Diplotypes of CYP2C9 gene is associated with coronary artery disease in the Xinjiang Han population for women in China

  • Zhenyan Fu1,

    Affiliated with

    • Qing Zhu1,

      Affiliated with

      • Yitong Ma1Email author,

        Affiliated with

        • Ding Huang1,

          Affiliated with

          • Shuo Pan1,

            Affiliated with

            • Xiang Xie1,

              Affiliated with

              • Fen Liu1 and

                Affiliated with

                • Erdenbat Cha1

                  Affiliated with

                  Contributed equally
                  Lipids in Health and Disease201413:143

                  DOI: 10.1186/1476-511X-13-143

                  Received: 29 April 2014

                  Accepted: 12 August 2014

                  Published: 2 September 2014

                  Abstract

                  Background

                  Cytochrome P450 (CYP) 2C9 is expressed in the vascular endothelium and metabolizes arachidonic acid to biologically active epoxyeicosatrienoic acids (EETs), which have the crucial role in the modulation of cardiovascular homeostasis. We sought to assess the association between the human CYP2C9 gene and coronary artery disease (CAD) in Xinjiang Han Population of China.

                  Methods

                  301 CAD patients and 220 control subjects were genotyped for 4 single-nucleotide polymorphisms (SNPs) of the human CYP2C9 gene (rs4086116, rs2475376, rs1057910, and rs1934967) by a Real-Time PCR instrument. The datas were assessed for 3 groups: total, men, and women via diplotype-based case–control study.

                  Results

                  For women, the distribution of genotypes, dominant model and alleles of SNP2 (rs2475376) showed significant difference between the CAD patients and control participants (p = 0.033, P = 0.010 and p = 0.038, respectively). The significant difference of the dominant model (CC vs CT + TT) was retained after adjustment for covariates in women (OR: 2.427, 95% confidence interval [CI]: 1.305-4.510, p = 0.005). The haplotype (C-T-A-C) and the diplotypes (CTAC/CTAC) in CYP2C9 gene were lower in CAD patients than in control subjects (p* = 0.0016, and p* = 0.036 respectively). The haplotype (C-C-A-T) was higher in the CAD patients than in the control subjects in women (p* = 0.016).

                  Conclusions

                  CC genotype of rs2475376 and C-C-A-T haplotype in CYP2C9 may be a risk genetic marker of CAD in women. T allele of rs2475376, the haplotype (C-T-A-C) and the diplotype (CTAC/CTAC) could be protective genetic markers of CAD for women in Han population of China.

                  Keywords

                  CYP2C9 Single-nucleotide polymorphism Haplotype Diplotype Case–control study

                  Introduction

                  CAD is a complex multifactorial and polygenic disorder thought to result from an interaction between an individual's genetic makeup and different environments [1]. Increasing evidence from animals and clinical and epidemiological studies has repeatedly supported the likelihood of a genetic contribution to CAD susceptibility [2, 3]. Cytochrome P450 (CYP) genes is a super family of cysteine-heme enzymes, which catalyze the oxidation of various drugs and endogenous substrates, such as vitamin D, steroids, and fatty acids, including arachidonic acid (AA) [4]. CYP enzymes of the P450 2C9 subfamily are found in the liver, vascular smooth muscle, endothelial cells of human aorta and coronary artery [57]. In human liver, CYP2C9 is responsible for 50% of the epoxygenase activity, and metabolizes a wide variety of clinically important drugs, including losartan and S-warfarin [8, 9]. In human heart, CYP2C9, as well as CYP2C8 and CYP2J2, participates in metabolizing arachidonic acid to epoxyeicosatrienoic acids (EETs) [10, 11]. EETs are supposed to play the key role in endothelial cell homeostasis, showing protective vascular effects including vasodilatation, anti-inflammtory, anti-apoptotic and anti-thrombotic effects [1214]. They are also involved in myocardial preconditioning and have cardioprotective effects by increasing postischemic function and reducing myocardial infarct size [15, 16]. Genetic polymorphisms might affect the activity of EETs, which determine susceptibility to the development of CAD. In recent years, many studies have shown the polymorphisms of CYP2C9 gene (rs1057910) were associated with the cardiovascular risk [17, 18]. Given this background, we sought to investigate the possible association between the genetic variation of CYP2C9 and CAD in Xinjiang Han population of China.

                  Methods

                  Ethical approval of the study protocol

                  This study was approved by the Ethics Committee of the First Affiliated Hospital of Xinjiang Medical University (Urumqi, China). It was conducted according to the standards of the Declaration of Helsinki. Written informed consent was obtained from all participants. All participants explicitly provided permission for DNA analyses as well as collection of relevant clinical data.

