Cardiac natriuretic peptides (NPs) including atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) play a critical role in the regulation of blood pressure and salt-water balance through natriuresis, diuresis, and vasodilatation (1). Their circulating levels have been associated with the risk of stroke which is the leading cause of long-term disability and mortality all over the world (2), China in particular (3), in various prospective studies (4–6). Corin, a trypsin-like protease highly expressed in the heart (7, 8), is the physiological activator of ANP (9) and could also activate BNP (10). It may be a switching regulator of the NP system and thereby contributing to the development of stroke. Indeed, the cardiovascular effect of corin has been suggested by basic and population studies. For example, the expression of corin was upregulated in atherosclerotic aorta intima and vascular endothelial cells stimulated by oxidative stress (11). Blood pressure was elevated in mice with corin gene knockout (12). In humans, single nucleotide variations (SNVs) in CORIN, the coding gene of corin protein, were associated with susceptibility to heart failure (13), cardiac hypertrophy (14), and hypertension (15). Circulating levels of corin have been associated with various cardiovascular disorders such as heart failure (16), atrial fibrillation (17), and myocardial infarction (18). Furthermore, our group previously found that decreased serum corin was significantly associated with prevalent stroke (19) and unfavorable poststroke outcomes (20). These findings suggest that corin could be a risk factor or drug candidate for the prevention and control of stroke. However, no inhibitor of corin function has been found in human plasma (21), which may increase the unsafety of its clinical translation. Therefore, a better understanding of the molecular mechanisms underlying the association between corin and stroke is urgent for clinical translation. As a mediator between the dynamic environment and fixed genome, DNA methylation may affect gene expression and function and thereby representing one of the candidate molecular mechanisms that we are seeking. To date, many DNA methylation markers of stroke have been identified by epigenome-wide association studies (22). DNA methylation levels of the global genome (23, 24) and some candidate genes, such as ATP-binding cassette G1 (25), matrix metalloproteinase-2 (26), estrogen receptor alpha (27), thrombomodulin (28), and tumor protein p53 (29) have been associated with stroke. As suggested above, we hypothesized that DNA methylation of the CORIN gene may also play a considerable role in stroke development, but lacking epidemiological evidence. Therefore, we aimed to examine the association between CORIN gene promoter methylation and the risk of stroke in two independent samples of Chinese adults. The identified methylation markers might be useful targets for the prevention and treatment of stroke because DNA methylation is a modifiable molecular modification.

Methods of selection of study participants and data collection were described in the Supplementary material (eMethods) in detail. Following we briefly introduced the methods of the current study.

In Chinese adults participating in two independent samples, we examined for the first time the association between DNA methylation at the CORIN gene promoter and stroke. We found that hypermethylation at three CpG sites (located at Chr4:47840038, Chr4:47839941, and Chr4:47839933) was not only associated with lower odds of prevalent ischemic stroke but also predicted a lower future risk of stroke. DNA methylation levels at these CpG loci, the CpG located at Chr4:47840038 in particular, could increase the prediction power for the risk of stroke over conventional risk factors including behavioral and metabolic factors. Our results suggest that CORIN promoter methylation may play a potential role in the development of stroke through mechanisms beyond metabolic factors. Because DNA methylation is modifiable, CORIN promoter methylation may serve as a potential predictor or even probably a therapeutic target for stroke.

In line with our study, the potential role of corin in stroke has also been studied in previous studies. For example, a cell-based study found that the corin level was increased in cultured vascular endothelial cells stimulated by oxidized low-density lipoprotein, which is a major risk factor for atherosclerosis and stroke (11). In humans, CORIN gene polymorphisms have been associated with susceptibility to heart failure (13), cardiac hypertrophy (14), and hypertension (15). Clinical studies have found that circulating levels of corin were associated with heart failure (16), atrial fibrillation (17), and myocardial infarction (18), as well as major adverse cardiovascular events after the onset of heart failure (36) and acute myocardial infarction (37), all of which shared many pathological mechanisms with stroke. Our previous case–control study including 597 stroke patients and 2,498 community-based healthy controls provided the first evidence for the association between serum corin levels and stroke (19). Recently, we further revealed a significant prospective association between serum corin at baseline and 10-year risk of stroke in the Gusu cohort (38). These findings consistently suggested a potential role of corin protein in stroke, but the molecular mechanisms through which corin contributes to stroke are not very clear.

