α-Adducin gene promoter DNA methylation and the risk of essential hypertension

This study was conducted to test the association between promoter DNA methylation of α-Adducin (ADD1) gene and the risk of essential hypertension (EH). A total of 150 EH patients and 100 aged- and gender-matched controls were investigated. DNA methylation levels of five cytosine-phosphate-guanine (CpG) dinucleotides on ADD1 promoter were measured employing bisulfite pyrosequencing technology. Our results showed that females have a higher ADD1 DNA methylation than males and a significantly lower CpG1 methylation level is associated with increased risk of EH among them. As for males, a significant association between lower CpG2-5 methylation levels and increased risk of EH was shown. In addition, CpG2-5 methylation was found to be a highly significant predictor for EH among males. In females, CpG1 methylation was considered a predictor of hypertension. No significant correlations were found with biochemical measures, apart from the concentration of aspartate aminotransferase which was inversely correlated with ADD1 CpG2-5 methylation levels among female controls (r = −0.703). These findings highlight that ADD1 methylation may have a contributing role in the pathogenesis of EH with varying implications for both genders.

Keywords: CPG dinucleotide; DNA methylation; essential hypertension; single nucleotide polymophism; α-Adducin gene

Essential hypertension (EH) is a common health problem in both the developed and developing worlds and it affects both sexes, mainly older patients ([1],[2]). This age-related condition affects about a quarter of the adult population with severe complications. It is also well recognized that males exhibit a higher prevalence. It is a complex disease resulting from interacting genetic and environmental factors. Twenty to fifty percent of the interindividual variability in blood pressure measure is heritable ([3]). Several genetic markers have been recognized as risk factors for EH. One of these loci studied is α-Adducin (ADD1) gene. Adducin, with its three α, β, and γ subunits, is a cytoskeleton constituent. It is a heterodimeric protein present in many tissues and plays a role in cell-to-cell contact and signal transduction ([4]). Importantly, it regulates Na+–K+ ATPase activity, thus, influencing the cell membrane ion transport ([5]). Accelerated renal tubular reabsorption was reported with adducin mutations by altering surface expression and maximum velocity of this pump ([6]). Both human and animal studies highlighted the significant implication of ADD1 in the pathogenesis of EH ([7]). Single nucleotide polymorphisms (SNPs) in the α and β subunits of adducin inflicted up to 50% blood pressure variation in hypertensive and normotensive rats ([8]). In humans, ADD1 is located on chromosome 4p16.3. There are several studies on the association between the ADD1 gene polymorphism and susceptibility to EH, but with inconclusive results. The common SNP of the ADD1 gene causing substitution of amino acid tryptophan in place of glycine (Gly460Trp) (rs4961) at position 460 was found to be associated with increased risk of hypertension ([9]) and its risk factors ([10]). Interestingly, gender difference of the association between hypertension and ADD1 Gly460Trp was observed in Caucasians ([11]). In a recent meta-analysis, Wang et al. ([11]) found a significant association between this Gly460Trp and salt sensitivity in Asians, but not in Caucasians, which could be attributed to the difference in the SNP frequency in these ethnic groups. In addition, an interaction between ADD1 Gly460Trp and other mutations such as ACE DD and CYP11B2 − 344CC may contribute to the blood pressure response to dietary salt ([11]).

Graph: Figure 1. ADD1 methylation difference between pre-menopausal and post-menopausal females.

Graph: Figure 2. High association between ADD1 CpG2-5 methylation levels and essential hypertension. This association is more significant among females than males.

DNA methylation is defined as the addition of a methyl group to the DNA molecule. This addition is associated with a change in the activity of DNA segment without the affection of its sequence. If this addition affects gene promoter, it will act to repress gene transcription. It was found to be associated with structural chromatin changes, causing access restriction of transcriptional factors to gene promoter ([12]). DNA methylation is a stable epigenetic mark and usually occurs at cytosine residues that present in the context of cytosine-phosphate-guanine dinucleotide (CpG) in mammalian cells ([13]). Promoter DNA methylation is linked to transcriptional silencing of protein-coding genes and thus regulates the function of protein ([14]). Atypical methylation has been shown to play important roles in the incidence and development of several common diseases including coronary artery disease ([15]) and malignancies such as colorectal cancer ([16]), breast cancer ([17]), and schizophrenia ([18]). Similarly, a significant decline in overall DNA methylation level was observed in EH patients and the trend continues along with the progression of hypertension ([19]). The methylation levels of α-Adducin CpG-1 and CpG 2-5 were lower in females after the age of menopause than in premenopausal period which raises the issue of effect of environmental factors such as decreased physical activity, obesity, and high sodium diet on DNA methylation in older females and the risk of hypertension ([4],[20],[21]). However, the association between DNA methylation and the risk of EH is not well studied.