                  Subjects

                  All patients and controls were enrolled from the First Affiliated Hospital of Xinjiang Medical University (Urumqi, China) from January 2011 to April 2013. The study involved 301 patients with CAD defined as the presence of at least one significant coronary artery stenosis of more than 50% luminal diameter on coronary angiography. 220 Control participants did not have coronary artery stenosis and did not show clinical or electrocardio-graphic evidence of myocardial infarction (MI) or CAD. Data and information about traditional coronary risk factors, including hypertension, diabetes mellitus (DM), and smoking, were collected from all study participants. The diagnosis of hypertension was established if patients were on antihypertensive medication or if the mean of 3 measurements of systolic blood pressure (SBP) >140 mmHg or diastolic blood pressure (DBP) >90 mmHg, respectively. Diabetes mellitus was defined by fasting plasma glucose >7.0 mmol/L and also if patients were taking antidiabetic medication or insulin therapy. “Smoking” was classified as smokers (including current or ex-smokers) or non-smokers. All patients with impaired renal function, malignancy, connective tissue disease, or chronic inflammatory disease were excluded.

                  Blood collection and DNA extraction

                  Blood samples were taken from all participants. The blood samples were drawn into a 5 ml ethylene diamine tetraacetic acid (EDTA) tube and centrifuged at 4000 × g for 5 min to separate the plasma content. Genomic DNA was extracted from the peripheral leukocytes using standard phenol–chloroform method. The DNA samples were stored at -80°C until use. When used, the DNA was diluted to 50 ng/ul concentration.

                  Genotyping

                  There are 1375 SNPs for the human CYP2C9 gene listed in the National Center for Biotechnology Information SNP database (http://​www.​ncbi.​nlm.​nih.​gov/​SNP). Using the Haploview 4.2 software and the HapMap phrase II database, we obtained four tag SNPs (rs4086116, rs2475376, rs1057910 and rs1934967) by using minor allele frequency (MAF) > =0.01 and linkage disequilibrium patterns with r2 > =0.5 as a cut off. We designated these SNPs as SNP1, SNP2, SNP3, and SNP4 (rs4086116, rs2475376, rs1057910 and rs1934967) in order of increasing distance from the CYP2C9 gene 5′end (Figure 1). SNP1, SNP2, and SNP4 are located in intron. SNP3(rs1057910) is located in exon7, and had a non-synonymous substitution amino acid change, which is defined by an A-to-C nucleotide substitution that leads to an exchange of leucine by isoleucine at amino acid position 359.
                  http://static-content.springer.com/image/art%3A10.1186%2F1476-511X-13-143/MediaObjects/12944_2014_Article_1157_Fig1_HTML.jpg
                  Figure 1

                  Structure of the human CYP2C9 gene. This gene consists of 9exons separated by 8 introns. Boxes indicate exons, and lines indicate introns and intergenic regions. Filled boxes indicate coding regions. Arrows mark the locations of polymorphisms.

                  Genotyping was undertaken using the TaqMan® SNP Genotyping Assay (Applied Biosystems) using Taq amplification, TaqMan® SNP Genotyping Assays were carried out. The primers and probes used in the TaqMan® SNP Genotyping Assays (ABI) were chosen based on information at the ABI website (http://​myscience.​appliedbiosystem​s.​com). Thermal cycling was done using the AppliedBiosystems 7900HT Fast Real-Time PCR System. Plates were read on Sequence Detection Systems (SDS) automation controller software v2.3 (ABI).PCR amplification was performed using 3.0 μl of TaqMan Universal Master Mix, 0.15 μl probes and 1.85 ddH2O in a 6-μl final reaction volume containing 1 μl DNA. Thermal cycling conditions were as follows: 95°C for 5 min; 40 cycles of 95°C for 15 s; and 60°C for 1 min. All 96 wells Plates were read on Sequence Detection Systems (SDS) automation controller software v2.3 (ABI).

                  Biochemical analysis

                  Serum concentrations of total cholesterol (TC), triglyceride (TG), glucose, high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), blood ureanitrogen (BUN), creatinine (Cr) and uric acid were measured using standard methods in the Central Laboratory of the First Affiliated Hospital of Xinjiang Medical University as described previously.

