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 significantly 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.
Association of NINJ2 polymorphisms and CHD risk
The genotype distribution of NINJ2 polymorphisms was presented in Table 2. All SNPs are in HWE (P > 0.05). In allelic model, no significant associations of NINJ2 polymorphisms and CHD risk were observed (P > 0.05). We further performed the association of NINJ2 polymorphisms and CHD risk in multiple models and stratification analysis (Table 3, Table 4, Table 5 and Supplemental table 2). The effects of NINJ2 rs118050317 on CHD risk were dependent on age and sex. For the individuals over than 60 years old, rs118050317 was significantly associated with CHD risk in allele (OR = 1.64, 95%CI = 1.13-2.40, P = 0.010), heterozygote (OR = 1.73, 95%CI = 1.11-2.70, P = 0.016), dominant (OR = 1.71, 95%CI = 1.11-2.65, P = 0.015) and additive (OR = 1.63, 95%CI = 1.08-2.46, P = 0.021) models. Meanwhile, rs118050317 significantly increased the risk of CHD among women (allele: OR = 1.75, 95%CI = 1.07-2.86, P = 0.026; heterozygote: OR = 1.99, 95%CI = 1.14-3.45, P = 0.015; dominant: OR = 1.92, 95%CI = 1.12-3.28, P = 0.018; additive: OR = 1.74, 95%CI = 1.06-2.86, P = 0.030). In CHD patients, rs118050317 increased the risk of hypertension (allele: OR = 1.66, 95%CI = 1.09-2.53, P = 0.017; heterozygote: OR = 1.72, 95%CI = 1.08-2.76, P = 0.024; dominant: OR = 1.70, 95%CI = 1.08-2.70, P = 0.023; additive: OR = 1.60, 95%CI = 1.04-2.46, P = 0.031), whereas rs7307242 was significantly associated with decreased hypertension risk (homozygote: OR = 0.35, 95%CI = 0.13-0.99, P = 0.049; recessive: OR = 0.35, 95%CI = 0.13-0.99, P = 0.047). In addition, rs75750647 and rs10849390 had strong associations with diabetes risk in CHD patients (P < 0.05).
We also conducted haplotype analysis of NINJ2 polymorphisms and CHD risk (Supplemental table 3). We did not find significant 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 significantly difference in rs118050317, rs11610368 and PLT level (Prs118050317 = 0.041, Prs11610368 = 0.040). There were no strong relationships between other polymorphisms and clinical indicators in CHD patients.
In this study, we found that NINJ2 polymorphisms were associated with the susceptibility of CHD. NINJ2 rs118050317 significantly 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. NINJ2 plays an important role in nerve, stroke and inflammation [17-20]. We explored the effects of NINJ2 polymorphism on susceptibility of CHD. It is the first 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-modifiable risk factor for CHD in both men (age > 46) and women (age > 55). The mortality of CHD is higher in men than in women at all stages, and it accelerates for women older than 60 years old. To investigate the effects of NINJ2 polymorphisms on CHD risk in different populations, we did stratification analysis. Our results showed the effect of rs118050317 on CHD risk was related to age and sex. Rs118050317 significantly 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 influences 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. Moreover, people are more likely to have hypertension or diabetes with aging. Our study confirmed 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 significantly different between CHD cases and controls. Especially, platelet plays an important role in CVD by involving in acute and chronic inflammatory. 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 five 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 findings.