Effects of Ninjurin 2 Polymorphisms on Susceptibility of Coronary Heart Disease

Objective: The aim of this study was to explore the effects of NINJ2 polymorphisms on susceptibility of coronary heart disease (CHD). Methods: We conducted a case-control study with 499 CHD cases and 505 age- and sex- matched controls. Five single nucleotide polymorphisms (SNP) in NINJ2 (rs118050317, rs75750647, rs7307242, rs10849390 and rs11610368) were genotyped by Agena MassARRAY platform. Odd ratios (OR) and 95% condence intervals (CI) were calculated using logistic regression to assess the association of NINJ2 polymorphism and CHD risk adjusting for age and gender.. Results: NINJ2 rs118050317 signicantly increased the risk of CHD in people over 60 years old (allele: P = 0.010; heterozygote: P = 0.016; dominant: P = 0.015; additive: P = 0.021) and women (allele: P = 0.026; heterozygote: P = 0.015; dominant: P = 0.018; additive: P = 0.030). Rs118050317 and rs7307242 were closely related to the risk of hypertension in CHD patients. Additionally, rs75750647 signicantly increased diabetes risk in multiple models among CHD cases (allele: P = 0.014; homozygote: P = 0.037; heterozygote: P = 0.044; dominant: P = 0.019; additive: P = 0.013), whereas rs10849390 could protect CHD patients from diabetes in allele (P = 0.035), homozygote (P = 0.047) and additive (P = 0.037) models. We also observed two block (block 1: rs118050317 and rs75750647; block 2: rs7307242, rs10849390 and rs11610368) in NINJ2. Conclusion: Our results suggested that the relationships of NINJ2 polymorphisms and CHD risk were dependent on age, sex or complications. that NINJ2 polymorphisms might be associated with susceptibility of CHD. In order to clarify the role of NINJ2 polymorphisms in the etiology of CHD, We carried out a genetic association study to examine the relationship between CHD risk and NINJ2 polymorphisms (rs118050317, rs75750647, rs7307242, rs10849390, and rs11610368) among the Chinese Han population.


Introduction
Coronary heart disease (CHD) is a common cardiovascular disorder (CVD) with rapidly increasing incidence in the recent year [1]. Moreover, the incidence of CHD varies by age and sex [2]. The World Health Organization (WHO) reported that approximately 7.4 million people died from CHD in 2012 [3]. In China, CHD has affected more than 10 million people [4]. CHD is a complex disease resulting from the interactions between genetic and environmental factors [5]. The risk factors of CHD include hypertension, diabetes, lifestyle and clinical indicators (the level of cholesterol, triglyceride and uric acid) [6]. Nevertheless, increasing studies revealed that genetic variants contribute to CHD susceptibility [7]. Ninjurin 2 (NINJ2) is a transmembrane protein belongs to the ninjurin family, expressed in glia that plays a role in neurite outgrowth [8]. The gene that encodes NINJ2 protein is located on chromosome 12p13. NINJ2 interacts with several substances (IL-1β, TNF-α, IL-8, IL-6, ICAM-1 and E-selectin) in leukocyte and endothelial cells and thus plays an important role in the regulation of in ammation [9,10]. NINJ2 could regulate NF-kB and c-jun through interacting with TLR4 [11]. Previous studies have reported that NINJ2 are associated with many diseases, including ischemic stroke, large artery atherosclerotic stroke, Alzheimer's disease and vascular dementia [11,12].
Rs11833579 and rs12425791 are the most two studied NINJ2 polymorphism, which are considered as potential risk variants for ischemic stroke in the four large cohorts [13]. These evidences showed that NINJ2 might be associated with pathology of atherosclerosis, suggesting a linkage of NINJ2 and CHD. However, no genetic polymorphisms have shown convincing evidence related to CHD.
Hence, we hypothesized that NINJ2 polymorphisms might be associated with susceptibility of CHD. In order to clarify the role of NINJ2 polymorphisms in the etiology of CHD, We carried out a genetic association study to examine the relationship between CHD risk and NINJ2 polymorphisms (rs118050317, rs75750647, rs7307242, rs10849390, and rs11610368) among the Chinese Han population.

Study population
This study consisted of 499 patients with CHD and 505 healthy controls. All participants were recruited from First A liated Hospital of Xi'an Jiaotong University in Shaanxi province, China. CHD patients were diagnosed by at least two cardiovascular physicians based on angiographically demonstrated stenosis (> 50%) in a major or main branch of the coronary artery [14]. CHD Patients with stroke, cardiomyopathy, chronic diseases or other serious physical diseases were excluded from this study. Controls were healthy people who receiving physical examinations in the same hospital with cases. Individuals who had CVD, immune diseases, cancer or disease history were excluded in the control group.
Five SNPs were genotyped using the Agena MassARRAY platform Agena, San Diego, CA, USA). The primers of NINJ2 polymorphisms were designed by the Agena MassARRAY Assay Design 3.0 Software (San Diego, California, USA) and were presented in Supplemental table 1. Data were managed and analyzed using Agena Typer 4.0 Software (San Diego, CA, USA).

Statistical analysis
Statistical analysis was analyzed with Microsoft Excel and SPSS version 21.0 software (SPSS, Chicago, IL, USA). Continuous data and categorical variables were compared by Student's t-test and chi-square test. We used Fisher's exact test to assess the Hardy-Weinberg equilibrium (HWE) for all SNPs in the control group. The association of NINJ2 polymorphisms and CHD risk in genetic models (co-dominant, dominant, recessive and additive) was assessed by logistic regression, and the relationship strength was evaluated by the odd ratios (OR) and 95% con dence intervals (CI). Then, we did linkage disequilibrium (LD) and haplotype analysis by Haploview software and the PLINK software [15]. All tests were two-sided, and P < 0.05 was considered as statistical signi cance.

