Genetic variants in CYP11B1 influence the susceptibility to coronary heart disease

Genetic factors are important risk factors to develop coronary heart disease (CHD). In this study, we mainly explored whether CYP11B1 mutations influence CHD risk among Chinese Han population. Six variants were genotyped using Agena MassARRAY system from 509 CHD patients and 509 healthy controls. The correlations between CYP11B1 mutations and CHD risk were assessed using odds ratio (OR) and 95% confidence interval (95% CI) by logistic regression. The haplotype analysis and were ultifactor dimensionality reduction (MDR) were conducted. In the overall analysis, CYP11B1 polymorphisms were not correlated with CHD susceptibility. In the stratified analysis, we found that rs5283, rs6410, and rs4534 are significantly associated with susceptibility to CHD dependent on age and gender (p < 0.05). Moreover, we also observed that rs5283 and rs4534 could affect diabetes/hypertension risk among CHD patients (p < 0.05). In addition, the Crs4736312Ars5017238Crs5301Grs5283Trs6410Crs4534 haplotype of CYP11B1 reduce the susceptibility to CHD (p < 0.05). We found that rs4534, rs6410 and rs5283 in CYP11B1 gene influence the susceptibility to CHD, which depend on age and gender.


Introduction
Coronary heart disease (CHD) is a heart disease caused by coronary artery atherosclerosis that causes stenosis or occlusion of the lumen, leading to myocardial ischemia, hypoxia, or necrosis. Epidemiological research showed that more than 8.14 million people died of CHD in 2013, accounting for 50% of the total deaths from cardiovascular disease (CVD) [1]. CHD is considered to be one of the leading causes of death in people worldwide. The World health organization (WHO) predicts that CHD will account for 13.1% of all deaths by 2030 [2]. In China, CHD is the second leading cause of death from CVD, accounting for 22% of urban and 13% of rural mortality. Moreover, the medical costs for CHD are expected to increase by 100%, posing a severe socio-economic burden on individuals and society [3]. Therefore, it is urgent to explore the pathogenesis and etiology of CHD.
It is widely known that CHD is a complex disease that is influenced by environmental and genetic factors [4]. Genetic factors are important risk factors to develop CHD, accounting for as 30 ~ 60% of the variation in the risk of CHD [5]. Large-scale studies have documented that genetic polymorphisms are significantly associated with CHD at genome-wide significance [6]. Moreover, previous studies also have reported that a significant association between candidate gene polymorphism and CHD susceptibility [7][8][9]. These results underscored the crucial role of genetic factors in the pathogenesis of CHD.

Open Access
*Correspondence: wangna20210527@163.com 1 The Department of Cardiovascology, Xi'an Hospital of Traditional Chinese Medicine, No. 69, Fengcheng Eighth Road, Weiyang District, Xi'an 710021, People's Republic of China Full list of author information is available at the end of the article The 11β-hydroxylase (CYP11B1) gene is located on chromosome 8q24.3, contains 9 exons and 8 introns, and consists of 503 amino acids [10]. It is a key enzyme responsible for the final step in cortisol biosynthesis [11]. At present, studies have found that the genetic variation of CYP11B1 was involved in the occurrence and development of various diseases. Rs6410 was significantly related to the secretion of aldosterone in the Chinese population [11]. However, rs4534, rs5283, rs4736312, rs5017238 and rs5301 in CYP11B1 have not been reported to be related to disease in the literature. The CYP11B family is involved in the synthesis of important steroid hormones. The genotypes of the CYP11B1/CYP11B2 loci are in strong linkage disequilibrium [12]. The two synthesize 11β hydroxylase and aldosterone synthase, and then synthesize steroids, which play an important role in myocardial fibrosis, hypertension and arteriosclerosis [10,13]. Studies have shown that steroid metabolism induces the development of coronary artery disease [14]. CYP11B1 activates the pituitary-adrenal axis by synthesizing 11β hydroxylase, promoting the accumulation of adrenal cortical hormones, and affecting the production of aldosterone [11], which can damage cardiac or renal organs and induce hypertension [15]. Aldosterone synthase inhibitors can efficiently regulate aldosterone levels [16] by targeting CYP11B1 with high selectivity. In addition, hypertension and diabetes are major risk factors for cardiovascular disease [15]. Based on the above studies, we hypothesized that CYP11B1 may be play a vital role in the occurrence of CHD.
Therefore, we explored the associations of CYP11B1 polymorphisms with CHD susceptibility. These genetic variants may provide a new screening strategy for CHD among Chinese Han population.