This is the first study of promoter DNA methylation of ADD1 gene in Egyptians. It was conducted to test its association with the risk of EH and to explore correlations between it and both clinical and laboratory profiles.

The study populations consisted of 150 patients with EH and 100 healthy individuals who were sex- and age-matched (age ranges: 44–65 years for the patients group versus 41–59 years for the healthy group). Patients were randomly selected from Suez Canal University Hospital, internal medicine out-clinic and clinical pathology departments. Controls were volunteers. The study spanned over the period from May 2014 to January 2015. All participants were Egyptians and of Egyptian descendants. Hypertensive patients were on antihypertensive drugs or have three consecutive records for systolic blood pressure (SBP) more than 140 mmHg and/or diastolic blood pressure (DBP) more than 90 mmHg ([22]). Individuals with SBP less than 120 mmHg and/or DBP less than 80 mmHg and had no family history of hypertension among first-degree relatives were considered healthy controls ([22]). All individuals did not have history of diabetes mellitus, secondary hypertension, myocardial infarction, stroke, renal failure, or any other serious diseases. The study was approved by the Ethics Committee of the Suez Canal University and carried out in accordance with the Helsinki Declaration. An informed written consent was obtained from each individual.

Three milliliters of whole blood were collected from each individual in a plain vacutainer sterile tube to have serum for the assessment of chemistry profiles, in addition to 3 ml whole blood in an EDTA sterile vacutainer tube for the extraction of DNA after 12 hours fasting. Serum total cholesterol, triglycerides, alanine transferase (ALT), aspartate transferase (AST), uric acid, and glucose were measured using fully automated auto-analyzer Cobas c501 (Roche Diagnostics, Germany).

DNA was extracted using commercially available Spin-column technique kit for DNA extraction from human whole blood (QIAamp®DNA Blood Mini Kit, QIAGEN, 28159 Avenue Stanford, Valencia, CA). The extracted DNA samples were stored at −20°C for further use. Bisulfite pyrosequencing technology was used for the determination of the five CpG dinucleotides methylation levels on the fragment within ADD1 promoter. The assays consisted of three consecutive steps: (a) sodium bisulfite DNA conversion (EpiTech Bisulphite Kit, Qiagen), (b) polymerase chain reaction (PCR) amplification (Pyromark PCR kit, Qiagen), and (c) sequencing by synthesis assay (Pyromark Gold Q24 reagents).

The PCR amplification and pyrosequencing assaysfor ADD1 gene promoter amplification were done using specific primers as follows: forward primer: #5ʹ-AAAATTAGGTTTGGGGATTGTATAAGG-3ʹ#, reverse primer: #5ʹ-CCAAATCATAACTTCACCATAACCAAATAAAC-3#, and sequencing primer: #5ʹ-TTTTAGGAGGAGGTTAATTATAATG-3ʹ# ([21]).

Pearson chi-square or Fisher exact test was used for the association of EH with categorical variables. Two sample t-test was applied for the association of EH with continuous variables. Receiver operating characteristic (ROC) curve was used to analyze the sensitivity of ADD1 DNA methylation in EH diagnosis. Logistic regression was implemented for the interaction of ADD1 methylation and age. A two-sided p-value <0.05 was considered statistically significant. Collected data were analyzed using SPSS version 16 program.

This study included 150 patients with EH and 100 age- and sex-matched healthy individuals. The demographic and laboratory characteristics of both groups are shown in Table 1. Since DNA methylation levels for the loci from two to five of the study gene were closely correlated (r > 0.80, p < 0.001), these correlations were considered as one and expressed together as CpG2-5. Table 1 shows no significant difference between cases and controls with respect to body mass index, smoking, and metabolic measures and they were within normal range. In the overall study population, higher DNA methylation levels of the ADD1 gene promoter were observed among females than males (CpG1: p = 0.012, pG2–5: p = 0.031). Our results found significant association between ADD1 CpG2-5 methylation levels and EH compared to healthy group (p = 0.021). However, this association was not expressed with CpG1 (p = 0.088) (Figures 1 and 2). Moreover, CpG1 methylation was inversely correlated with age among females (r = −0.481, p = 0.023). Methylation was more evidently higher in premenopausal (≤50 years) women in comparison to postmenopausal age (≥50 years) (CpG1: p = 0.008, CpG2-5: p = 0.041).

Table 1. Demographic and laboratory characteristics.

