|Year : 2021 | Volume
| Issue : 1 | Page : 56-64
FURIN promoter methylation predicts the risk of incident hypertension: A prospective analysis of the Gusu cohort
Sheng-Qi Ma1, Jin-Hua Zhu2, Lei Wu3, Yan He1, Li-Yun Ren1, Bin Shen2, Jia Yu1, Rong-Yan Zhang2, Jing Li1, Ming-Zhi Zhang1, Hao Peng4
1 Department of Epidemiology, School of Public Health, Medical College of Soochow University, Suzhou, Jiangsu Province, China
2 Department of Chronic Disease Management, Center for Disease Prevention and Control of Wujiang District, Suzhou, Jiangsu Province, China
3 Department of Maternal and Child Health, Suzhou Industrial Park Center for Disease Control and Prevention, Suzhou, Jiangsu Province, China
4 Department of Epidemiology, School of Public Health, Medical College of Soochow University; Jiangsu Key Laboratory of Preventive and Translational Medicine for Geriatric Diseases, Soochow University, Suzhou, Jiangsu Province, China
|Date of Submission||12-Dec-2020|
|Date of Acceptance||25-Feb-2021|
|Date of Web Publication||30-Mar-2021|
Department of Epidemiology, School of Public Health, Medical College of Soochow University, Suzhou, Jiangsu Province
Department of Biostatistics, School of Public Health, Medical College of Soochow University, Suzhou, Jiangsu Province
Source of Support: None, Conflict of Interest: None
Objectives: Furin has been associated with hypertension through unclear underlying mechanisms. FURIN promoter methylation may participate in the underlying mechanisms, but no evidence supports this possibility. Here, we performed a prospective analysis to study the association between FURIN promoter methylation and incident hypertension. Methods: DNA methylation levels in the FURIN promoter were quantified by target bisulfite sequencing using peripheral blood from 1043 participants in the Gusu cohort (mean age: 50 years, 30% men) who were free of hypertension at baseline. After an average of 4 years of follow-up, 149 (14.3%) participants developed hypertension. Multiple testing was controlled for by measuring the false-discovery rate. Results: Of the eight CpG loci assayed, DNA methylation levels at Chr15: 91416118 were significantly associated with incident hypertension after adjusting for covariates and multiple testing (hazard ratio [HR] = 1.38, 95% confidence interval [CI]: 1.17–1.64, q = 0.001). The weighted truncated-product method, which combines single CpG associations, revealed that DNA methylation at multiple CpG sites was jointly associated with incident hypertension (P < 0.001). Using the average methylation level of all CpG sites as a surrogate for FURIN promoter methylation revealed a similar association (HR = 1.36, 95% CI: 1.08–1.72, P = 0.009). Almost all CpG methylations negatively correlated with serum furin levels, which mediated approximately 29.44% of the association between FURIN promoter methylation and incident hypertension. Conclusions: These results suggest that FURIN promoter hypermethylation is associated with an increased risk for hypertension in Chinese adults, partially through suppressing furin expression or excretion.
Keywords: DNA methylation; Hypertension; Mediation analysis; Prospective Studies
|How to cite this article:|
Ma SQ, Zhu JH, Wu L, He Y, Ren LY, Shen B, Yu J, Zhang RY, Li J, Zhang MZ, Peng H. FURIN promoter methylation predicts the risk of incident hypertension: A prospective analysis of the Gusu cohort. Cardiol Plus 2021;6:56-64
|How to cite this URL:|
Ma SQ, Zhu JH, Wu L, He Y, Ren LY, Shen B, Yu J, Zhang RY, Li J, Zhang MZ, Peng H. FURIN promoter methylation predicts the risk of incident hypertension: A prospective analysis of the Gusu cohort. Cardiol Plus [serial online] 2021 [cited 2021 Apr 16];6:56-64. Available from: https://www.cardiologyplus.org/text.asp?2021/6/1/56/312596
Authors Sheng.Qi Ma, Jin.Hua Zhu and Lei Wu contributed
equally to this work.
