Introduction

Stroke is the leading cause of death and disability worldwide (Feigin et al. 2015; Hankey 2017). In China, stroke is currently the leading cause of death and contributes to a heavy disease burden. The National Epidemiological Survey of Stroke in China (NESS-China), which was conducted in 2012–2013 and involved 480,687 individuals (aged ≥20 years) from 31 provinces, reported age-standardized prevalence, incidence, and mortality rates for stroke of 1115 per 100,000 people, and 247 and 115 per 100,000 person-years, respectively (Wang et al. 2017). Stroke burden in China has increased over the past 30 years, and remains particularly high in rural areas (Wu et al. 2019). Ischemic stroke (IS) is the most common type, accounting for 80% of all cases of stroke. Epidemiological studies have shown that older age, smoking, alcohol intake, hypertension, diabetes mellitus, hyperlipidemia, atrial fibrillation, and low sex hormones are major risk factors for IS (Boehme et al. 2017; Meschia and Brott 2018). In addition to the environmental factors, genetic factors have been demonstrated to play an important role in the pathology of IS (Boehme et al. 2017). Many genes associated with IS risk have been investigated, revealing numerous significant associations, such as variants in ALOX5 (He et al. 2016), AHSG (Li et al. 2018), and MMP-2 (Niu et al. 2018).

Cytochrome P450, family 2, subfamily B, polypeptide 6 (CYP2B6) is a member of the cytochrome P450 superfamily, which plays a vital role in the degradation of various endogenous metabolites, including therapeutic drugs, carcinogens, and other environmental chemicals, as well as many endogenous genotoxic compounds (Hodgson and Rose 2007; Lamba et al. 2003; Wang et al. 2006). In addition, it was found that CYP2B6 expression is brain region-specific, and was observed in both neurons and astrocytes (Miksys et al. 2003). One of the polymorphisms (516G > T) in exon 4 the of CYP2B6 gene is associated with a pronounced decrease in gene expression and CYP2B6 activity in the liver (Hofmann et al. 2008). The genetic variants in CYP2B6 are known to decrease the activity of the CYP2B6 enzyme, contributing to an increased breast cancer risk (Justenhoven et al. 2014). Daraki et al. suggested that the CYP2B6 polymorphism (785A > G) and specific CYP2B6 haplotypes may contribute to acute myeloid leukemia and its specific chromosomal aberrations (Daraki et al. 2014). Moreover, several studies have shown that polymorphisms of the CYP2B6 gene were associated with the risk of prostate cancer (Kurosaki et al. 2009), Hirschsprung disease (Xu et al. 2015), and bronchopulmonary dysplasia in preterm neonates (Zachaki et al. 2017).

However, little is known about the association between the CYP2B6 polymorphisms and the risk of IS. Therefore, we carried out a case–control study including 477 patients with IS and 495 healthy controls to investigate the relationship between CYP2B6 polymorphisms (rs2099361, rs4803420, rs1038376, and rs12979270) and the susceptibility to IS in a Chinese Han population. The effects of the genetic polymorphisms of CYP2B6 on risk of IS were investigated which may provide new insights into understanding the mechanisms of IS.

Materials and Methods

Study Subjects

A total of 477 patients with IS from the Haikou Hospital Affiliated to Xiangya School of Medicine, Central South University, were enrolled in the study. The IS patients had been newly diagnosed and confirmed according to the International Classification of Disease using brain computed tomography (CT) and magnetic resonance imaging (MRI) combined with history interview and neurological examination. The 495 healthy controls were randomly recruited from people who requested general health examinations in the same hospital during the same period. Inclusion and exclusion criteria of participants were as follows: All participants were of a genetically unrelated Chinese Han population; Patients with a history of other types of stroke (hemorrhagic stroke, subarachnoid hemorrhage, and transient ischemic attack), traumatic brain injuries, cerebral vein thrombosis, or brain aneurysm were excluded in this study.

Medical records were used to collect information on patient demographic characteristics (age, gender, and ethnicity) and clinical indicators including fasting serum levels of total protein, total bilirubin, direct bilirubin, indirect bilirubin, uric acid, total cholesterol, triglyceride, high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), glucose, white blood cell (WBC) count, neutrophil absolute value (NEUT), eosinophils absolute value (EO), basophilic absolute value (BASO), platelet count, plateletcrit, red blood cell (RBC) count, hemoglobin (HGB), erythrocyte mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), red blood cell distribution width (RDW), and red blood cell distribution width coefficient of variation (RDW-CV) were collected from medical records review.

