Role of CYP19A1 Loci (rs28757157 and rs3751591) with Ischemic Stroke Risk in the Chinese Han Population

Introduction

Stroke is a multifactorial and polygenic disease, which is known to be the main cause of death and adult disability.1 In general, most stroke patients cannot fully recover in the later stage. In severe cases, hemiplegia, lack of living ability, and inability to take care of themselves will occur, which brings great mental and economic burdens to their families. Stroke is divided into ischemic and hemorrhagic, of which 85% of stroke patients are due to ischemic.2 Moreover, hemorrhagic stroke may occur in ischemic brain, but it is not clear how much bleeding begins from ischemic stroke (IS). In China, stroke has become the leading cause of death.3 Up to 2013, stroke was the leading cause of death in 27 out of 33 provinces in China.4 The incidence of stroke in China is increasing year by year, but its pathogenesis is complex and has not yet been elucidated. Through recent years of research, factors such as hypertension,5 diabetes,6 obesity,7 hypercholesterolemia,8 smoking,9 and alcohol consumption10 were found to be likely to influence IS burden in China. However, numerous scientific studies have shown that IS was highly influenced by gene polymorphisms. Therefore, loci should also be paid attention to in the study of the pathogenesis of IS.

Cytochrome P450s are important oxidative metabolic enzymes that play a well-established role in the metabolism of endogenous compounds and the metabolic clearance of drugs and other exogenous substances.11 Cytochrome P450s enzymes can catalyze the metabolism of arachidonic acid into epoxyeicosatrienoic acids, dihydroxyeicosatetraenoic acids, and hydroxyeicosahexaenoic acids (HETEs). Advances in many disciplines have clarified the emerging role of cytochrome P450 enzymes and their metabolic substrates and end-products in the pathogenesis of central nervous system diseases, including Alzheimer’s disease, Parkinson’s disease, and stroke.12 Now, studies have shown that CYP candidate genes and variant loci played a non-negligible role in the pathogenesis of IS.13,14 Cytochrome P450 family 19 subfamily A member 1 (CYP19A1), one of the members of cytochrome P450 genes, which encodes an aromatase enzyme that can catalyze the conversion of C19 androgen, androstenedione, and testosterone to C18 estrogen, estrone, and estradiol, respectively. In an article reported by Zheng et al,15CYP19A1 variants were associated with Alzheimer's disease, but the mechanism of CYP19A1 loci in IS remains unclear.

In this research, we used to explore the association between CYP19A1 variants and the incidence of IS in the Chinese population by logistic-regression analysis with OR and 95%, to provide the theoretical basis for the role of CYP19A1 in ischemic stroke.

Methods Study Subjects

In the present study, we used G*power 3.1.9.7 software to estimate the sample size of the case group and the control group through an independent-sample t-test. The parameters were set as follows: Tail = 2, Effect size = 0.20, α = 0.05, Power = 0.95, Allocation ratio N2/N1=1. Finally, 1302 subjects (651 controls and 651 cases) were recruited from Haikou People’s Hospital. All participants were Han Chinese and signed informed consent. The study was approved by the ethics committee of Haikou People’s Hospital.

Based on the World Health Organization’s diagnostic criteria, all patients with IS underwent a series of diagnostic tests, including standardized blood tests, as well as computed tomography (CT) and/or magnetic resonance imaging (MRI). And each patient’s diagnosis was confirmed by at least two independent neurologists. Patients with a history of transient ischemic attack, systemic inflammatory disease, or tumor were excluded from this study. In the controls, they were examined at the physical examination center of the hospital at the same time. They had no history of hypertension, diabetes, coronary heart disease, cerebrovascular disease, etc.

SNPs Selection and Genotyping

Based on the 1000-genome project (https://www.internationalgenome.org/) in the global population with minor allele frequencies (MAFs) >0.05 and Hardy-Weinberg equilibrium (HWE) >0.01, we selected variants of CYP19A1 gene. In addition, we applied online software-Agena MassARRAY Assay Designer 3.1 to design amplification primers and extension primers (Supplementary Table 1). By removing non-specific primers and ineffective primers, we finally selected four loci (rs28757157, rs3751592, rs3751591 and rs59429575) on CYP19A1 gene. Using our previously extracted genomic DNA as a template, we completed SNPs genotyping for CYP19A1 gene on the basis of the Agena MassARRAY nucleic acid mass spectrometry platform. Data sorting and analysis of the genotyping results were finished by Agena software Bioscience TYPER, version 4.0.