| Introduction|| |
The incidence of hypertension, a leading modifiable risk factor for cardiovascular disease (CVD), has nearly doubled in recent decades and affects approximately one-third of the world's population., A better understanding of the pathogenesis and mechanisms of hypertension is essential for preventing and managing this debilitating disorder. Furin, an enzyme that belongs to the proprotein convertase subtilisin/Kexin family, has been suggested to participate in blood pressure regulation. For example, furin activated the precursor of brain natriuretic peptide (NP) into its biologically active form that is involved in the NP system. Importantly, the NP system plays critical roles in maintaining blood pressure homeostasis through diuresis, natriuresis, and vasodilation. In addition to the NP system, furin also participates in the RAAS system by activating epithelial sodium channels and transforming growth factor-β1, thereby resulting in increased renin release and angiotensin II expression. In humans, genetic polymorphisms of FURIN, the gene encoding the furin protein, have been associated with susceptibility to hypertension,, stroke, and coronary artery disease. The levels of the furin protein in the circulation were previously associated with diabetes, atrial fibrillation, and CVD. Previously, we found that furin-deficient serum at baseline could predict the future risks for hypertension and obesity in Chinese adults in the Gusu cohort. However, the underlying molecular mechanisms remain unclear, although understanding these mechanisms would undoubtedly assist in the clinical development of novel drug agents against hypertension and related complications. DNA methylation, an important epigenetic modification that links the environment to the fixed genome, regulates gene functions and expression levels. Indeed, data from some case-control studies have revealed aberrant DNA methylation patterns in patients with hypertension.,,
Therefore, we hypothesized that FURIN promoter methylation may regulate furin expression/excretion and thereby participate in the molecular mechanisms underneath the association between serum furin and hypertension, but this has not been studied in humans. Because the temporal sequence is of considerable importance for causal inferences, we aimed to examine the prospective association of FURIN promoter methylation with incident hypertension and whether this association was mediated through serum furin. We also applied a gene-based approach to test whether DNA methylation at multiple CpG sites could jointly predict the risk of hypertension.
| Materials and Methods|| |
The Gusu cohort is a community-based, prospective longitudinal study of CVD and its risk factors in middle-aged and elderly Chinese adults in eight communities residing in a traditional, but economically developed district of Suzhou, China. The study design, survey methods, and laboratory techniques have been described previously. Briefly, the first clinical examination was conducted in 2010 by enrolling 2706 community members, aged over 30 years. All surviving participants (n = 2671) were invited to participate in the follow-up examination in 2014 and are currently being followed up with through 2020. The protocols of the current study were approved by the Soochow University Ethics Committee (Approval No. SUDA20200601H02). Written informed consent was obtained from all study participants. After excluding participants who had a history of CVD at baseline (n = 101), provided no blood samples at baseline (n = 107), had prevalent hypertension at baseline (n = 1280), died during follow-up (n = 35), and declined to participate in the follow-up examination (n = 140), 1043 participants were included in the current analysis. All participants were free of CVD and chronic kidney disease at baseline.
Quantification of FURIN promoter methylation
DNA methylation levels in the FURIN promoter were quantified by targeted bisulfite sequencing as previously described, using genomic DNA isolated from peripheral blood mononuclear cells drawn at baseline. Briefly, based on the genomic coordinates of the FURIN promoter in Genome Reference Consortium Human Build 37, we carefully designed primers to detect the maximum CpG loci within the CpG islands. The targeted sequence (Chr15: 91415936-91416189, forward strand, relative to the transcription start site [TSS]: +914 bp to +1168 bp) are illustrated in [Figure 1]. Following primer validation, genomic DNA was bisulfite treated using the EZ DNA Methylation-Gold Kit (Zymo Research, Inc., CA, United States) according to the manufacturer's protocol, which converts unmethylated cytosine into uracil and leaves methylated cytosine unchanged. The treated samples were amplified, barcoded, and sequenced with an Illumina HiSeq 2000 instrument (Illumina, Inc., CA, United States) using the paired-end sequencing protocol, according to the manufacturer's guidelines. The methylation levels at each CpG dinucleotide were calculated as the percentage of the methylated alleles over the sum of methylated and unmethylated alleles. For quality control purposes, we filtered out samples with a bisulfite conversion rate of <98% and cytosine sites with an average coverage of <20×. DNA methylation levels were finally quantified at eight different CpG loci in the FURIN promoter.