Study population
This study recruited 509 patients with CHD and 509 healthy controls from Xi'an Hospital of Traditional Chinese Medicine. The patients were diagnosed as CHD by two experienced imaging specialists according to coronary angiography examination. CHD was defined as more than 1 ( ≥) atherosclerotic plaque in a major coronary artery (≥ 1.5 mm lumen diameter) causing ≥ 50% luminal diameter stenosis by coronary angiography examination. Patients suffered from congenital heart disease, cardiomyopathy, malignancy, chronic inflammatory disease, and liver or kidney disease were excluded. The healthy controls were selected from the same hospital during the same period. The subjects without cardiovascular disease, autoimmune disease, malignancy, and other known disease were included in this study. The study got approval of the ethics committee of the hospital and the experimental procedures were in accordance with the Declaration of Helsinki. Meanwhile, the informed consent signed by the subjects was obtained. Dyslipidemia is a key factor leading to the development of coronary heart disease, and coronary heart disease is related to various indicators such as triglycerides (TG), total cholesterol (TC), low density lipoprotein (LDL) and high density lipoprotein (HDL) in blood lipids [17]. There is an association between elevated serum UA levels and cardiovascular diseases such as coronary heart disease and stroke [18]. In addition, WBC often serves as an effective marker of inflammation, and elevated WBC counts are associated with common risk factors for coronary heart disease, including hypertension. An automatic blood analyzer was used to detect TC, TG, LDL, HDL and other indicators at 4 degrees Celsius. EDTA-treated anticoagulated samples are used to measure blood rheology parameters such as red blood cells (RBC) and white blood cells (WBC).

Statistical analysis
Student t-test and χ 2 test were used to assess the difference in age and gender of study population. The Hardy-Weinberg equilibrium (HWE) in controls was evaluated by χ 2 test. The associations between CYP11B1 SNPs and CHD susceptibility was analyzed using odds ratio (OR) and 95% confidence interval (CI) by logistic regression. In addition, linkage disequilibrium (LD) and haplotype analysis were evaluated by the Haploview software and the PLINK software. Multifactor dimensionality reduction (MDR) was conducted to assess the SNP-SNP interactions in the risk of CHD. The differences in clinical parameters in CHD patients were tested by one-way analysis of variance (ANOVA). Statistical power and false positive report probability (FPRP) values were calculated by the Excel spreadsheet which was offered on Wacholder's website [21]. A p < 0.05 was used as the threshold of significance.

General characteristics of CHD patients and healthy controls
A total of 509 CHD patients (335 males and 174 females) and 509 healthy controls (335 males and 174 females) included in this study were of Han Chinese ethnicity. The basic characteristics of participants were summarized in Table 1. No significant difference was observed in terms of age (p = 0.094), gender (p = 1.000), low-density lipoprotein (LDL, p = 0.157), urea (p = 0.056) between the case and control groups, with their mean age being 62.16 ± 10.30 and 61.14 ± 9.02 years, respectively. Among all the CHD patients, 318 patients had hypertension, and 147 patients had diabetes. Compared with the normal control group, the levels of TG, TC, HDL, UA, RBC, WBC, HGB and Platelet were significantly decreased in the CHD group.

Association of CYP11B1 polymorphisms and CHD risk
Additional File 1: Table S1 exhibited the primary information of CYP11B1 polymorphisms. The genotype frequency distributions of the selected polymorphisms of CYP11B1 were in line with HWE (p > 0.05).
We investigated the association between CYP11B1 polymorphisms and CHD risk among Chinese Hans. As shown in Table 2, CYP11B1 polymorphisms were not correlated with CHD susceptibility in total population under five heritance models (p > 0.05).

Clinical characteristics and SNPs
We also investigated the correlation between clinical characteristics and SNPs (Additional File 1: Table S2).  The results have shown that there was no significant correlation between the genetic variation of CYP11B1 and clinical parameters (p > 0.05).

FPRP analysis
FPRP and statistical power were calculated for all positive results. As shown in Additional File 1: Table S3, at the prior probability of 0.1 and FPRP threshold of 0.2, the significant results of rs5283 remained noteworthy.

