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Associations of NPPA promoter true methylation and hydroxymethylation with ischemic stroke and its functional outcome

Epigenetics Communications volume 4, Article number: 6 (2024) Cite this article

Abstract

Background

Atrial natriuretic peptide (ANP) has been associated with ischemic stroke (IS), but the underlying molecular mechanisms are not clear. The objective of this study was to examine whether DNA methylation and hydroxymethylation in the coding gene of ANP (NPPA) were associated with IS, as well as its functional outcome.

Methods and results

DNA methylation and hydroxymethylation in NPPA promoter region were quantified by targeted bisulfite sequencing and APOBEC-coupled epigenetic sequencing, respectively, in 615 IS patients and 610 age- and sex-matched healthy controls. True methylation was calculated as the quantified methylation level minus the quantified hydroxymethylation level. The functional outcome of IS was evaluated by the modified Rankin Scale (mRS) at a 3-month follow-up visit after onset. Multiple testing was controlled by the false discovery rate approach. Of the nine CpG sites assayed, hypomethylation at eight CpGs was not only associated with an increased risk of having IS but also predicted a higher mRS score after onset (all q < 0.05) and hydroxymethylation at one CpG located at Chr1:11908348 was positively associated with IS (OR = 1.39, 95%CI:1.15–1.68, q < 0.05), after adjusting for covariates and multiple testing. While, hydroxymethylation levels at none of the CpGs assayed were significantly associated with the mRS score.

Conclusions

DNA methylation and hydroxymethylation in NPPA promoter were associated with the risk of IS and its functional outcome in Chinese adults.

Background

Atrial natriuretic peptide (ANP), a hormone synthesized and released mainly from myocytes with pleiotropic cardiovascular and metabolic properties [1], plays a critical role in hemodynamic homeostasis through natriuresis, diuresis, and vasodilation [2] and cardiovascular remodeling due to its antiproliferative, antifibrotic, and antiangiogenic functions [3]. All of these processes constitute the physiopathology of ischemic stroke (IS) [4]. Admittedly, circulating ANP levels have been associated with IS [5] as well as related cardiovascular disorders, e.g., hypertension [6], heart failure [7], and myocardial infarction [8]. In addition to IS development, plasma ANP levels could also predict 90-day mortality and functional outcomes after IS onset [9, 10]. However, the clinical implementation of ANP in the management of heart failure with ANP-targeted agents, such as sacubitril/valsartan [11] and recombinant carperitide [12, 13] has encountered significant challenges. These include the occurrence of hypotension, angioedema, and fatalities in hospital settings. A better understanding of the molecular mechanisms underlying the association between ANP and the occurrence and development of IS may help its drug development and application in the prevention and control of this debilitating disorder.

As an interface between the fixed genome and dynamic environment, epigenetic modifications such as DNA methylation regulate gene expression without changing the DNA sequence [14], thereby participating in the mechanisms of complex phenotypes. For example, epigenome-wide association studies and candidate gene studies have identified many methylation markers of IS [15,16,17], as well as its prognosis [18, 19], with a lack of genome-widely significant epigenetic markers related to ANP. Considering the contribution of genetic variants of NPPA, the coding gene of ANP, to the susceptibility of IS [20, 21], we hypothesized that DNA methylation in the NPPA promoter region may explain, at least partially, the molecular mechanisms underlying the role of ANP in IS development and prognosis. Further, our previous studies have already found that NPPA promoter hypomethylation predicted an increased risk of cardiovascular disease (CVD) [22], central obesity [23], and hypertension [24].