|Figure 1: Schematic illustration of the target sequence and the primers used for targeted bisulfite sequencing.|
The red text represents the CpG loci in the FURIN promoter that were assayed in this study (+914 to +1168 bp from TSS). TSS, transcriptional start site
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Blood pressure measurement and the definition of incident hypertension
Blood pressure was measured three times by trained staff using a standard mercury sphygmomanometer and a cuff of appropriate size according to a standard protocol, after the participants had been resting for at least 5 min in a relaxed, sitting position. The first and fifth Korotkoff sounds were recorded as the systolic blood pressure (SBP) and diastolic blood pressure (DBP), respectively. The means of the three measurements were used for statistical analyses. According to the Chinese guidelines for the management of hypertension, hypertension was defined as an SBP of ≥140 mmHg and/or a DBP of ≥90 mmHg, or by the use of antihypertensive medications in the last 2 weeks. Incident hypertension was defined as a lack of hypertension at baseline, with an initiation of antihypertensive medications during follow-up, or an SBP of ≥140 mmHg, or a DBP of ≥90 mmHg at the follow-up examination.
Assessment of conventional risk factors
Demographic data including age, sex, and education level, were obtained using questionnaires administered by trained staff. In terms of cigarette smoking, the subjects were classified as current smokers or nonsmokers. Current smokers were defined as those who have smoked at least 100 cigarettes in the entire lifetime, smoke cigarettes regularly, and smoke currently. In terms of alcohol consumption, patients were classified as being current drinkers or not current drinkers. Current drinkers were those who had consumed any alcohol during the past year. Body weight (kg) and height (cm) were measured by trained staff, with the participants wearing light clothes and no shoes. The body mass index (BMI) was calculated by dividing the weight in kilograms by the square of height in meters (kg/m2). Fasting glucose and blood lipids, including total cholesterol, triglycerides, high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) were measured by standard laboratory methods.
We simultaneously conducted single-CpG and gene-based-association analyses to examine the relationship between FURIN promoter methylation and incident hypertension, followed by causal mediation analysis to examine whether serum furin levels were linked to FURIN promoter methylation and incident hypertension. Log2 transformation was applied to maximize the normal distribution of serum furin levels, and the generated values (log2 furin levels) were used in downstream analyses. All statistical analyses were conducted using R software, version 3.6.1.
Single-CpG association analysis
To examine the association between DNA methylation at each CpG site and incident hypertension, we constructed a logistic regression model where incident hypertension was the dependent variable and each CpG-methylation status was the independent variable, after adjusting for conventional baseline risk factors, including age, sex, education level, current smoking status, current drinking status, BMI, LDL-C, HDL-C, and fasting glucose. Similarly, we also constructed a linear regression model with baseline serum furin as the dependent variable to examine the association between FURIN promoter methylation and serum furin. Multiple testing was controlled by adjusting for the total number of CpG sites tested using the false-discovery rate (FDR) method, and an FDR-adjusted P value (i.e., q value) of <0.05 was considered to reflect a statistically significant difference.