Statistical Analyses

With the support of SPSS 19.0 and Microsoft Excel 22.0, we completed the data analysis. Among them, each SNP in the control group was detected using the chi-square test to compare the expected and actual genotype frequencies of the SNPs to test whether these loci were consistent with HWE. Subsequently, the allele and genotype frequencies of the selected SNPs were analyzed in the case group and the control group using the chi-square test. The association between CYP19A1 polymorphisms and IS risk was assessed by logistic regression analysis with odds ratios (OR) and 95% confidence intervals (CIs). FPRP tested for significant positive results at the power level (OR = 1.5) and a prior probability level “0.25, 0.1, 0.01, 0.001, 0.0001”. Moreover, MDR software predicted the correlation between CYP19A1 polymorphisms and IS risk from the perspective of SNP interaction (synergy or antagonism). p < 0.05 indicated statistical significance.

Results The Information of Study Subjects and Variants

Totally, 651 controls (437 males/214 females) and 651 cases (434 males/217 females) were randomly recruited in the study. In the Supplementary Table 2, the basic information of subjects’ characteristics was shown in the table. The average age of the controls and cases was 60.55 ± 0.23 years old and 63.76 ± 0.39 years old, respectively. Although the groups were not matched in age (p = 0.000), they were matched in gender (p = 0.860). In addition, the average height, weight and BMI of the controls (168.51 ± 0.29, 65.43 ± 0.43 and 23.01 ± 0.13) and cases (168.56 ± 0.28, 65.65 ± 0.38 and 23.09 ± 0.12) were displayed in the table, respectively. Also, there was no significant difference in height, weight, BMI index, smoking status and drinking status between the two groups (p = 0.910, p = 0.700, p = 0.633, p = 0.121 and p = 0.579).

In Table 1, the basic information of the selected variants (rs28757157, rs3751592, rs3751591 and rs59429575) located in CYP19A1, including the chromosome, position, allele, minor allele frequency (MAF) in cases and controls, consequence, HWE p-value and the function of loci predicted by HaploReg v4.1.

Table 1 The Basic Information of the Selected Variants Located in CYP19A1

The Association Between CYP19A1 Polymorphisms and Ischemic Stroke Risk (Overall Analysis)

In Table 2 and Figure 1, we performed the correlation between CYP19A1 polymorphisms and IS risk shown in the overall analysis. In the allele model, rs28757157C and rs59429575C were associated with an increased risk of IS (OR: 1.23, padj = 0.030; OR: 1.27, padj = 0.020). A significant association of the rs28757157 was also observed in the heterozygous C/T model with OR: 1.42 and padj  =  0.004, the dominant model with OR: 1.39 and padj  =  0.005 and the log-additive model with OR: 1.26 and padj  =  0.018. Meanwhile, rs28757157 and rs59429575 were providing risk for IS in the codominant (OR: 1.42 and padj  =  0.004; OR: 1.47 and padj  =  0.003), dominant (OR: 1.39 and padj  =  0.005; OR: 1.45 and padj  =  0.003) and log-additive (OR: 1.26 and padj  =  0.018; OR: 1.34 and padj  =  0.006) models. Subsequently, FPRP analysis was completed to verify the significant above results in the allele model (rs28757157: Power = 0.982, FPRP values = 0.080, 0.206; rs59429575: Power = 0.944, FPRP values = 0.067, 0.178), the heterozygous model (rs28757157: Power = 0.671, FPRP values = 0.020, 0.058; rs59429575: Power = 0.564, FPRP values = 0.011, 0.033) and the dominant model (Power = 0.747, FPRP values = 0.016, 0.047; Power = 0.610, FPRP values = 0.011, 0.032) at the prior probabilities are 0.25 and 0.1 (Supplementary Table 3).

Table 2 The Association Between CYP19A1 Polymorphisms and Ischemic Stroke Risk (Overall Analysis)

Figure 1 The forest map showed the association between CYP19A1 polymorphisms and ischemic stroke risk (overall analysis).

Association Between CYP19A1 Polymorphisms and Ischemic Stroke Risk Stratified by Age and Gender

In Table 3, we did the age and gender stratified analysis to assess the association between CYP19A1 polymorphisms and IS risk. In people older than 60 years old, the variant-rs28757157 showed a significant association with IS risk with OR: 1.34 and padj = 0.023 (allele), OR: 1.46 and padj = 0.029 (codominant), OR: 1.45 and padj = 0.028 (dominant). As for rs59429575, an increased association of the variant was also observed in the dominant model with OR: 1.43 and padj = 0.043 and the log-additive model with OR: 1.46 and padj = 0.034. The two variants were not found to be associated with IS risk in people younger than 60 years old.