DNA methylation is a dynamic process in response to the environment. DNA methyltransferase binds a methyl group to cytosine 5’ carbon atom at a CpG site forming 5-methylcytosine (5mC) [25]. When DNA methylation occurs at CpG islands of the gene promoter region, it is normally associated with the repression of gene expression [26]. Under the action of the TET enzyme, 5mC can be further oxidized to 5-hydroxymethyl cytosine (5hmC) by incorporating a single oxygen atom from molecular oxygen into its substrate, 5-methylcytosine [27]. This process is hydroxymethylation, a known demethylation intermediate that may upregulate gene expression [28]. The demethylation process also includes the oxidation of 5hmC to 5-formylcytosine (5fC), 5-carboxylcytosine (5caC), and further conversion to an unmodified cytosine by replication-dependent dilution or thymine DNA glycosylase (TDG)-dependent base excision repair [29]. Of these epigenetic modifications, 5mC and 5hmC are the most common but have conflicting functions on the regulation of gene expression [30]. Nevertheless, DNA methylation levels are currently quantified based on bisulfite sequencing which cannot discriminate between 5mC and 5hmC [31]. Our previous study has found that NPPA promoter methylation assayed using such a method was associated with IS [32], but the methylation level assayed actually reported a composite level including both 5mC and 5hmC and may therefore be prone to information bias. Moreover, mice experiments have demonstrated that 5hmC could protect the brain from ischemic injury [33, 34]. DNA hydroxymethylation of the NPPA gene may also be associated with the development and prognosis of IS, but this association was scarcely studied in humans. Therefore, we quantified the levels of DNA methylation and hydroxymethylation simultaneously at identical CpG sites in the NPPA promoter region for patients with IS and age- and sex-matched healthy controls. We then calculated true methylation levels by subtracting the hydroxymethylation level from the methylation level at corresponding site to examine whether NPPA promoter true methylation and hydroxymethylation are associated with IS, as well as its functional outcome after IS.

Results

Baseline characteristics of study participants

A total of 615 patients with IS (mean aged 62 years, 47% males) and 610 healthy controls (mean aged 62 years, 48% males) were included in the current study. Their baseline characteristics are presented in Table 1. As expected, patients with IS had more metabolic risk factors including hypertension, diabetes, obesity, hyperglycemia, and dyslipidemia than healthy controls (all P < 0.05). Additionally, the difference in education level was also statistically significant between two groups (P < 0.05).

Table 1 Baseline characteristics of study participants in ischemic stroke cases and healthy controls
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Association between NPPA promoter true methylation and IS

As shown in eTable 1, eight of the nine CpG sites assayed were hypomethylated, as suggested by true methylation levels, in patients with IS, in comparison to healthy controls (all q < 0.05). The single CpG association analysis using binary logistic regression with log2-transformed methylation level as the independent variable showed that true methylation levels at eight CpG sites (except for CpG9) were negatively associated with the risk of having IS (all P < 0.05), after adjusting for age, sex, education level, current smoking, current drinking, BMI, LDL-C, HDL-C, hypertension, and diabetes (Fig. 1). After further correction of multiple testing, these associations were still statistically significant (all q < 0.05).

Fig. 1
figure 1

A forest plot showing the single-CpG associations of true methylation and hydroxymethylation of NPPA promoter with ischemic stroke. The black squares and line segments illustrate the ORs and 95% CIs of having ischemic stroke associated with DNA methylation and hydroxymethylation levels (after log2-transformation), adjusting for age, sex, education level, current smoking, current drinking, BMI, LDL-C, HDL-C, hypertension, and diabetes. q value indicates the significance level after correction of multiple testing by false discovery rate approach. CI: confidence interval; OR: odds ratio

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By combing P-values of the single CpG associations, wTPM revealed significant gene-based associations between true methylation of NPPA promoter and IS (P < 0.05), suggesting that DNA methylation of the nine CpG sites may be jointly associated with IS. The impact of the mean of true methylation levels on the risk of IS was visualized in Fig. 2a, the mean methylation level was also significantly associated with IS in the same direction as the single CpG associations (OR = 0.94, 95%CI:0.91–0.97, P < 0.001). In subgroup analyses, the association between the mean of true methylation levels of the nine CpG sites and IS was more pronounced among elders, females, non-smokers, non-drinkers, and those with a low level of education (eTable 2), which may have practical guiding significance for the clinical application of epigenetic markers. Further, we did not find a statistically significant group difference regarding metabolic risk factors of IS, such as obesity and hypertension, which have been associated with NPPA methylation in our previous studies. The results of the subgroup analysis suggested a robust association between NPPA true methylation and IS and this association may not be driven by metabolic factors.

