CDKN2B‐AS1 gene rs4977574 A/G polymorphism and coronary heart disease: A meta‐analysis of 40,979 subjects

Abstract It has been implied that there is a possible relationship between cyclin‐dependent protein kinase inhibitors antisense RNA 1 (CDKN2B‐AS1) gene rs4977574 A/G polymorphism and coronary heart disease (CHD) susceptibility. However, as the research results are discrepant, no distinct consensus on this issue has been reached so far. In order to further elaborate the latent association of the CDKN2B‐AS1 gene rs4977574 A/G polymorphism and CHD, this present meta‐analysis was conducted. There were 40,979 subjects of 17 individual studies in the present meta‐analysis. The pooled odds ratios (ORs) and their corresponding 95% confidence intervals (CIs) were estimated to determine the association strength. Considering the significant heterogeneity among the individual studies, the random‐effect models were used. In the current meta‐analysis, a significant association between CDKN2B‐AS1 gene rs4977574 A/G polymorphism and CHD was found under allelic (OR: 1.18, 95% CI: 1.08–1.29, p = 4.83×10−4), recessive (OR: 1.36, 95% CI: 1.11–1.67, p = 0.003), dominant (OR: 0.71, 95% CI: 0.58–0.86, p = 6.26×10−4), heterozygous (OR:1.210, 95% CI: 1.076–1.360, p = 0.001), homozygous (OR: 1.394, 95% CI: 1.163–1.671, p = 3.31×10−4) and additive (OR: 1.180, 95% CI: 1.075–1.295, p = 4.83×10−4) genetic models. A more significant association between them was found in the Asian population than that in the whole population under these genetic models (p < 0.05). However, no significant association between them was found in the Caucasian population (p > 0.05). CDKN2B‐AS1 gene rs4977574 A/G polymorphism was associated with CHD susceptibility, especially in the Asian population. G allele of CDKN2B‐AS1 gene rs4977574 A/G polymorphism is the risk allele for CHD.


| INTRODUC TI ON
Cardiovascular disease (CVD) due to atherosclerosis (AS) is the leading cause of death in humans. In Europe, the United States and other developed countries, deaths caused by coronary heart disease (CHD) account for nearly half of the total number of deaths. Approximately 5.7 million Americans, or 2.5% of the population, have CHD, and one in five American men have a chance of acquiring CHD before the age of 60. In accordance with the CVD report in China in 2019, the prevalence of CVD in China is on the rise. The number of CVD patients has reached 330 million, of which CHD is up to 11 million. In 2017, CVD mortality was the highest, higher than that of cancer and other diseases. 1 The main pathological change of CHD is AS, which is marked by the infiltration of inflammatory cells in the artery wall and the abnormal proliferation of vascular smooth muscle cells (VSMCs), leading to vascular endothelial hyperplasia. 2 The coronal AS progression is influenced by genetic and environmental factors. Under the coaction of mechanical and chemical injury, vascular endothelial cells are damaged and inflammatory cells stick together, thus promoting the proliferation and migration of vascular endothelial cells and forming AS plaque. Although the aetiology of CHD is incompletely clear, genetic and environmental factors are recognized as the main pathogenesis. 3,4 Environmental factors include gender, age, obesity, high cholesterol, high blood pressure and unhealthy living habits, which can explain 50%-60% of the aetiology of CHD. The remaining 40%-50% of the aetiology of CHD is due to genetic factors. 5 As a result, the CHD genetic cause factors become the hot spot in pathogenesis research.
Previous research has confirmed that the cyclin-dependent kinase inhibitor 2A/2B (CDKN2A/2B) gene is associated with the occurrence of coronal AS in the general population. 6,7 Cyclin-dependent TA B L E 1 Characteristics of the association between theCDKN2B-AS1 gene rs4977574 A/G polymorphism and CHD To verify whether CDKN2B-AS1 gene rs4977574A/G polymorphism is susceptible to CHD development, a meta-analysis of 40,979 subjects from 17 separate studies was conducted (Supplements S1).

| Publication search and inclusion criteria
The terms "CDKN2A/2B," "CDKN2B-AS1," "rs4977574," "coronary heart disease," "myocardial infarction," "coronary artery disease" and "polymorphism" were used to perform a search. The collected studies must meet the next inclusion criteria in the current meta-analysis. The selected studies must evaluate the association of CHD and CDKN2B-AS1gene rs4977574 A/G polymorphism. The CHD diagnosis was defined as that the coronary artery stenosis was over 50% in at least one main coronary artery.
The coronary artery stenosis was calculated through dual-source coronary computed tomography or coronary angiography. The selected studies must conform to the Hardy-Weinberg equilibrium (HWE) in genotype distribution in the control group. The included studies were cohort or case-control studies that were publicly released.

| Data extraction
In accordance with a standardized protocol, the information of individual studies was drawn out by three investigators together. Two of them selected the eligible studies. The third one acted as an intercessor to solve any ambiguous issues between them. Studies that deviated from the inclusion criteria were repeatedly published or did not TA B L E 2 Summary of meta-analysis of association between CDKN2B-AS1 gene rs4977574 A/G polymorphism and CHD provide valid data were excluded. If analogical data were presented in different papers by an identical author group, the data could be merely used once in the current meta-analysis. The definition of controls included the source of controls, that is either hospital-based or population-based. The quality of studies was assessed by using the Newcastle-Ottawa scale scores.

| Statistical analyses
In this study, six genetic models as allelic (G allele frequency distribu- evaluated through the chi-square-based Q-tests. 12 If there was obvious heterogeneity, the random-effect model would be applied. 13 Otherwise, the fixed-effect model would be utilized. 14 The significance was set at p < 0.05 level in all of the above tests.
The potential publication bias was assessed by using the funnel plot and Egger's linear regression test. 15 All of the statistical analyses were conducted by utilizing Review Manager 5.3 and Stata 12.0 software.

| Pooled analyses
In the current meta-analysis, a significant association between  Figures 1-6).
In the subsequent subgroups analysis stratified by the ethnicity, the relationship between the CDKN2B-AS1 gene rs4977574 A/G polymorphism and CHD was found more prominent in the Asian subgroup than that in the whole population. In the Asian sub-

| Bias diagnostics
In the funnel plot under the allelic genetic model, no publication bias was visualized (Figure 7). Moreover, no significant difference was found by utilizing Egger's test under the additive genetic model.

| Sensitivity analysis
Sensitivity analysis was performed under the additive genetic model. After any individual study was removed from the current meta-analysis, the primary analysis result was unaffected. This result was comparatively steady (Figure 9).

| DISCUSS ION
In this meta-analysis, a remarkable association between CDKN2B- Huang et al. 30 performed a case-control study and a meta-analysis on the association between them. They found a strong association between rs4977574 and CHD risk. However, their case-control study was against HWE. Hence, their conclusion was not as reliable as that deduced from the current meta-analysis. In 2018, Xu et al. 49 performed a meta-analysis and found that rs4977574 polymorphism was associated with the CHD risk in Asian population. The G allele might contribute to a high risk of CHD. Although they obtained a similar conclusion to that of the current meta-analysis, they limited the studies in the Asian population. They also included studies In sum, CDKN2B-AS1 gene rs4977574 polymorphism was evidently relevant to CHD risk, particularly in the Asian population.
The G allele of CDKN2B-AS1 gene rs4977574 polymorphism might confer the CHD risk to the population. Additional large-scale studies on the association of CDKN2B-AS1 gene rs4977574 polymorphism and CHD should be conducted to verify this viewpoint in the near future.

CO N FLI C T O F I NTE R E S T
The authors have no other conflicts of interest to disclose.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.