Table 3 The Association Between CYP19A1 Polymorphisms and Ischemic Stroke Risk (Age and Gender Stratified Analysis)

In the results of gender stratification, the allelic association of rs28757157 showed a significant association with IS risk in females, with an observed OR of 1.46 (1.02–1.98 at 95% CI) and padj = 0.038 (allele). In the codominant and dominant models, the locus provided an increased risk for IS in females (codominant: OR: 1.70, padj = 0.015; dominant: OR: 1.62, padj = 0.020). In males, a significant association between rs59429575 and IS risk were found in the allele (OR: 1.33, padj = 0.025), codominant (OR: 1.69, padj = 0.001), dominant (OR: 1.64, padj = 0.001) and log-additive (OR: 1.44, padj = 0.006) models.

Association Between CYP19A1 Polymorphisms and Ischemic Stroke Risk Stratified by BMI

In Table 4, BMI stratification analysis was also completed to evaluate the association between CYP19A1 loci and IS risk. In subjects with BMI greater than 24, variant-rs59429575 was found to be associated with IS risk in the allele (OR: 1.45, padj = 0.041), codominant (OR: 1.87, padj = 0.008), dominant (OR: 1.84, padj = 0.007) and log-additive (OR: 1.59, padj = 0.014) models. In subjects with BMI less than 24, the locus showed a significant association with IS risk in the allele model with OR: 1.27 and padj = 0.035, the codominant model with OR: 1.45 and padj = 0.012, the dominant model with OR: 1.41 and padj = 0.015, the log-additive model with OR: 1.29 and padj = 0.037.

Table 4 The Association Between CYP19A1 Polymorphisms and Ischemic Stroke Risk (BMI Stratified Analysis)

Association Between CYP19A1 Polymorphisms and Ischemic Stroke Risk Stratified by Smoking and Drinking

In Table 5, smoking and drinking stratification analysis was also completed to evaluate the association between CYP19A1 loci and IS risk. In the results of smoking stratification, variant rs28757157 was found to be significantly associated with IS risk in smokers and non-smokers. Meanwhile, a significant association was observed between rs59429575 and IS risk in the allele (OR: 1.48, padj = 0.011), codominant (OR: 1.97, padj = 0.000), dominant (OR: 1.94, padj = 0.000) and log-additive (OR: 1.70, padj = 0.001) models.

Table 5 The Association Between CYP19A1 Polymorphisms and Ischemic Stroke Risk (Smoking and Drinking Stratified Analysis)

In the results of drinking stratification, rs28757157 was related to the risk of IS (allele C: OR: 1.36, padj = 0.026; Heterozygous C/T: OR: 1.36, padj = 0.026; dominant C/T-C/C: OR: 1.45, padj = 0.025; log-additive: OR: 1.35, padj = 0.042) in drinkers. The variant-rs59429575 provided an increased risk for IS in the allele (OR: 1.48, padj = 0.010), codominant (OR: 1.87, padj = 0.001), dominant (OR: 1.87, padj = 0.001) and log-additive (OR: 1.65, padj = 0.002) models. However, the two variants were not found to be associated with IS risk in drinkers and non-drinkers.

Association Between CYP19A1 Polymorphisms and Ischemic Stroke Risk (Clinical Information Analysis)

In Table 6, we further performed comparative analyses of hypertensive patients versus non-hypertensive patients, coronary heart disease patients versus non-coronary heart disease patients, and diabetic patients versus non-diabetic patients in the case group. In the patients with and without diabetes, we found that rs28757157 was associated with an increased risk of IS in the allele (OR: 0.66, padj = 0.025), dominant (OR: 0.65, padj = 0.047) and log-additive (OR: 0.65, padj = 0.023) models. However, the loci did not show a significant association with IS risk in patients with hypertension or coronary heart disease.

Table 6 The Association Between CYP19A1 Polymorphisms and Ischemic Stroke Risk (Clinical Information Analysis)

SNP-SNP Interaction Models of Candidate SNPs Analyzed by the MDR Software

Then, MDR software was used to analyze the impact of potential SNP–SNP interaction on IS risk (Supplementary Table 4). The three-locus model containing CYP19A1 loci (rs28757157, rs3751591 and rs59429575) was regarded as the best model for predicting the impact of SNP–SNP interactions on IS risk (cross-validation consistency 10/10, testing balanced accuracy 54.07%, OR: 1.37 (1.33–2.07 at 95% CI), p < 0.000). In Figure 2, the interaction between rs28757157 and rs3751591 was synergistic, with the information gain value 0.35%.

Figure 2 The circle graph showed that the interaction between rs28757157 and rs3751591 was synergistic, with the information gain value 0.35%.