Fig. 2
figure 2

Cubic spline curves visualizing the impacts of the average levels of true methylation and hydroxymethylation in the NPPA promoter region on ischemic stroke. Red lines indicate odds ratios (solid) and their 95% confidence intervals (dotted) of ischemic stroke associated with the mean levels of true methylation (a) and hydroxymethylation (b), adjusting for age, sex, education level, current smoking, current drinking, BMI, LDL-C, HDL-C, hypertension, and diabetes. The histogram shows the distribution of participants according to increasing levels of true methylation and hydroxymethylation. CI: confidence interval; OR: odds ratio

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Association between NPPA promoter hydroxymethylation and IS

In contrast to methylation, the median levels of hydroxymethylation at the nine CpG sites assayed were generally higher in patients with IS than that in healthy controls, although no statistically significant group-difference was found (eTable 1). After adjusting for covariates, hydroxymethylation level at one CpG site (CpG2, located in Chr1:11908348) was positively associated with IS (OR = 1.39, 95%CI: 1.15–1.68, P < 0.05 after log2-transformation, Fig. 1). This association persisted after further correction of multiple testing (q < 0.05). The wTPM revealed a significant gene-based association (P < 0.05), suggesting that NPPA promoter hydroxymethylation as a whole was significantly associated with IS. Nevertheless, we failed to observe a significant association between the mean of hydroxymethylation levels of the nine CpG sites and IS (Fig. 2b and eTable 3).

Association between NPPA promoter true methylation and the functional outcome of IS

Of the 615 IS patients with available true methylation data of NPPA promoter at baseline, 17 patients were lost of follow-up and the 598 patients remained were included in the prospective analysis to examine whether true methylation of NPPA promoter was associated with the functional outcome of IS. Ordered logistic regression found that the true methylation levels at eight CpG sites assayed at baseline were significantly associated with a lower risk of having a higher mRS score at the 3-month visit, after multivariate adjustment and multiple testing correction (all q < 0.05, Table 2). To ease data interpretation, binary logistic regression with poor functional outcome defined as mRS ≥ 3 as the dependent variable found similar associations (eFigure 1). Gene-based association between true methylation of NPPA promoter and mRS (all P < 0.05) was also significant, suggesting that DNA methylation of the nine CpG sites may be jointly associated with functional outcome of IS.

Table 2 Associations of true methylation and hydroxymethylation of NPPA promoter with the functional outcome at 3-month follow up after ischemic stroke onset
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Association between NPPA promoter hydroxymethylation and the functional outcome of IS

DNA hydroxymethylation levels at none of the CpG sites assayed were significantly associated with the mRS (Table 2) and the poor functional outcome (eFigure 1) at the 3-month follow-up visit. In addition, no gene-based association was found that NPPA promoter hydroxymethylation as a whole was jointly associated with functional outcome of IS (P = 0.453).

Discussion

In a large sample of patients with IS and healthy controls, we simultaneously examined for the first time whether DNA true methylation and hydroxymethylation in NPPA promoter were associated with IS as well as its functional outcome. We found that NPPA promoter hypomethylation was not only associated with an increased risk of having IS but also predicted poorer neurologic recovery at 3-month follow up visit after onset and hydroxymethylation was elevated in patients with IS but failed to predict the functional outcome of IS, independent of conventional risk factors. These results suggest that DNA methylation and hydroxymethylation in NPPA promoter may participate in the development and prognosis of IS. The identified CpG sites could be used as epigenetic markers and even therapeutic targets for IS.