DNA Extraction

Vacutainers containing ethylene diamine tetraacetic acid (EDTA) were used to collected peripheral venous blood samples (5 ml) from IS patients and controls, which were then stored at −80 °C in a refrigerator for DNA extraction. We used the GoldMag-Mini Whole Blood Genomic DNA Purification Kit (GoldMag Co., Ltd., Xi’an City, China) to extract DNA from whole blood, according to the manufacturer’s instructions. We used a spectrophotometer (NanoDrop 2000; Thermo Fisher Scientific, Waltham, MA, USA) to determine the purity and concentration of the extracted DNA and for sample quality control.

SNP Selection and Genotyping

We selected the tag SNPs of the CYP2B6 gene with a minor allele frequency (MAF) >5% in the global population from the 1000 Genomes Project. Four SNPs (rs2099361 (C > A, intron), rs4803420 (G > T, 3′ prime UTR), rs1038376 (A > T, 3′ prime UTR), and rs12979270 (A > C, 3′ prime UTR)) were selected using a pairwise Tagger method with r2 > 0.8 to capture other SNPs. We used the online software Agena Bioscience Assay Design Suite version 2.0 (https://agenacx.com/online-tools/) to design the primers. SNP genotyping was performed using the Agena MassARRAY platform with iPLEX Gold chemistry (Agena Bioscience, San Diego, CA, USA) according to the protocol described. Data management and analysis of genotyping results were conducted using Agena Bioscience Typer software (version 4.0).

Statistical Analysis

Microsoft Excel (Microsoft Corp., Redmond, WA, USA) and SPSS version 20.0 statistical software (IBM Corp., Armonk, NY, USA) were used for statistical analysis. We used the chi-squared test to evaluate the Hardy–Weinberg equilibrium of SNP genotype frequencies in the controls and the differences between allelic frequencies of these polymorphisms in the case and control groups. We evaluated the association between polymorphism genotype and IS risk by logistic regression to calculate odds ratios (ORs) and 95% confidence intervals (CIs) under four genetic models (codominant, dominant, recessive, and additive). Haploview 4.2 software was used to conduct linkage disequilibrium (LD) haplotype block and calculate the linkage strength between each pair of SNPs with D′ and R-squared values. The association between haplotypes and IS risk was assessed by logistic regression. P values less than 0.05 were considered statistically significant, and all statistical tests were two-sided.

Results

Demographic and Clinical Characteristics

This case–control study recruited 477 IS patients and 495 healthy controls. The demographic and clinical characteristics of the subjects are listed in Table 1. There were no significant differences in gender between case (316 male and 161 female) and control (326 male and 169 female) study populations (P = 0.898). There was a significant difference in age between the IS patients (mean ± SD: 64.1 ± 10.8) and controls (mean ± SD: 60.1 ± 6.6; P < 0.001). There were no significant differences in HDL (P = 0.879) or MCV (P = 0.658) between patients and controls. There were also significant differences in biochemistry and blood test indicators (total protein, total bilirubin, direct bilirubin, indirect bilirubin, uric acid, total cholesterol, LDL-C, glucose, WBC, NEUT, EO, BASO, platelet count, plateletcrit, RBC, HGB, MCH, RDW, and RDW%) between cases and controls (P < 0.05). At the same time, we found that the mean direct bilirubin, glucose, BASO, plateletcrit, RBC, MCH, and RDW% levels in the case group were higher than the clinical reference values and those of the control group, suggesting that these factors may increase the risk of IS.

Table 1 Characteristics of case and control subjects
Full size table

Overall Analysis of CYP2B6 Polymorphisms and IS Risk

Basic information regarding the SNPs in the CYP2B6 gene, including SNP ID, chromosome, position, minor and major alleles, MAF and HWE-p, is presented in Table 2. The call rate of genotyping was more than 95%. The genotype distribution of CYP2B6 polymorphisms (rs2099361, rs4803420, rs1038376, rs12979270) were in accordance with HWE in the control group (P > 0.05). However, there were no significant differences in the allelic distribution of the four SNPs in CYP2B6 between the patients and control group (P > 0.05). No significant association was found between CYP2B6 polymorphisms and the risk of IS under the allele model (Table 2).

