationship between IL-7/7R variants and CHD susceptibility among the Chinese Han population. Previous studies have shown that HDL-C and LDL-C levels are considered independent risk factors for the development of CHD [29,30]. In our study, we found that the total cholesterol and LDL-C level were different between CHD patients and the controls. We also found that rs969129, rs6451231 and rs117173992 variants associated with HDL-C concentrations, while rs118137916 and rs10053847 associated with LDL-C levels. Moreover, subjects carrying the rs6451231 “TT” genotype had a higher leukocyte level than those carrying “CT” and “CC” genotypes, suggesting that this polymorphism might be related to the function of IL-7R. However, more functional studies are required to corroborate this. 6

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Inevitably, there are several potential limitations. Firstly, the selection bias cannot be avoided in a hospital based case–control study. Secondly, the biological mechanism of IL-7/7R genetic variants was not explored, and require further research. Thirdly, these results need must be handled with caution because SNPs were not significant after multiple correction. This may be due to a small sample size or low frequency of the minor allele. Further studies, with a larger sample size are necessary to confirm our results. Despite the limitations mentioned above, the results of this study provided scientific evidence of IL-7/7R polymorphisms with CHD in the Chinese Han population.

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5. Conclusion In conclusion, this study showed that IL-7/7R polymorphisms and haplotype Ars10213865Grs969129Ars118137916Grs10053847 were the risk factor of CHD in the Chinese Han population. It was also suggested that age, gender, and diabetes had an effect on the association of IL-7/7R polymorphisms and CHD risk. These findings improved our knowledge concerning the effect of IL-7/7R on CHD processes. However, more population-based and functional studies are necessary. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements We thank all authors for their contributions and support. We are grateful to all study participants who provided blood samples. We would also like to thank the hospital staff for their contribution to data collection for this study. Appendix A. Supplementary material Supplementary data to this article can be found online at https:// doi.org/10.1016/j.intimp.2019.106084. References [1] P. Libby, P. Theroux, Pathophysiology of coronary artery disease, Circulation 111 (25) (2005) 3481–3488. [2] Global, regional, and national age-sex specific all-cause and cause-specific mortality for 240 causes of death, 1990–2013: a systematic analysis for the Global Burden of Disease Study 2013, Lancet (London, England), 2015, 385(9963):117–171. [3] C. Shen, J. Ge, Epidemic of cardiovascular disease in China, Circulation 138 (4) (2018) 342–344. [4] N.D. Wong, Epidemiological studies of CHD and the evolution of preventive cardiology, Nat. Rev. Cardiol. 11 (5) (2014) 276–289. [5] J.E. Dalen, J.S. Alpert, R.J. Goldberg, R.S. Weinstein, The epidemic of the 20(th) century: coronary heart disease, Am. J. Med. 127 (9) (2014) 807–812. [6] W. Chang, R. Ma, X. Zhang, Y. Yan, W. Zhang, B. Zhao, G. Li, H. Wang, Y. Zhu, Y. Song, Impact of PPAP2B and TCF21 gene polymorphisms on risk of coronary heart disease in Chinese Han population, Int. J. Clin. Exp. Pathol. 9 (2) (2016) 1968–1974. [7] X. Li, H. Ma, F. Li, Y. Li, Z. Xie, H. Bai, B. Gao, Q. Liang, F. Gao, T. Jin, CDKN2BAS polymorphisms are associated with coronary artery disease in Chinese Han population, Int. J. Clin. Exp. Pathol. 10 (1) (2017) 804–810. [8] R. Ma, X. Zhu, B. Yan, SCARB1 rs5888 gene polymorphisms in coronary heart disease: a systematic review and a meta-analysis, Gene 678 (2018) 280–287. [9] H. Li, K. Sun, R. Zhao, J. Hu, Z. Hao, F. Wang, Y. Lu, F. Liu, Y. Zhang, Inflammatory biomarkers of coronary heart disease, Front. Biosci. (Scholar edition) 10 (2018) 185–196.

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