In line with our study, the role of ANP and its coding gene in IS has been extensively studied. For example, previous studies have identified ANP as a potential biomarker for early detection of IS [35] and a therapeutic target for disease due to its biological functions of natriuresis, diuresis, vasorelaxation, and inhibition of renin and aldosterone secretion [2, 36]. Researches on the role of ANP in the pathogenesis of cerebrovascular diseases have been conducted in great depth, yet the clinical application of ANP-related drugs is still very limited [11,12,13]. Genetic polymorphisms in the NPPA gene could not only determine the circulating levels of ANP [37] but also indicate the susceptibility of IS [20, 21]. A genetic association study including 2660 participants revealed that the NPPA rs5063 was associated with a reduced risk for having IS [20]. Another study conducted in a Korean population found that rs5065 and rs5067 of the NPPA gene were associated with the susceptibility for cardioembolic stroke [21]. As an interface between the fixed genome and dynamic environment, DNA methylation in the NPPA gene may affect its function and subsequent ANP synthesis and excretion. DNA methylation in NPPA promoter may explain the underlying molecular mechanisms of the relationship between ANP and IS as well as its functional outcome. Importantly, since such epigenetic modifications are reversible processes, they could assist the development of new therapeutic approaches for the treatment of human diseases [38].

In recent years, with the continuous development of technology, the correlation between DNA methylation markers and IS as well as its functional outcome have been gradually identified. In line with our study, animal studies have found that dysregulated DNA methylation could cause the aggravation of central ischemic injury in mice [39, 40]. In humans, methylation markers at some candidate genes such as SLC6A4 [41] and CRY1 [42] were related to the long-term adverse outcomes in patients with IS. A comprehensive review suggested that and DNA methylation at hundreds of methylation markers, in addition to global methylation, covering a wide range of genes but none was related to the NPPA gene, were associated with IS [43]. However, DNA methylation levels reported by these studies were measured utilizing bisulfite treatment-based methods, which cannot distinguish between the methylated and hydroxymethylated forms of cytosine [44]. In our study, we calculated true methylation levels by quantifying hydroxymethylation levels at the same CpG sites. We are the first, to the best of our knowledge, to examine and found that DNA true methylation in NPPA promoter was associated with IS and its functional outcome. In our study, participants with IS were tended to have lower true methylation levels, and hypomethylation was associated with increased risk of having IS and poor neurologic recovery. It is well known that methylation status at the gene promoter region is inversely correlated to the gene expression level generally [45], and many studies have also found that participants with IS had higher levels of serum ANP than those without and the severity of the disease could be distinguished in the light of ANP levels [46,47,48]. Our results were consistent with the findings of existing studies, though the causal association between NPPA promoter methylation and ANP expression level has not been verified yet.

As an important member of epigenetic modifications, DNA hydroxymethylation has conflicting functions of regulating gene expression compared with DNA methylation so that we cannot ignore its role in the occurrence and development of disease. Therefore, we further explored the associations of NPPA promoter hydroxymethylation with IS and its functional outcome. The prevailing evidence has shown that DNA hydroxymethylation is involved in the pathogenesis and prognosis of IS while the elucidation of the link between them is still in its infancy, especially in humans. In animal models of focal ischemia, TET2/3, the enzyme that catalyzed the formation of 5hmC, knockdown further increased brain degeneration in mice, demonstrating a role of 5hmC in endogenous protection against stroke [33, 49]. In humans, there was only a small sample of 24 participants showed a significant increase in the abundance of 5hmC in blood samples from patients with IS [33]. In our study, we found that NPPA promoter hydroxymethylation levels at only one CpG site (CpG2 located at Chr1:11908348) seemed to be positively associated with the risk of having IS, revealing the possibility of DNA hydroxymethylation in NPPA promoter as a potential biomarker and a therapeutic target for IS.