Haplotype analysis and MDR analysis
The haplotype analysis of CYP11B1 polymorphisms and CHD risk was conducted. The results of Table 5 presented that the C rs4736312 A rs5017238 C rs5301 G rs5283 T rs6410 C rs4534 haplotype was correlated with a decreased risk of CHD compared to C rs4736312 A rs5017238 C rs5301 G rs5283 C rs6410 T rs4534 (OR = 0.72, 95%CI = 0.54-0.96, p = 0.024). And we observed an LD plot consisted of six SNPs (rs4736312, rs5017238, rs5301, rs5283, rs6410 and rs4534), as exhibited in Fig. 1. Then, the SNP-SNP interaction was performed by MDR analysis. As shown in Fig. 2, the Fruchterman-Reingold (Fig. 2) described the interactions between these SNPs. The results of MDR model analysis of the SNP-SNP interactions are demonstrated in Table 6. The six-locus model including rs4736312, rs5017238, rs5301, rs5283, rs6410, rs4534 was the best model and driving the high-risk combinations for CHD (CVC = 10/10, OR = 1.51, 95% CI = 1.18-1.93, p = 0.0011).

Discussion
This is the first study to explore the effect of CYP11B1 polymorphisms on CHD susceptibility. The results of overall analysis revealed that CYP11B1 polymorphisms were not correlated with CHD susceptibility. In the stratified analysis, we found that rs5283, rs6410, and rs4534 are significantly associated with susceptibility to CHD in females and individuals aged ≤ 60 years old. Moreover, we also observed that rs5283 and rs4534 could affect diabetes/hypertension risk among CHD patients. In addition, the C rs4736312 A rs5017238 C rs5301 G rs5283 T rs6410 C rs4534 haplotype of CYP11B1 reduce the susceptibility to CHD. These data highlight the crucial role of CYP11B1 genetic variants in the development of CHD.
CYP11B1 gene is located on chromosome 8q24.3, containing 9 exons and 8 introns. It catalyzes the final step of cortisol biosynthesis. Some studies have documented that elevated cortisol is associated with a number of metabolic changes, such as hyperlipidaemia, diabetes, hypertension and abdominal adiposity [22,23], which are correlated with CHD risk. Moreover, cortisol had a direct impact on the heart and blood vessels and played an important role in the process of atherogenesis and cardiovascular disease [24]. These results implied that CYP11B1 may be involved in pathophysiology of CHD through regulating cortisol. Recently, some reports have studied the role of CYP11B1 polymorphisms in disease. For example, Deng et al. indicated that rs4534 showed no significant association with autism in Chinese children [25]. Zhang et al. have shown that rs6410 was related to primary hyperaldosteronism, which is a common form of secondary hypertension [11]. Meanwhile, Wang et al. indicated that rs6410 and rs6387 haplotype is correlated with persistent postoperative hypertension in Chinese patients undergoing adrenalectomy with aldosterone-producing adenoma [13]. However, no study has investigated the effect of CYP11B1 genetic variants on CHD. Rs6410 in CYP11B1 can affect skeletal      maturation through variable shearing [26] and was significantly associated with trabecular bone mineral density and cross-sectional area in Caucasian elderly men [27]. However, rs5283, rs4736312, rs5017238 and rs5301 in CYP11B1 have not been reported to be related to disease in the literature.
In the present study, we found that rs6410 reduced the susceptibility to CHD individuals aged ≤ 60 years old and females. Rs5283 not only increased the susceptibility to CHD in females, but also enhanced the risk of diabetes among CHD patients. Rs4534 also correlated with increased risk of CHD subjects younger than 60 years. Besides, in CHD patients, rs4534 enhanced the susceptibility to diabetes, whereas reduced the risk of hypertension. The rs4534 polymorphism is a missense variant located in exon 9 of the CYP11B1 gene. Rs5283 and rs6410 are, synonymous nucleotide polymorphisms, located on the exon region. Missense and synonymous mutations have been widely studied in the development of disease by causing changes in protein expression, conformation and function [28][29][30][31]. Therefore, we presumed that these three polymorphisms can affect CYP11B1 gene mRNA and protein by altering translation, mRNA stability or protein folding, thereby affecting CHD susceptibility. However, it should be confirmed in further functional studies.

Conclusions
To sum up, we found that a missense mutation (rs4534) and two synonymous variants (rs6410 and rs5283) in CYP11B1 gene influence the susceptibility to CHD, which depend on age and gender. It indicated that CYP11B1 gene variant plays an important role in the development of CHD. These mutations may provide new ideas for exploring the pathogenesis of CHD.