Gene-based association analysis
To examine the joint association between DNA methylation levels at multiple CpG sites in the FURIN promoter and incident hypertension, we treated the average methylation level of multiple CpG sites as a substitute for the methylation level of the targeted region and examined its association with incident hypertension. The weighted truncated product method (wTPM) was also employed. This method combines P values of all CpGs that reaches a predetermined threshold (e.g., raw P < 0.1 in this study). The regression coefficient of each individual CpG methylation generated from the above single-CpG association analysis was included as the weight in the wTPM statistic. This method has been evaluated in simulation studies and has been applied to epigenetic analysis.
Causal mediation analysis
Causal mediation analysis was used to focus on CpG sites whose DNA methylation levels showed significant associations with both baseline serum furin and incident hypertension during follow-up. To test whether serum furin mediates the association between FURIN promoter methylation and incident hypertension, we constructed a causal mediation model based on the following conditional regressions:
- The relationship between baseline DNA methylation (X) and incident hypertension (Y) (Model Y = βTotX, βTot: Total effect);
- The relationship between baseline DNA methylation (X) and baseline serum furin (M) (Model M = β1X, β1: Indirect effect 1);
- The relationship between baseline DNA methylation (X) and incident hypertension (Y) controlling for baseline serum furin (M) (Model Y=β2M + βDirX, β2: Indirect effect 2, βDir: Direct effect).
We then calculated the mediating effect (βMed = β1× β2) and the proportion of mediation (βMed/βTot × 100%). Mediation analysis was performed using the “mediation” of R software, after adjusting for the covariates listed above. The 95% confidence interval (CI) of the mediating effect was estimated by determining Monte Carlo CI values.
To examine the stability of our results, we also performed stratification analysis for men and women.
| Results|| |
Baseline characteristics of the study participants
The current study included 1043 middle-aged and elderly Chinese adults (mean aged 50.5 years, 30.4% men). The baseline characteristics of the study participants are shown in [Table 1]. Participants who developed hypertension during follow-up were more likely to be older, men, current drinkers, current smokers, with higher BMI, SBP, and DBP levels, and lower HDL-C and serum furin levels at baseline than those who were free of hypertension.
Association between FURIN promoter methylation and incident hypertension
Among the 1043 participants free of hypertension at baseline, 149 participants developed new hypertension after an average of 4 years of follow-up. Of the eight CpG loci assayed, DNA methylation levels at five CpG loci seemed to be lower in participants who developed hypertension than those who did not [P < 0.05, [Figure 2]]. [Table 2] shows their associations with the risk of incident hypertension, independent of conventional risk factors. Hypermethylation at four of eight CpG sites was nominally associated with a higher risk of incident hypertension (all P < 0.05), but only one association (the CpG located at Chr15: 91416118) withstood after correction for multiple testing (hazard ratio [HR] = 1.38, q = 0.001 for each 5% increase in methylation level).
|Figure 2: Baseline DNA methylation levels of the FURIN promoter in participants who developed hypertension (high blood pressure) versus those who did not (non-high blood pressure) during follow-up.|
non-HBP: non-high blood pressure, HBP: high blood pressure
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|Table 2: The association between baseline FURIN promoter methylation and incident hypertension|
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The intercorrelations among the eight CpG sites methylation showed a high correlation between the neighboring CpG sites [Supplementary Figure 1]], suggesting a comethylation pattern. Therefore, we examined whether DNA methylation within a region, rather than at a single CpG site, was associated with hypertension. Although not all CpG methylation levels were significantly associated with hypertension, gene-based association analysis revealed that DNA methylation at multiple CpG sites in the FURIN promoter may be jointly associated with hypertension. The average methylation level of the eight CpGs was significantly associated with a higher risk of incident hypertension (HR = 1.36, 95% CI: 1.08–1.72, P = 0.009). The wTPM further demonstrated a joint association of the eight CpG methylation levels with incident hypertension by combining their single associations with hypertension (P < 0.001).