In this study, it can be seen that methylation levels and hydroxymethylation levels differed substantially between CpG sites assayed, and their effects on the IS were also different. Of the nine CpG sites assayed, one CpG site (CpG2 located at Chr1:11908348) exhibited a strong correlation with IS, as both methylation level and hydroxymethylation level of this site were significantly associated with IS. This CpG site is highly expected to be a precise intervention target for IS if the relevant technology is feasible in the future. Nevertheless, the methylation and hydroxymethylation levels at these CpG sites were highly correlated. In addition, the contribution of methylation or hydroxymethylation at a single CpG site to the complex phenotypes was generally small [50], and some CpG sites may be overlooked due to a lack of statistical significance before and after multiple testing corrections. Thus, we further tested the combined effects of multiple CpG sites and found that NPPA promoter methylation and hydroxymethylation as a whole were jointly associated with IS. Our research suggested that testing the combined effects of multiple CpG sites was another important approach in epigenetic analysis for complex diseases.

Several strengths in our study deserved to be mentioned. First, careful and systematic analyses were made to examine the associations of true methylation and hydroxymethylation in NPPA promoter with IS and its functional outcome. In addition, associations of NPPA promoter methylation and hydroxymethylation with pathogenesis and prognosis of IS were taken into consideration simultaneously in this article, providing a comprehensive basis for further development of the clinical application of ANP. Some limitations also characterized the present study. First, an observational study is incapable of deducing whether aberrant methylation and hydroxymethylation in NPPA promoter is a risk factor, outcome or just a concomitant phenomenon of IS. Second, although our results remained significant after adjusting for a variety of known risk factors, residual confounding may still exist. Third, the generalizability of our results to other ethnic populations or younger populations is unknown, since our findings were derived from Chinese adults whose cardiovascular profiles could be different from other populations with different ethnic backgrounds or other age groups. Fourth, although our results supported that DNA methylation and hydroxymethylation in NPPA promoter might explain the molecular mechanisms underlying the relationship between ANP and IS as well as its functional outcome, whether or to what extent the contribution of ANP to IS could be explained is unknown due to lacking data on circulating ANP levels. In our previous study [22], we examined the associations between methylation levels at the same CpG sites of the NPPA gene and serum proANP, but it is with regret that no CpG sites reached statistical significance. Although the association between NPPA promoter methylation and ANP has not been confirmed yet, many studies have already found that the variants of the NPPA gene were associated with circulating levels of ANP.

Conclusions

Levels of DNA methylation and hydroxymethylation in NPPA promoter was associated with the risk of IS and its functional outcome in Chinese adults. Dysregulated methylation and hydroxymethylation in the NPPA gene may participate in the development and prognosis of IS. Due to epigenetic modifications are modifiable, the CpG sites identified may be epigenetic markers and even potential therapeutic targets for IS.

Methods

Study participants

As described in Fig. 3, cases of IS and their healthy controls included in the current case-control study were selected from the China Antihypertensive Trial in Ischemic Stroke (CATIS) and the Prevention of Metabolic syndrome and multi-metabolic disorders Study (PMMS), respectively. The detailed methods of the selection of study participants have been detailed elsewhere [32]. In brief, the CATIS was a multicenter randomized controlled clinical trial designed to test the effect of blood pressure reduction on the prognosis of IS. A total of 4071 patients with IS confirmed by brain tomography or MAGNETIC resonance imaging were recruited. Among them, 3013 patients had DNA samples and 1000 patients were randomly selected as candidate cases. A total of 1000 healthy controls were randomly selected by frequency matching based on age (± 4 years) and sex with cases from 3999 participants with available DNA samples in the PMMS, which was a community-based prospective cohort study of CVD and its risk factors in Chinese adults. Of the 2000 candidate participants whose DNA samples were assayed, 615 IS cases and 610 healthy controls with successful quantification of hydroxymethylation and further calculation of true methylation in NPPA promoter were finally included in the current analysis, after excluding samples that failed in quantification of methylation and hydroxymethylation.