                  Statistical analysis

                  All continuous variables (e.g. age, BMI, cholesterol levels) are presented as means ± standard deviation (S.D.). The continuous variables conform to normal distribution, and the study is a large sample data, so the differences of all continuous variables between the CAD and the Control groups were analyzed using T-test. The differences in the frequencies of smoking, hypertension, DM, and CYP2C9 genotypes were analyzed using Fisher’s exact test. Chi-square analysis was used to test the deviations of genotype distribution from the Hardy-Weinberg equilibrium and to determine the differences of allele or genotype frequencies between patients and controls. Logistic regression analyses were used to assess the contribution of the major risk factors. And adjusted estimations of conditioned relative risk and 95 % confidence intervals (CIs) were done. All statistical analyses were performed using SPSS 17.0 for Windows (SPSS Institute, Chicago, USA). P-values were considered to be significant at the 0.05 level. Haplotypes were estimated using the expectation maximization algorithm and the software SNPAlyze version 3.2 (Dynacom, Yokohama, Japan), and using the SHEsis platform to verify reliability [19, 20]. The estimated diplotypes (combinations of two haplotypes) in each subject were analyzed using the software SNPAlyze version 3.2 (Dynacom, Yokohama, Japan), The P value of haplotype and diplotype were revised by False discovery rate.

                  Results

                  Table 1 showed the clinical characteristics of the CAD patients (n = 301) and control participants (n = 220). For total, men and women subjects, there was no significant difference in age between CAD patients and control subjects. It meant the study was age-matched case–control study. We observed several differences between the two groups of patients. As expected, several common risk factors for CAD were significantly different between the two subgroups: Glu, low HDL-C, high LDL-C, EH, DM. For total, the serum concentrations of glucose (Glu), LDL-C were significantly higher for CAD patients than for control participants (p < 0.05), and the serum concentrations of HDL-C were significantly lower for CAD patients than for control participants (p < 0.05). The prevalence of DM was significantly higher for patients with CAD than for control participants. For men, the serum concentration of LDL-C was significantly higher for CAD patients than for control participants (p < 0.05). The prevalence of EH, DM, and smoking were significantly higher for patients with CAD than for control participants. For women, the prevalence of EH and DM were significantly higher for patients with CAD than for control participants.
                  Table 1

                  Characteristics of study participants

                    

                  Total

                    

                  Men

                    

                  Women

                   
                   

                  CAD patients

                  Control subjects

                  p Value

                  CAD patients

                  Control subjects

                  p Value

                  CAD patients

                  Control subjects

                  p Value

                  Number (n)

                  301

                  220

                   

                  202

                  126

                   

                  99

                  94

                   

                  Age (years)

                  59.13 ± 8.97

                  57.64 ± 8.78

                  0.092

                  60.73 ± 9.12

                  58.66 ± 8.43

                  0.077

                  62.53 ± 8.45

                  61.39 ± 7.81

                  0.364

                  BMI (kg/m2)

                  25.74 ± 3.39

                  25.44 ± 3.51

                  0.353

                  25.16 ± 5.16

                  25.46 ± 3.81

                  0.591

                  24.68 ± 6.32

                  25.58 ± 5.01

                  0.294

                  Pulse (beats/min)

                  74.05 ± 10.13

                  74.13 ± 11.58

                  0.933

                  74.34 ± 10.84

                  74.53 ± 11.04

                  0.874

                  73.46 ± 8.52

                  73.59 ± 12.30

                  0.937

                  BUN(mmol/L)

                  5.46 ± 1.58

                  5.53 ± 1.63

                  0.620

                  5.67 ± 2.12

                  5.55 ± 1.70

                  0.627

                  5.40 ± 1.83

                  5.36 ± 1.51

                  0.855

                  Cr(umol/L)

                  75.07 ± 20.99

                  74.09 ± 22.71

                  0.625

                  77.50 ± 18.01

                  76.05 ± 10.00

                  0.790

                  74.54 ± 12.90

                  79.26 ± 10.66

                  0.762

                  Glu (mmol/L)

                  6.10 ± 2.04

                  5.73 ± 1.91

                  0.043*

                  6.03 ± 1.99

                  5.65 ± 1.92

                  0.096

                  6.32 ± 2.53

                  5.84 ± 1.89

                  0.139

                  TG (mmol/L)

                  1.75 ± 1.02

                  1.81 ± 1.23

                  0.545

                  1.71 ± 0.978

                  1.79 ± 1.35

                  0.498

                  1.94 ± 1.46

                  1.83 ± 1.01

                  0.306

                  TC (mmol/L)

                  4.09 ± 1.08

                  4.24 ± 0.98

                  0.137

                  4.05 ± 1.70

                  4.14 ± 1.01

                  0.595

                  4.38 ± 1.07

                  4.38 ± 0.93

                  0.430

                  HDL (mmol/L)

                  1.04 ± 0.37

                  1.15 ± 0.33

                  0.001*

                  1.10 ± 0.94

                  1.07 ± 0.289

                  0.737

                  1.08 ± 0.32

                  1.26 ± 0.34

                  0.368

                  LDL (mmol/L)

                  2.44 ± 0.77

                  2..25 ± 0.80

                  0.004*

                  2.52 ± 2.20

                  2.29 ± 1.91

                  0.007*

                  2.36 ± 0.80

                  2.35 ± 0.88

                  0.461

                  EH (%)