DNA methylation is a modifiable chemical modification of the genome without changing the gene sequence and could repress gene expression by recruiting proteins associated with gene suppression or by preventing transcription factors from binding to DNA (39). Indeed, many risk factors of stroke have been associated with the level of DNA methylation, such as smoking (40), obesity (41), and hypertension (42). Compared with questionnaires, DNA methylation may better capture the influence of environmental factors and individual behavioral habits (which are difficult to quantify accurately) on the risk of stroke, therefore, DNA methylation may provide a more accurate prediction of stroke risk (43). It has been suggested to be involved in the pathogenesis of stroke in some small case–control studies. For example, epigenome-wide association studies (EWAS) have identified multiple epigenetic markers for stroke (44–46). Clinical studies found that DNA promoter methylation at some candidate genes, such as tumor necrosis factor (47), estrogen receptor alpha (27), and matrix metalloproteinase-2 (26) were associated with stroke. In a study including a discovery sample of 511 patients with first-ever acute ischemic stroke and a replication sample of 85 patients with the same disease, biological age has been estimated based on DNA methylation at 71 CpG sites, and was found to be an independent predictor of 3-month ischemic stroke outcome evaluated by 3-month modified Rankin Scale (48), another study reported that biological age calculated by DNA methylation was associated with death within 3 months after ischemic stroke (49). Therefore, DNA methylation of the CORIN gene may exist as a potential molecular mechanism that regulates the expression or excretion of corin protein and thereby participates in the pathogenesis of stroke. Indeed, a case–control study including 731 hypertension patients and 731 controls has demonstrated that CORIN gene methylation mediated the effect of CORIN SNVs on corin protein level (31). To the best of our knowledge, no study examined the association between DNA methylation at the CORIN gene and the risk of stroke. Leveraging two independent samples, our study provided initial evidence that CORIN promoter methylation may play an important role in the development of stroke.

Previous studies introduced that the contribution of methylation at single CpG sites to a complex phenotype was relatively small (50, 51). This phenomenon was also observed in our study. DNA methylation levels of individual CpG sites at baseline could only explain 0.04–2.46% of the risk of stroke during follow-up. Although DNA methylation levels at the 9 CpG sites assayed were strongly correlated, only three CpG sites were associated with stroke, and none of them survived correction for multiple testing. Although a small effect size is not easy to be identified, the joint effect of multiple CpG sites may be stronger and more suitable for stroke risk prediction. Therefore, we examined the joint association between methylation of multiple CpG sites in the CORIN promoter with incident stroke and found a significant joint association. Our results indicated the importance of detecting the combined effect of methylation at multiple CpG sites on complex disorders.

The strengths of our study include independent replication, comprehensive measurement and adjustment for confounding factors, and application of weighted truncated product methods to test the joint association between CORIN promoter methylation and stroke. Some limitations need to be acknowledged. First, as an observational study, unknown confounders may influence the association that we found. The causality between CORIN promoter methylation and stroke is still unclear. Second, our participants only included Chinese adults. The generalizability of our results to other populations with different ethnic backgrounds is uncertain. Third, the methylation we detected came from peripheral blood genomic DNA. It is not clear whether it can reflect the effect of methylation in the target organs of stroke, such as the brain and arteries. However, there is increasing evidence that epimutations may not be limited to affected tissues, but can also be detected in peripheral blood (52). Fourth, whether CORIN promoter methylation affects gene expression and protein synthesis is still unknown and needs further investigation. Fifth, natriuretic peptide, the final activation hormone of the natriuretic peptide system to exert cardiovascular protection, may be affected by the methylation level of the CORIN gene, however, we did not have data on natriuretic peptides and could not include the levels of natriuretic peptide in this study.

In summary, hypermethylation of the CORIN gene promoter was not only associated with prevalent ischemic stroke but also predicted a lower future risk of incident stroke in Chinese adults. DNA methylation level of the CORIN promoter, the CpG located at Chr4:47840038 in particular, could be a predictor of incident stroke. Because DNA methylation is a modifiable molecular modification, CORIN promoter methylation could be a candidate therapeutic target for the prevention and management of stroke, although the underlying causality is still unclear.

The original contributions presented in the study are publicly available. This data can be found at: https://doi.org/10.5061/dryad.k3j9kd5bx.

The studies involving human participants were reviewed and approved by the Soochow University Ethics Committee. The patients/participants provided their written informed consent to participate in this study.

LC and JJ performed the data analysis and drafted the manuscript. HP and QZ developed the concept of the study design and revised the manuscript. JY, YL, and XZ obtained the clinical data and critically reviewed the manuscript. JJ, QZ, and HP contributed to the interpretation of the results. All authors read and approved the final manuscript.

This study was supported by the National Natural Science Foundation of China (nos. 82173596, 81903384, and 81872690), the Youth Program of Science and Technology for Invigorating Health through Science and Education in Suzhou (no. GSWS2019091), 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.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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