Fig. 3
figure 3

A flowchart illustrating the study design and selection of study participants. A case-control analysis was performed in 615 patients with ischemic stroke and 610 healthy controls to examine the associations of true methylation and hydroxymethylation of NPPA promoter with ischemic stroke. Leveraging the follow-up data for the patients with ischemic stroke, a prospective analysis was performed to examine whether true methylation and hydroxymethylation of NPPA promoter predicted the functional outcome after ischemic stroke onset

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Standard protocol approvals

The protocols of the CATIS were approved by the institutional review boards at Tulane University in the United States and Soochow University in China, as well as ethical committees at the participating hospitals. The protocols of the PMMS were approved by the Soochow University Ethics Committee. Written informed consent was obtained from all study participants or their immediate family members in both the CATIS and the PMMS.

Quantification of NPPA promoter methylation

As illustrated in Fig. 4, targeted bisulfite sequencing [51] was applied to quantify DNA methylation levels in the promoter region of the NPPA gene. The targeted region was selected from Chr1:11908380 to Chr1:11907840, relative to the transcriptional start site − 540 bp to -270 bp. Primers were carefully designed based on the targeted sequence coordinated in the Genome Reference Consortium Human Build 37 (GRCh37). The genomic DNA, extracted from peripheral blood mononuclear cells, was first bisulfite treated using the EZ DNA Methylation-Gold Kit (Zymo Research, Inc., CA, USA) and then amplified by polymerase chain reaction (PCR). Using the paired-end sequencing protocol according to the manufacturer’s guidelines, the PCR products were ultimately sequenced by Illumina Hiseq 2000 (Illumina, Inc., CA, USA). After bisulfite treatment, unmethylated cytosines were deaminated and converted to uracil while methylated cytosines, containing traces of hydroxymethylated cytosines, were unchanged. The methylation level at each CpG site was calculated as the percentage of methylated alleles over the total alleles. In the aspect of quality control, we took the percentage of converted non-CpG cytosines as the conversion rate of each sample and excluded samples whose conversion rate was lower than 98%. In addition, samples with an average coverage of less than 20× were further excluded.

Fig. 4
figure 4

A schematic illustration of the targeted sequence, primers, and quantification of DNA methylation and hydroxymethylation. A total of 9 CpG sites (red colored) were finally assayed in the NPPA gene promoter (-540 ~ -276 bp from TSS). ACE-seq: APOBEC-coupled epigenetic sequencing; BS-seq: Bisulfite sequencing; TSS: transcriptional start site; 5mC: 5-methylcytosine; 5hmC: 5-hydroxymethyl cytosine; 5gmC: 5hmc labeled with glucose; C: cytosine; U: uracil; T: thymine

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Quantification of NPPA promoter hydroxymethylation

Using the same DNA samples, we additionally quantified the hydroxymethylation levels at the same CpG sites with APOBEC-coupled sequencing (Fig. 4). With 0.2% synthetic full methylated and full hydroxymethylated control DNA spiked in, the genomic DNA was treated using T4 β-glucosyltransferase (β-GT, NEB) and APOBEC3A. β-GT was used for selective labeling of hydroxymethylated cytosines with glucose which can prevent them from being deaminated by APOBEC3A. As a result, this enzymatic conversion converts cytosines and methylated cytosines into uracils and leaves hydroxymethylated cytosines unchanged. Using the paired-end sequencing protocol according to the manufacturer’s guidelines, the enzyme-treated samples were then sequenced by Illumina Hiseq 2000 (Illumina, Inc., CA, United States). The hydroxymethylation level at each CpG site was calculated as the percentage of the hydroxymethylated alleles. The same procedures of quality control as used in methylation quantification were applied.

Calculation of NPPA promoter true methylation

In the methods of assessment for methylation, bisulfite conversion does not allow for distinguishing between methylation and hydroxymethylation. As a result, the amounts of methylated alleles detected in fact comprise hydroxymethylated alleles. Therefore, the true methylation level at each CpG site could be calculated as the corresponding quantified methylation level minus the quantified hydroxymethylation level.