Association between FURIN promoter methylation and serum furin
[Table 3] shows the association between FURIN promoter methylation and serum furin at baseline. Hypermethylation levels at seven of eight CpG loci assayed were significantly associated with lower serum furin levels, after multivariate adjustment and multiple testing correction (all q < 0.05). The average methylation level of the eight CpG sites was also negatively associated with serum furin (P < 0.001). Consistently, the wTPM also revealed a joint association between FURIN promoter methylation and serum furin (P < 0.001).
|Table 3: The association between FURIN promoter methylation and serum furin levels|
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Results of mediation analysis
Mediation analysis focused on CpG7 (located at Chr15: 91416118) and the average methylation level of FURIN promoter, both of which were significantly associated with serum furin levels and incident hypertension. The mediating effects are illustrated schematically in [Figure 3]. The results showed that the mediating effect of serum furin did not achieve statistical significance in terms of the association between CpG7 methylation and incident hypertension, but statistical significance was observed regarding the association between the average FURIN promoter methylation level and incident hypertension. Serum furin mediated approximately 29.44% (95% CI: 1.27%–59.54%, P < 0.05) of the association between FURIN promoter methylation and incident hypertension.
|Figure 3: Schematic illustration of the results of mediation analyses.|
A, The mediating effect of serum furin on the associations between DNA methylation at CpG7 located in Chr15:91416118 and incident hypertension. B, The mediating effect of serum furin on the association between the average methylation level in the FURIN promoter and incident hypertension.
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Results of sensitivity analysis
Stratification analysis showed that the significant single CpG association persisted among all male and female participants after adjusting for covariates, whereas this association did not survive multiple testing in women [Supplementary Table 1]. The effect size of this association did not change substantially, indicating a robust association between FURIN promoter methylation and hypertension.
| Discussion|| |
In an unselected population of middle-aged and elderly Chinese adults in the Gusu cohort, we demonstrated for the first time that hypermethylation of the FURIN promoter was associated with lower serum furin levels and was significantly associated with a higher risk of future incident hypertension. This association persisted after controlling for metabolic risk factors. These results suggest that FURIN promoter hypermethylation may serve as a potential predictor of the risk for hypertension.
To the best of our knowledge, no previous study has been conducted to examine the association between FURIN gene methylation and hypertension, as well as its complications. The potential role of furin in hypertension has been suggested by several previous studies with different layers. For example, mice that overexpressed profurin, a furin precursor, exhibited reduced atherosclerosis and vascular remodeling, which are the fundamental for the pathogenesis of hypertension. In humans, genome-wide association studies have identified many SNPs in the FURIN' gene that were associated with the susceptibility to hypertension, such as rs4702, rs2521501, and rs8032315. Further, the circulating levels of the furin protein have been associated with hypertension and related complications. For example, a case-control study found that serum furin was significantly higher in diabetic patients with CVD than that in those without CVD. Our group previously demonstrated that serum furin was significantly associated with hypertension, obesity, and microalbuminuria. However, the underlying molecular mechanisms remain unclear, and gaining insight into these mechanisms would help in translating furin (or associated targets) into clinical settings for treating hypertension and related complications. Considering the expression quantitative trait loci (eQTL) found in the FURIN gene, factors regulating FURIN gene function may be potentially involved in the molecular mechanisms underlying the association between serum furin and hypertension. As a mediator between the fixed genome and environment, DNA methylation can regulate gene functions and transcription levels. Indeed, some hypertension-related methylation markers have been identified in humans in epigenome-wide association studies. Therefore, we tested whether FURIN promoter methylation could be a potential molecular modification that regulates furin expression/excretion and influences the pathogenesis of hypertension. Here, we found that hypermethylation of a CpG locus in the FURIN promoter located at Chr15:91416118 was not only associated with lower serum furin levels but also predicted a higher risk of hypertension during an average 4-year follow-up period in Chinese adults. Our study may provide initial evidence for the potential role of FURIN promoter methylation in the pathogenesis of hypertension. However, the association between FURIN promoter methylation and hypertension still requires further replication in populations with other ethnic backgrounds.
