A male-specific association between AGTR1 hypermethylation and coronary heart disease

The AGTR1 gene encodes angiotensin II receptor type 1, which is involved in cardiovascular diseases such as coronary heart disease (CHD). In the current study, we analyzed AGTR1 promoter methylation level in a Han Chinese population by SYBR green-based quantitative methylation-specific PCR (qMSP). We collected blood samples from 761 CHD patients and 398 non-CHD controls at the Ningbo First Hospital. A data mining analysis was also performed to explore the association between AGTR1 methylation and AGTR1 gene expression, using datasets from the cBioPortal for Cancer Genomics and the Gene Expression Omnibus (GEO) database. Our results showed a significantly higher percentage of methylated reference (PMR) of AGTR1 in male CHD patients compared with male non-CHD controls (median PMR: 2.12% vs. 0.59%, p = 0.037). The data mining analysis showed that AGTR1 expression was significantly increased in human hepatoma HepG2 cells treated with the demethylation agent 5-aza-2’-deoxycytidine (fold = 3.12, p = 0.009). Further data mining analysis using the cholangiocarcinoma (TCGA, PanCancer Atlas) data indicated an inverse association between AGTR1 methylation and AGTR1 expression (r = -0.595, p = 1.29E-04). Overall, our results suggest that AGTR1 methylation is involved in the regulation of AGTR1 gene expression and that AGTR1 hypermethylation is associated with CHD in males. These findings may provide new clues about the pathogenesis of CHD.


INTRODUCTION
Coronary heart disease (CHD) is a type of cardiovascular disease, which remains a significant public health problem globally [1,2]. CHD is a complex disease influenced by both environmental and genetic factors. Previous studies suggested that epigenetic modifications are involved in the pathophysiology of CHD [3,4].
An important epigenetic mechanism of gene expression regulation is DNA methylation, which represents the methylation of cytosine to 5-methylcytosine, primarily at the CpG sites [5]. Recent studies showed that the methylation level of FOXP3, BAX, and PON1 is significantly associated with www.bjbms.org The relationship between AGTR1 methylation and CHD is still not clear. Therefore, in this study, we aimed to explore the association of AGTR1 promoter methylation with CHD in a Han Chinese population.

Patients
We collected blood samples from 761 CHD patients (501 men and 260 women, median age: 62 years) and 398 non-CHD controls (220 men and 178 women, median age: 60 years) at the Ningbo First Hospital. CHD patients were diagnosed with coronary artery stenosis greater than 50% in one or more of the major coronary arteries. The control group consisted of inpatients with coronary artery stenosis less than 50% in the major coronary arteries and without any atherosclerotic vascular disease. Standardized coronary angiography with Seldinger' s technique was performed in all patients [21,22]. The results of the angiography were independently evaluated by at least two cardiologists. None of the control inpatients had histories of congenital heart disease, cardiomyopathy, and severe liver or kidney disease. The Institutional Review Board of Ningbo First Hospital and Ningbo University approved the study (NBU-CHD-20180305). All participants provided written informed consent form.
SYBR green-based quantitative methylationspecific PCR (qMSP) We extracted genomic DNA from peripheral blood samples as previously described [23]. The details of bisulfite conversion and qMSP procedures are available from our previous studies [24,25]. To measure the methylation of AGTR1 promoter we calculated the percentage of methylated reference (PMR) [24,25]. The primer sequences used in qMSP were forward 5'-GGAGGAGGAGGGAATGTAA-3' and reverse 5'-CCTATCACTCGCTACTACCT-3'.

Data mining analysis
A data mining analysis was performed to explore the association between AGTR1 methylation and AGTR1 gene expression. We obtained the cholangiocarcinoma dataset (TCGA, PanCancer Atlas) from the cBioPortal for Cancer Genomics (http://www.cbioportal.org) [26]; AGTR1 methylation and AGTR1 gene expression were determined with Infinium HumanMethylation450 BeadChip and RNAseq, respectively. In addition, we used the data for human hepatoma HepG2 cells from the Gene Expression Omnibus (GEO) database (GSE5230). Statistical analysis PASW statistics 18.0 software (SPSS Inc., Somers, NY, USA) was used for statistical analysis. We compared continuous variables between the two groups by a t-test or a non-parametric test. A univariate linear regression analysis was used to determine the individual risk factors, then a multivariate regression analysis was performed to explore the association between these factors and CHD. A Spearman' s correlation test was used to assess the association between AGTR1 methylation and each of the 8 metabolic parameters, as follows low-density lipoprotein (LDL), total cholesterol (TC), triglyceride (TG), apolipoprotein A1 (ApoA1), apolipoprotein B (ApoB), apolipoprotein E (ApoE), lipoprotein A [Lp(a)], and high-sensitivity C-reactive protein (hs-CRP). A two-tailed p < 0.05 was considered to be significant.