Table 2 Alleles of CYP2B6 polymorphisms and association with ischemic stroke risk
Full size table

We performed genetic model analyses (codominant, dominant, recessive, and additive) to further explore the association between CYP2B6 polymorphisms and the risk of IS (Table 2). The CC genotype of rs2099361 was significantly associated with an increased risk of IS compared with the wild homozygous AA genotype before and after adjusting for age and gender (crude OR = 1.87, 95% CI: 1.19–2.93, P = 0.007; adjusted OR = 1.85, 95% CI: 1.16–2.93, P = 0.010). In the recessive model, the CC genotype was also found to be significantly associated with an increased the risk of IS compared with the AA/CA genotype (crude OR = 1.89, 95% CI: 1.23–2.92, P = 0.004; adjusted OR = 1.81, 95% CI: 1.16–2.83, P = 0.009). The GT genotype of rs4803420 was associated with a decreased risk of IS compared with the TT genotype both before and after adjusting for age and gender (crude OR = 0.75, 95% CI: 0.57–0.98, P = 0.033; adjusted OR = 0.74, 95% CI: 0.57–0.98, P = 0.036). The other two SNPs (rs1038376 and rs12979270) were not found to be associated with IS risk under the four genetic models (Table 3).

Table 3 Genetic analysis of CYP2B6 polymorphisms and ischemic stroke risk
Full size table

Stratification Analysis of CYP2B6 Polymorphisms and IS Risk

Age stratification analysis showed that rs2099361 was associated with an increased risk of IS for age ≤ 60 years (CC vs. AA: OR = 1.94, 95% CI: 1.02–3.70, P = 0.045; recessive CC vs. AA/CA: OR = 1.90, 95% CI: 1.03–3.53, P = 0.041), while rs1038376 was associated with a decreased risk of IS for age ≤ 60 years (AT vs. TT: OR = 0.63, 95% CI: 0.40–0.99, P = 0.046) (Table 4). In addition, we found that rs4803420 was significantly associated with reduced IS risk in those aged > 60 years (G vs. T: OR = 0.69, 95% CI: 0.50–0.93, P = 0.015; GT vs. TT: OR = 0.56, 95% CI: 0.37–0.86, P = 0.008; dominant GT/GG vs. TT: OR = 0.61, 95% CI: 0.40–0.92, P = 0.017). Stratification analysis based on gender indicated that rs2099361 was significantly associated with an increased risk of IS in male (CC vs. AA: OR = 2.17, 95% CI: 1.22–3.86, P = 0.009; recessive CC vs. AA/CA: OR = 2.05, 95% CI: 1.18–3.57, P = 0.011; additive: OR = 1.33, 95% CI: 1.04–1.70, P = 0.024). However, rs4803420 was associated with an increased risk of IS in women (GG vs. TT: OR = 2.31, 95% CI: 1.00–5.31, P = 0.049; GG vs. TT/GT: OR = 2.39, 95% CI: 1.06–5.37, P = 0.035) (Table 4). These results suggest that age and gender are important factors influencing the risk of IS.

Table 4 Stratification analysis of CYP2B6 polymorphisms and ischemic stroke risk
Full size table

LD and Haplotype Analysis

Linkage analysis indicated that there was strong linkage disequilibrium among the three SNPs (rs4803420, rs1038376, and rs12979270) in the CYP2B6 gene, as shown in Fig. 1. The results of the association between the haplotypes and IS risk are listed in Table 5. However, no association was found between the haplotypes (GAC, GTA, TAA, and GAA) and risk of IS before and after adjusting for age and gender.

Fig. 1
figure 1

Haplotype block map for the four SNPs in CYP2B6. A standard color frame is used to show LD pattern. One block in the figure shows higher LD. The value in each cell is the value of D′ (multiplied by 100). Bright red represents very strong LD. Pink represents intermediate LD

Full size image
Table 5 Association between CYP2B6 haplotypes and ischemic stroke risk
Full size table

SNPs and Clinical Indicators

To further explore the impact of SNPs in CYP2B6 on the risk of stroke, we compared the levels of clinical indicators under different genotypes of CYP2B6 polymorphisms (Table 6). We found that the level of I-BIL levels was significantly different between the three genotypes (AA: 6.28 ± 3.45 umol/L; CA: 5.37 ± 3.39 umol/L; CC: 6.36 ± 3.29 umol/L) of rs2099361 (P = 0.025). Relative to the three genotypes (TT, GT, and GG) of rs4803420, statistically significant differences were found for the levels of BASO (P = 0.009), plateletcrit (P = 0. 030), RBC (P = 0.011), MCV (P = 0.031), MCH (P = 0.021), and RDW (P = 0.007) in IS patients. Moreover, there was a significant difference in the distribution of patient glucose levels between the three genotypes (TT: 6.3 ± 2.13 mmol/L; AT: 6.19 ± 1.76 mmol/L; AA: 9.35 ± 7.11 mmol/L) of rs1038376 (P = 0.001).