To further examine whether FURIN promoter methylation constitutes a molecular mechanism underlying the association between serum furin and hypertension, we conducted causal mediation analysis and found that serum furin mediated approximately 29.44% of the association between FURIN promoter methylation and incident hypertension. Our results increased the probability that FURIN promoter methylation may represent a candidate molecular mechanism underlying the association between serum furin and hypertension. FURIN promoter methylation can potentially serve as a drug target for treating hypertension and related complications.
In line with previous studies,,, the contribution of a single-CpG methylation to a complex phenotype, e.g., diabetes, hypertension, and CVD, was relatively very small. Of the eight CpG sites assayed in our study, DNA methylation at three CpGs was nominally associated with incident hypertension (raw P < 0.05), but only one site withstood multiple-testing correction (q < 0.05). In relative terms, a small effect may not be very easily identified in a study with a relatively low sample size. However, the combined effect of multiple CpG sites could be much larger. Therefore, we tested and found a significant joint association of multiple CpG-methylation sites in the FURIN promoter with incident hypertension. Our results may highlight the importance of testing the joint epigenetic effect of multiple CpGs on human complex diseases, e.g., hypertension.
Our study is the first to prospectively examine the association between FURIN promoter methylation and incident hypertension in Chinese adults. The strengths of this study include its prospective study design; a comprehensive adjustment for environmental factors including lifestyles, metabolic factors, and medical history; the application of a gene-based analytical approach to test the combined effect of multiple CpG-methylation sites in the FURIN promoter on hypertension; and causal mediation analysis to delineate the potential role of FURIN promoter methylation underlying the association between furin protein and hypertension.
Our study also has several limitations that should be acknowledged. First, as in all observational studies, unobserved confounders may exist in our study. We did not establish the causal association between FURIN promoter methylation and the risk for hypertension, even though a prospective study design and causal mediation analysis were applied. Second, we only included middle-aged and elderly Chinese adults in our study. The generalizability of our findings to other age groups or populations with different ethnic backgrounds is uncertain. Third, given that DNA methylation is tissue- and cell-type specific, it is unclear whether or to what extent the results derived from peripheral blood could reflect methylation changes in the target organs of hypertension, e. g., cardiac tissue and arteries. However, accumulating evidence has indicated that epimutations may not be limited to the affected tissue but could also be detected in peripheral blood.,, Fourth, some important variables related to the risks of hypertension such as a family history of hypertension and increased homocysteine were not available in our study. It remains unclear whether the observed association between FURIN promoter methylation and hypertension was independent of family history of hypertension and homocysteine levels. Fifth, during the follow-up period, blood pressure was only measured at face-to-face and follow-up examinations. Misclassification of incident hypertension cannot be prevented.
| Conclusions|| |
The results of our study demonstrate that FURIN promoter hypermethylation at baseline predicted an increased risk for future hypertension in Chinese adults, and this association was mediated, at least partially, by serum furin. These findings indicate that FURIN promoter methylation may serve as a predictor and probable drug target for hypertension, but the causality of FURIN promoter methylation in hypertension development requires further investigation.
We are deeply appreciative of the participants in this study and thank all staff members for their support and assistance. We especially thank the clinical staff at all participating hospitals for their support and contribution to this project. Without their contribution, this research would not have been possible.
Financial support and sponsorship
This study was supported by the National Key Research and Development Program of China (grant number 2017YFC1307600), the National Natural Science Foundation of China (grant numbers 81903384 and 81872690), the Natural Science Foundation of Jiangsu Province (grant number BK20180841), the Suzhou Municipal Science and Technology Bureau (grant number SS201853), the Suzhou Science and Education Project for Youth Scholars (grant number KJXW2018078), a project of Suzhou Key Technologies of Prevention and Control of Major Diseases and Infectious Diseases (grant number Gwzx201803), and a project of the Priority Academic Program Development of Jiangsu Higher Education Institutions. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3]