RESULTS
In this study, we included 761 CHD patients (mean age: 58.52 ± 9.52) and 398 controls [mean age: 61.41 ± 9.29] ( Table 1). As shown in Figure 1, the selected region for the AGTR1 methylation assay is located in a transcription factor-binding site. The methylation level of an AGTR1 CpG was measured, representing a 142-bp amplified fragment in the CpG island of AGTR1 ( Figure 1).
Demographic characteristics and laboratory parameters, including LDL, TC, TG, ApoA1, ApoB, ApoE, Lp(a), and hs-CRP are shown in Table 1. Our univariate regression analyses showed that age, male sex, cigarette smoking, diabetes, TG, and hs-CRP were the risk factors of CHD (all p < 0.05, Table 1). A multivariate regression analysis showed that 4 factors (age, male sex, diabetes, and TG) were the risk factors and Lp(a) was the protective factor of CHD (all p < 0.05, Table 2). However, AGTR1 methylation was not associated with CHD in the subgroup analyses by diabetic status (p > 0.05, data not shown). We found no significant relationships between AGTR1 methylation and clinical indexes, including age, TG, and Lp(a) (p > 0.05, data not shown).
In our previous study, we found no association of CHD with CpG-SNP rs275653 on the AGTR1 promoter [17]. Here, we performed an interaction test between rs275653 and AGTR1 methylation. We did not observe a significant interaction between the rs275653 SNP and methylation level (data not shown). www.bjbms.org

DISCUSSION
In the present study, we explored the association between AGTR1 methylation and CHD. We found four risk factors (age, male sex, diabetes, and TG) and one protective factor [Lp(a)] of CHD. Although there was no association of AGTR1 methylation with CHD in the total sample, we found elevated AGTR1 methylation in male CHD patients compared with   www.bjbms.org male non-CHD controls. Moreover, the data mining analyses showed that a demethylation agent could induce AGTR1 expression and that AGTR1 expression was inversely associated with AGTR1 methylation. These results suggest that AGTR1 methylation contributes to the risk of CHD in males through the regulation of AGTR1 gene expression. Sex differences exist in the incidence and development of CHD as well as in the frequency of CHD-related polymorphisms [27]. The incidence of CHD is higher in men than in women, in all age groups [28]. Men have a higher prevalence of ST-elevation myocardial infarction but are less likely to have heart failure compared with women [29]. The difference in the distribution of CHD between males and females is possibly due to different lifestyles, including smoking, alcohol consumption, high sodium diet, and physical activity [30][31][32][33].
A previous study showed that males with the deletion/ deletion (D/D) angiotensin-I converting enzyme (ACE) gene polymorphism have a higher risk of premature myocardial infarction compared with women [34], suggesting a sex difference in the effect of ACE gene polymorphisms on CHD risk. In addition, sex hormones were suggested to regulate   29E-04). The x-axis shows AGTR1 methylation levels. The y-axis shows log-transformed AGTR1 gene expression values.   [35]. A study investigating the association of AGTR1 promoter methylation with the risk of essential hypertension showed that CpG1 hypomethylation in the AGTR1 promoter is likely associated with the risk of essential hypertension, with significantly lower CpG1 methylation levels in males than in females [20]. In the current study, we showed that male sex was a risk factor of CHD, and we detected AGTR1 hypermethylation only in male patients with CHD. Therefore, the sex differences in AGTR1 methylation levels in CHD patients may reflect the differences in lifestyle and sex hormone levels. AGTR1 mediates the cardiovascular effects of angiotensin II [36], and AGTR1 promoter hypermethylation suppresses the gene expression. Our results indicated that sex-dependent difference exist in CHD and that AGTR1 hypermethylation increases the risk of CHD by regulating AGTR1 gene expression. Some studies showed that an elevated Lp(a) level is an independent risk factor for CHD [37,38]. High levels of Lp(a) are independently associated with CHD-induced recurrent heart failure in patients with chronic heart failure [39]. Interestingly, contrary to the previous findings, our results suggest that Lp(a) may be a protective factor for CHD. Therefore, the role of Lp(a) in CHD should be further explored in larger-sample studies.
Although we demonstrated an association between AGTR1 methylation and CHD in males, the cause-effect relationship remains unclear. In addition, we measured AGTR1 methylation only in peripheral blood samples and DNA methylation profiles may vary among different tissues. Further studies with longitudinal cohort design and that include different tissues should confirm our findings.

CONCLUSION
Overall, our results suggest that AGTR1 methylation is involved in the regulation of AGTR1 gene expression and that AGTR1 hypermethylation is associated with CHD in males. These findings may provide new clues about the pathogenesis of CHD.