Discussion

In the research, CYP19A1 loci (rs28757157 and rs3751591) were associated with the occurrence of IS. The two variants conferred an increased susceptibility to IS in the subjects aged over 60 years old, smokers and drinkers. Rs28757157 was related to the risk of IS in females, non-smokers and subjects with BMI less than 24, while rs59429575 was related to the risk of IS in males and subjects with BMI greater than 24. The findings suggest that CYP19A1 loci (rs28757157 and rs3751591) may affect IS risk in individuals from China.

Previous researchers have detected the expression of CYP450 aromatase in the human cerebral cortex,16 and even found that it can regulate the metabolism of androgen and estrogen. For example, aromatase encoded by CYP19A1 played an important role in androgen metabolism.17 Gaella et al18 found that Cyp19a1 was post-transcriptionally upregulated in Celf1−/− testis, resulting in high aromatase activity. And excess aromatase partly caused defects in spermatogenesis. In addition, aromatase encoded by CYP19A1 converts testosterone to estradiol. There is increasing evidence that local estrogen synthesized by aromatase can also prevent cerebral ischemic injury. Among them, the aromatase encoded by CYP19A1 was thought to be beneficial to the injured brain.12,19 Compared with young male rodents, young female rodents were less possibility to develop cerebral injury, because estrogen played a major role in neuroprotection.20,21 Also, CYP19A1 SNPs were also related to the occurrence of nervous system diseases, such as Alzheimer’s disease15 and astrocytoma.22 In addition, Zhang et al23 observed the relationship between CYP19A1 loci (rs4646 and rs17601876) and a reduced risk of stroke. Although the above loci were not found to be associated with the risk of stroke in our study, we first found that CYP19A1 loci (rs28757157 and rs3751591) conferred an increased susceptibility to IS.

Previous studies have found that the prevalence of stroke gradually increases with age and that stroke patients are more common in the elderly population.24 Gender is another key factor in the occurrence of stroke. According to the subgroup analysis results of age, we observed that rs28757157 and rs59429575 showed an increased association with IS risk in people older than 60 years old. However, the two loci were not found to be related to IS stroke in people younger than 60 years old. Besides, our research showed that rs28757157 was related to the risk of IS in females, while rs59429575 was related to the risk of IS in males. Therefore, our study suggested that the effect of CYP19A1 polymorphisms (rs28757157 and rs59429575) on IS risk was age- and sex-dependent, but the underlying mechanism remains unclear.

Smoking is a risk factor for ischemic stroke. Previous studies have shown a strong dose-dependent relationship between smoking and IS risk.25 As cigarette consumption increased, the incidence of stroke increased linearly. According to the stratified analysis of smoking and alcohol consumption, CYP19A1 loci (rs28757157 and rs3751591) were associated with the occurrence of IS in smokers. Combined with the results of gender stratification, we found that rs28757157 was related to the risk of is in females, while rs59429575 was related to the risk of is in males. We believed that this may be closely related to participant’s living habits, such as smoking. With the change of people’s life concept, drinking has become an indispensable way of communication in the process of interpersonal communication. Alcohol consumption is also the risk factor for ischemic stroke. Ischemic stroke risk increased with increasing frequency of alcohol consumption.26 According to the stratified analysis of alcohol consumption, CYP19A1 loci (rs28757157 and rs3751591) were associated with the occurrence of IS in drinkers. The above correlation obtained was dependent on drinking and smoking. Thus, this may be the reason why we strongly advocate people to actively quit smoking and alcohol.

In the present study, we observed that CYP19A1 loci (rs28757157 and rs3751591) may be a risk factor for ischemic stroke. However, this study has some shortcomings. First, the selected samples are mainly from one hospital, and the sample size is not large. We further expand the sample size from different hospitals in the follow-up study to make the obtained results credible. Second, the role of CYP19A1 and its sites in ischemic stroke is unknown. Molecular experiments need to be further explored to provide a theoretical basis for the mechanism of CYP19A1 in ischemic stroke.

Data Sharing Statement

The data used to support the findings of this study are available from the corresponding author upon request.

Human and Animal Ethics

This study was approved by the ethics committee of Haikou People’s Hospital. All participants signed informed consent forms before participating in this study.

Ethics Approval and Consent to Participate

This study was approved by the ethics committee of Haikou People’s Hospital and conformed to the ethical principles of the Declaration of Helsinki. All participants signed informed consent forms before participating in this study.

Acknowledgments

We thank all authors for their contributions and supports. We are also grateful to all subjects for providing blood samples. Kang Huang and Tianyi Ma are the co-first authors. Jianghua Zhong and Shijuan Lu are the co-corresponding authors.

Funding

The study was supported by the specific research fund of The Innovation Platform for Academicians of Hainan Province (YSPTZX202032).

Disclosure

The authors have no conflict of interest.

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