• 5 Values were expressed in mean ± SD, M: males, F: females, BMI: body mass index, T.: total, FBS: fasting blood sugar.
• 6 p-Value is considered significant if less than 0.05.

In Table 2, ADD1 CpG1 methylation level in females was significantly associated with EH (9.78 ± 1.33% versus 11.18 ± 2.89% for cases and controls, respectively, p = 0.037), but not so among males (p = 0.148). On the other hand, lower levels of ADD1 CpG2-5 methylation were associated with increased risk of EH among males (21.51 ± 5.94 versus 32.17 ± 6.21 for cases and controls, respectively, p = 0.003), while it was absent among females with p = 0.471.

Table 2. Comparison between EH and non-EH by gender.

• 7 Values were expressed in mean ± SD, M: males, F: females, BMI: body mass index, T.: total, FBS: fasting blood sugar.
• 8 p-Value is considered significant if less than 0.05.

Also, our results showed a significant interaction between CpG1 methylation and age to affect the presence of EH among females (p = 0.032). ROC analysis showed that CpG2-5 methylation can be considered a significant predictor for EH among males with area under curve = 0.781, p < 0.001, but not among females with area under curve = 0.745, p = 0.603. No correlation was found between ADD1 DNA methylation and any of the laboratory-assessed parameters. Post hoc test analysis showed a significant correlation between CpG2-5 and AST among females (r = −703, p = 0.032), while no other correlations were found in the rest of the parameters.

EH is an age-related chronic condition affecting males more than females. It requires lifelong management and is a risk factor for other serious cardiovascular complications. Genetic predisposition is a well-recognized contributor in EH etiology. This study was carried out to evaluate incrimination of ADD1 gene promoter DNA methylation in EH. The results showed that ADD1 gene DNA methylation was higher among females than among males. Importantly, it showed that DNA methylation can be considered as a risk factor for EH among males (CpG2-5) as well as among females (CpG1). These results are comparable to the results of a study that was done among Chinese population ([21]). However, this study is the first one to show this association between Adducin gene methylation and EH among Egyptians. Adducin was reported to be incriminated in the pathogenesis of EH, and this was explained by its modulation to Na–K ATPase activity ([23]). It was suspected that adducin might act as a candidate protein to explain genetic alterations in ion transport associated with EH ([24]). This study suggested that altered ADD1 methylation may contribute to the risk of EH. A plausible explanation could be that lower ADD1 methylation may lead to higher expression of α-Adducin which results in an increase in the expression and activity of Na+–K+ pump resulting in higher Na reabsorption and hence elevated blood pressure. This explanation is reflected by our results of lower level of ADD1 gene promoter methylation among EH cases in comparison to controls.

We report a significant association between CpG1 methylation and EH among females. On the other hand, CpG2-5 methylation was significantly associated with EH among males. In the overall study population, ADD1 methylation was found to be higher among females than among males.

Gender dimorphism was found in association studies of hypertension. ADD1 Gly460Trp polymorphism was observed among female Caucasians ([11]), CYP19A1 polymorphisms among male Japanese ([25]), and SELE T1559C polymorphism ([26]) and PNMT G390A polymorphism among male Chinese ([27]), as well as P2RY2 polymorphism ([28]).

Our study found that ALT and AST were associated with EH among females, and also that ADD1 CpG1 and CpG2-5 methylation levels were associated with EH among females and males, respectively. Also, CpG2-5 was correlated with AST among females. This notice may be due to the differences in the sex hormones. Roles of sex hormones were described in mediating the epigenetic effects on cardiomyocytes ([29]) and endocrinal system ([30]). Significant higher ADD1 methylation levels were found in premenopausal women than in postmenopausal ones. Our results provide new clues to explain the sexual dimorphism of EH.

Changes in the DNA methylation status were found to be caused by environmental and different dietary habits ([31]). Also, many of the traditional risk factors were known to be implicated in the causation of hypertension such as physical inactivity, high sodium intake, and obesity ([32]). The link between alteration in DNA methylation levels and these risk factors can explain the causation of EH over time. As the traditional risk factors are different between both genders, our results of gender-dependent ADD1 methylation may reflect the difference in these risk factors of EH.

In conclusion, this study found that lower ADD1 promoter methylation increases the risk of EH. CpG2-5 methylation can predict EH among males and CpG1 methylation does among females. Lesser effect of ADD1 DNA methylation among females may be due to the interaction between CpG1 and age. Our analysis on the role of ADD1 methylation and the risk of EH will help provide insight on the pathogenesis and management of EH.

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the article.