Characteristics of study subjects
The characteristics of study subjects were listed in Table 1. The distribution of age and sex was similar for cases and controls (age: P = 0.191, sex: P = 0.896). The mean ages of cases (318 men and 181 women) and controls (323 men and 182 women) were 61.34 ± 11.69 and 60.51 ± 8.11 years old, respectively. Additionally, we collected clinical characteristics of all subjects, including uric acid (UA), total cholesterol (TC), triglyceride (TG), high-density lipoprotein (HDL-C), low-density lipoprotein (LDL-C), platelet (PLT), platelet distribution width (PDW), mean platelet volume (MPV), and plateletcrit (PCT). There were signi cantly differences between two groups in the level of UA, TC, LDL, PLT and PCT. In CHD patients, 294 (59%) had hypertension and 100 (20%) had diabetes.

Haplotype analysis
We also conducted haplotype analysis of NINJ2 polymorphisms and CHD risk (Supplemental table 3). We did not nd signi cant association between haplotype of NINJ2 polymorphisms and susceptibility to CHD (P > 0.05). As it shown in Figure 1, there are two blocks in NINJ2 (block 1: rs118050317 and rs75750647; block 2: rs7307242, rs10849390 and rs11610368).

Association of genotypes of NINJ2 polymorphisms and clinical indicators of CHD patients
In Supplemental table 4, we showed the association of different genotypes of NINJ2 polymorphisms (rs118050317, rs75750647, rs7307242, rs10849390 and rs11610368) and clinical indicators (UA, TC, TG, HDL, LDL, PLT, PDW, MPV and PCT) of CHD patients. All patients with different genotypes had signi cantly difference in rs118050317, rs11610368 and PLT level (P rs118050317 = 0.041, P rs11610368 = 0.040). There were no strong relationships between other polymorphisms and clinical indicators in CHD patients.

Discussion
In this study, we found that NINJ2 polymorphisms were associated with the susceptibility of CHD. NINJ2 rs118050317 signi cantly increased CHD risk in elderly person (age > 60) and woman. Among CHD patients, NINJ2 polymorphisms (rs118050317, rs75750647, rs7307242 and rs10849390) had strong relationship with risk of hypertension and diabetes. Five SNPs were intonic, and they might be affected CHD by regulating Promoter histone marks, Enhancer histone marks, DNAse, Motifs changed and Selected eQTL hits. In addition, we observed two blocks (block 1: rs118050317 and rs75750647; block 2: rs7307242, rs10849390 and rs11610368).
NINJ2 is a cell surface adhesion protein that is upregulated after nerve injury [16]. NINJ2 plays an important role in nerve, stroke and in ammation [17][18][19][20]. We explored the effects of NINJ2 polymorphism on susceptibility of CHD. It is the rst study to show the strong association of NINJ2 polymorphisms and CHD risk among the Chinese Han population. Age and sex differences are obvious in the development of CHD. Advancing age is a non-modi able risk factor for CHD in both men (age > 46) and women (age > 55) [21]. The mortality of CHD is higher in men than in women at all stages, and it accelerates for women older than 60 years old [22]. To investigate the effects of NINJ2 polymorphisms on CHD risk in different populations, we did strati cation analysis. Our results showed the effect of rs118050317 on CHD risk was related to age and sex. Rs118050317 signi cantly increased risk of CHD for the elderly people and women in multiple genetic models, it may be attributed to the level of hormone. The exact mechanism of these in uences needs further studies. It gives us a clue for diagnosis, treatment and prevention of CHD in clinic.
Hypertension and diabetes are closely related to CHD. Previous studies showed each 10 mm Hg increase in systolic blood pressure is associated with an increased risk of CHD [23]. Moreover, people are more likely to have hypertension or diabetes with aging. Our study con rmed the association of CHD and some diseases (hypertension and diabetes). For CHD patients, rs118050317 and rs75750647 were associated with higher risk of hypertension and diabetes, individually. Rs7307242 and rs10849390 could protect CHD patients from hypertension and diabetes in genetic models. Additionally, many clinical factors (UA, TC, LDL, PLT and PCT) were signi cantly different between CHD cases and controls. Especially, platelet plays an important role in CVD by involving in acute and chronic in ammatory [24]. We further explored the association of NINJ2 polymorphisms and clinical indicators among CHD patients. We found the level of PLT was different in the genotypes of rs118050317 and rs11610368, it suggests the level of PLT may affect the development of CHD. However, the mechanism of CHD still needs further study.
Several limitations in the present study should be noted. Firstly, we recruited all study subjects from hospital, a selection bias may exist.
Secondly, we only focused ve polymorphisms of NINJ2, the association of other NINJ2 polymorphisms and CHD risk should be further studied. Thirdly, due to the limitation of information we collected, we could not take more risk factors into this manuscript, such as family history, lifestyle factors, and diet habits. Hence, further investigations in larger populations and functional experiments are necessary to validate our ndings.

Conclusions
In conclusion, the effects of NINJ2 polymorphisms on CHD susceptibility were related to age, sex or complications (hypertension and diabetes). It revealed that NINJ2 polymorphisms might be involved in the development of CHD among elder people (age > 60) and women. Our results will be helpful for the individualized diagnosis and testing of CHD. Additionally, further studies are needed to con rm the results obtained in this study.

Declarations Ethics approval and consent to participate
This study was approved by the Ethical Committee of First A liated Hospital of Xi'an Jiaotong University and conducted in accordance with the Declaration of Helsinki. And, informed consents were collected from all participants in this study.

Consent for publication
We all agree to publish this study.
Availability of data and material All data and material are available.