Assessment of functional outcome of IS

The modified Rankin Scale (mRS) was applied to assess the functional outcome of patients with IS at 3 months after onset. It is rated on a scale of 0–6, with 0–2 indicating no symptoms to a slight disability, 3–5 indicating moderate to severe disability, and 6 indicating death. The mRS score was evaluated by trained neurologic physicians blinded to the baseline characteristics of the patients with IS.

Collection of risk factors for IS

As detailed in eMethods (supplementary data), demographic data (age, sex, education level), lifestyle factors (current smoking, current drinking), disease history, body mass index (BMI), blood pressure, fasting glucose, and blood lipids, including total cholesterol, triglycerides, low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C), were collected for all study participants. For patients with IS, subtypes including thrombotic, embolism, lacunar infarction, and other subtypes were assessed based on the symptoms and imaging data [52]. The severity of IS was assessed by trained neurologists using the National Institutes of Health Stroke Scale (NIHSS) [53].

Statistical analysis

All statistical analyses were performed using R version 4.1.3. The baseline characteristics of study participants were presented in patients with IS and healthy controls, respectively. Log2-transformation was applied to maximize the normality of epigenetic data and the generated values were used in logistic regression analyses.

Analysis of the associations of NPPA true methylation and hydroxymethylation with IS

To examine whether true methylation of NPPA promoter was associated with IS, we first conducted a single CpG association analysis followed by a gene-based association analysis. In specific, the single CpG association analysis was performed by constructing a binary logistic regression model in which IS (y/n) was the dependent variable and true methylation level at each CpG site was the independent variable, adjusting for age, sex, education level, current smoking, current drinking, BMI, LDL-C, HDL-C, hypertension, and diabetes. Multiple testing was controlled by adjusting for the total number of CpG loci tested using the false discovery rate (FDR) approach, and an FDR-adjusted P value (i.e., q value) of less than 0.05 was considered statistically significant. Based on the results of the single CpG association analysis, the weighted truncated product method (wTPM) [54] was used to test the gene-based association between true methylation at multiple CpG sites and IS. This method combined P values of all CpGs that reached a preselected threshold (e.g., raw P < 0.1 in this study). The regression coefficient of each individual CpG methylation was included as weights in the wTPM statistic. To visualization of the effect of DNA methylation on IS, the mean of true methylation levels at multiple CpG sites was considered a surrogate of the true methylation level of the target region and was also used to examine the gene-based association by constructing a restricted cubic spline regression model with 4 knots (5th, 35th, 65th, and 95th percentiles) [55]. The association between DNA hydroxymethylation and IS was similarly examined.

Analysis of the associations of NPPA true methylation and hydroxymethylation with the functional outcome of IS

The associations of true methylation and hydroxymethylation of NPPA promoter with the functional outcome of IS were similarly examined. In brief, an ordered logistic regression model with mRS score at 3-month follow-up as an ordinal response variable was constructed. In addition to the variables listed above, ischemic stroke subtypes, hours from onset to hospitalization, NIHSS score, and treatment group at admission were also adjusted for in this model. To ease data interpretation, the associations of true methylation and hydroxymethylation of NPPA promoter with poor functional outcome defined as mRS score ≥ 3 were also examined by constructing a binary logistic regression model.

Data availability

The data that supports the finding of this study was publicly available from Dryad: https://doiorg.publicaciones.saludcastillayleon.es/10.5061/dryad.h18931zvn. This link is permanent and will be active once the paper is published. Before publication, we can access the data using the temporary URL below: https://datadryad.org/stash/share/if0mcxAb7wcCTZNgkI690VuxPDY-k5LweCABJJnTbf0.