Table 6 Distribution differences of clinical indicators in different genotypes
Full size table

Discussion

This study investigated the association between SNPs in CYP2B6 and IS risk in a Chinese Han population. The results demonstrated that the rs2099361 was associated with an increased risk of IS overall and for age ≤ 60 years and male gender. The GT genotype of rs4803420 was associated with a reduced risk of IS overall and for age > 60 years, but the GG genotype was found to be associated with increased IS risk in women. The AT genotype of rs1038376 was associated with a reduced IS risk in those aged ≤ 60 years. Notably, there was significant difference in the distribution of clinical indicator levels in patients between the case and control groups, and genotypes of each SNP.

CYP2B6, a member of the human CYP family of pharmacogenes expressed mainly in the liver, accounts for 2–10% of total hepatic CYP content and contributes to the biotransformation of many drugs and xenobiotics (Wang and Tompkins 2008; Zanger and Klein 2013). It was estimated that CYP2B6 is involved in the metabolism of 4% of the top 200 drugs (Zanger et al. 2008). CYP2B6 is also expressed in the brain and may play an important role in the metabolism of drugs by acting on the central nervous system, resulting in neurological side effects of drug treatments (Miksys and Tyndale 2004; Thorn et al. 2010). SNPs are the most common form of genetic variation in CYP genes (Daly 2004). The CYP2B6 gene is highly polymorphic, with a large number of allelic variants (http://www.cypalleles.ki.se/cyp2b6.htm). High inter-individual variation is seen in CYP2B6 mRNA expression and activity, ranging from 20- to 250-fold, which is considered to be attributable mainly to the highly inducible and polymorphic nature of this gene (Code et al. 1997; Lamba et al. 2003; Lang et al. 2001).

Our study is first to find that rs2099361, rs4803420, and rs1038376 are significantly associated with the risk of IS. Interestingly, we found that the mean levels of direct bilirubin, glucose, BASO, plateletcrit, RBC, MCH, and RDW-CV in the case group were higher than those in the control group and the normal values. Moreover, the mean levels of I-BIL, BASO, plateletcrit, RBC, MCV, MCH, and RDW, and the glucose levels showed statistically significant differences between the genotypes of rs2099361, rs4803420, and rs1038376. Bilirubin is the ultimate product of heme metabolism, and when accumulated with high concentrations in tissues, it becomes highly toxic. Perlstein et al. demonstrated that higher total serum bilirubin levels were associated with reduced stroke prevalence and improved stroke outcomes (Perlstein et al. 2008). However, Arsalan et al. found that higher serum bilirubin levels were associated with increased stroke severity and poor prognosis in patients (Arsalan et al. 2011). Blood glucose is often elevated in acute stroke, and higher admission glucose levels are associated with larger lesions, greater mortality, and poorer functional outcome (Olsen 2009). One study reported that plateletcrit was significantly correlated with poor outcome of acute ischemic stroke (Mohamed et al. 2019). Panwar et al. demonstrated that lower and higher hemoglobin concentrations were associated with a higher risk of incident stroke in women (Panwar et al. 2016). RDW was found to be associated with the risk of IS and outcome in patients (Lappegard et al. 2016; Turcato et al. 2017). Therefore, these findings should be confirmed with detailed association studies in larger samples.

Although the results are promising, some limitations of our study should be mentioned. First, the small sample size in our study may result in insufficient power to detect the association of the CYP2B6 polymorphisms with IS risk. Therefore, further detailed association studies in larger samples are needed to confirm these findings. Second, this study did not take into consideration the association of conventional IS risk factors, such as smoking, alcohol intake, hypertension, or diabetes mellitus, with CYP2B6 polymorphisms. Third, other SNPs in CYP2B6 were not assessed for their potential association with IS risk. Finally, this study did not elucidate the specific mechanism of the CYP2B6 polymorphisms that affect the risk of IS.

Conclusion

In conclusion, our results indicated that polymorphisms (rs2099361, rs4803420, and rs1038376) in CYP2B6 were significantly associated with the risk of IS in a Chinese Han population. The results provide evidence that CYP2B6 polymorphisms may be a genetic determinant of IS susceptibility. However, this is the first study to report this phenomenon, and it is unclear whether these findings are reproducible in other populations. Therefore, further detailed association and functional studies in larger samples are needed to validate the association between CYP2B6 polymorphisms and risk of IS.