Abbreviations

ANP:

Atrial natriuretic peptide

ACE-seq:

APOBEC-coupled epigenetic sequencing

BMI:

Body mass index

BS-seq:

Bisulfite sequencing

CATIS:

China Antihypertensive Trial in Acute Ischemic Stroke

CI:

Confidence interval

CVD:

Cardiovascular disease

DBP:

Diastolic blood pressure

FDR:

False discovery rate

GRCh37:

Genome Reference Consortium Human Build 37

HDL:

High-density lipoprotein

IS:

Ischemic stroke

LDL:

Low-density lipoprotein

mRS:

Modified Rankin Scale

NIHSS:

National Institutes of Health Stroke Scale

OR:

Odds ratio

PMMS:

Prevention of Metabolic syndrome and multi-metabolic disorders Study

SBP:

Systolic blood pressure

TDG:

Thymine DNA glycosylase

TSS:

Transcriptional start site

wTPM:

Weighted truncated product method

5caC:

5-carboxylcytosine

5fC:

5-formylcytosine

5hmC:

5-hydroxymethyl cytosine

5mC:

5-methylcytosine

β-GT:

β-glucosyltransferase

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Acknowledgements

We gratefully acknowledge the cooperation and participation of our study. 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. DNA methylation and hydroxymethylation were quantified by Genesky Biotechnologies.

Funding

This study was supported by the National Natural Science Foundation of China (NO. 82173596, 81903384, and 81872690), the Suzhou Municipal Science and Technology Bureau (NO. SYS2020091), Suzhou Key Technologies of Prevention and Control of Major Diseases and Infectious Diseases (NO. GWZX202001), and a Project of the Priority Academic Program Development of Jiangsu Higher Education Institutions.

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Author notes

  1. Jialing Yao, Linghua Song and Jun Jiang contributed equally to this work.

Authors and Affiliations

  1. Department of Medical Insurance Price, Suzhou Ninth Hospital affiliated to Soochow University, Suzhou, China

    Jialing Yao

  2. Department of Neurology, Dushu Lake Affiliated Hospital of Soochow University, 9 Chongwen Road, Industrial Park District, Suzhou, 215123, China

    Linghua Song & Yongang Hao

  3. Department of Tuberculosis Control, Suzhou Center for Disease Control and Prevention, Suzhou, China

    Jun Jiang & Xiaolong Zhang

  4. Department of Chronic Disease, Taicang Center for Disease Control and Prevention, Suzhou, China

    Jianan Zhang

  5. Department of Epidemiology, School of Public Health, MOE Key Laboratory of Geriatric Diseases and Immunology, Suzhou Medical College of Soochow University, Suzhou, China

    Linan Chen, Wenxiu Fan, Ying Lu, Jiexiang Jing, Yibing Jin, Mingzhi Zhang & Hao Peng

  6. Department of Neurology, Taixing Second People’s Hospital, Suzhou Medical College of Soochow University, Suzhou, China

    Hao Peng

  7. Department of Neurology, Taixing Second People’s Hospital, Suzhou Medical College of Soochow University, 588 Yinxing West Road, Taixing City, Taizhou, China

    Hao Peng

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Contributions

JY and LS performed the statistical data analysis and drafted the manuscript. HP and YH developed the concept of the study design and contributed to draft the manuscript. JZ, LC, WF, YL, XZ, JJ (Jiexiang), YJ, and MZ obtained the clinical data and critically reviewed the manuscript. JJ (Jun), YH, and LS contributed to the interpretation of the results. All authors contributed to draft the final versions of the manuscript. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Yongang Hao or Hao Peng.

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Ethics approval

The protocols of the CATIS were approved by the institutional review boards at Tulane University in the United States and Soochow University in China, as well as ethical committees at the participating hospitals. The protocols of the PMMS were approved by the Soochow University Ethics Committee. Written informed consent was obtained from all study participants or their immediate family members in both the CATIS and the PMMS.

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The authors declare no competing interests.

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Yao, J., Song, L., Jiang, J. et al. Associations of NPPA promoter true methylation and hydroxymethylation with ischemic stroke and its functional outcome. Epigenetics Commun. 4, 6 (2024). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s43682-024-00029-5

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Epigenetics Communications

ISSN: 2730-7034