                  63.6

                  51.54

                  0.072

                  61.4

                  48.7

                  0.032*

                  65.6

                  54.1

                  0.110*

                  DM (%)

                  19.95

                  10.15

                  0.029*

                  17.8

                  8.9

                  0.043*

                  22.1

                  11.4

                  0.014*

                  Smoke (%)

                  21.32

                  12.56

                  0.143

                  37.6

                  23.0

                  0.008*

                  5.05

                  2.13

                  0.278

                  BMI, body mass index; BUN, blood urea nitrogen; Cr, creatinine; Glu, glucose; TG, triglyceride; TC, total cholesterol; HDL, high density lipoprotein; LDL, low density lipoprotein; EH, essential hypertension; DM, diabetes mellitus.

                  Continuous variable were expressed as mean ± standard deviation. P value of continuous variables was calculated by independent T-T test. The P value of categorical variable was calculated by Fisher's exact test. *P>0.05.

                  Table 2 showed the distribution of genotypes and alleles of SNP1, SNP2, SNP3, and SNP4 of CYP2C9 gene. The genotype distributions for each of the SNPs were in agreement with the predicted Hardy-Weinberg equilibrium values (data not shown). For total, the distribution of the four SNPs genotypes and alleles showed no difference between the CAD patients and control participants. For men, the distribution of the dominant model of SNP2 (rs2475376) (CC vs CT + TT) was higher in CAD patients than in control participants (p = 0.045). For women, the distribution of genotypes, of SNP2 (rs2475376) showed significant difference between the CAD patients and control participants (p = 0.033). The dominant model (CC vs CT + TT) was significantly higher for CAD patients than for control subjects (p = 0.010). The frequency of T allele (rs2475376) was lower for CAD patients than for control subjects (p = 0.038).
                  Table 2

                  Genotype and allele distributions in patients with CAD and control subjects

                   

                  Total

                  Men

                  Women

                  CAD

                  Control

                  p

                  CAD

                  Control

                  p

                  CAD

                  Control

                  p

                   

                  n = 301

                  n = 220

                  n = 202

                  n = 126

                  n = 99

                  n = 94

                   

                  rs4086116

                  Genotype

                   

                  C/C

                  237

                  176

                  0.356

                  158

                  97

                  0.464

                  79

                  79

                  0.559

                  (SNP1)

                    

                  C/T

                  56

                  42

                  39

                  28

                  17

                  14

                     

                  T/T

                  8

                  2

                  5

                  1

                  3

                  1

                    

                  Dominant model

                  CC

                  237

                  176

                  0.726

                  158

                  97

                  0.194

                  79

                  79

                  0.444

                    

                  CT + TT

                  64

                  44

                  44

                  29

                  20

                  15

                    

                  Recessive model

                  TT

                  8

                  2

                  0.151

                  5

                  1

                  0.269

                  3

                  1

                  0.338

                    

                  CT + CC

                  293

                  218

                  197

                  125

                  96

                  93

                   

                  Allele

                   

                  C

                  530

                  394

                  0.449

                  355

                  222

                  0.932

                  175

                  172

                  0.312

                     

                  T

                  72

                  46

                  49

                  30

                  23

                  16

                  rs2475376

                  Genotype

                   

                  C/C

                  104

                  76

                  0.834

                  60

                  51

                  0.122

                  44

                  25

                  0.033*

                  (SNP2)

                    

                  C/T

                  142

                  108

                  102

                  56

                  40

                  52

                     

                  T/T

                  55

                  36

                  40

                  19

                  15

                  17

                    

                  Dominant model

                  CC

                  104

                  76

                  0.999

                  60

                  51

                  0.045*

                  44

                  25

                  0.010*

                    

                  CT + TT

                  197

                  144

                  142

                  75

                  55

                  69

                    

                  Recessive model

                  TT

                  55

                  36

                  0.571

                  40

                  19

                  0.279

                  15

                  17

                  0.584

                    

                  CT + CC

                  246

                  184

                  162

                  107

                  84

                  77

                   

                  Allele

                   

                  C

                  350

                  260

                  0.758

                  222

                  158

                  0.051

                  128

                  102

                  0.038*

                     

                  T

                  252

                  180

                  182

                  94

                  70

                  86

                  rs1057910

                  Genotype

                   

                  A/A

                  237

                  178

                  0.282

                  156

                  96

                  0.76

                  81

                  82

                  0.174

                  (SNP3)

                    

                  A/C

                  49

                  37

                  37

                  26

                  12

                  11

                     

                  C/C

                  15

                  5

                  9

                  4

                  6

                  1

                    

                  Dominant model

                  AA

                  237

                  178

                  0.543

                  156

                  96

                  0.829

                  81

                  82

                  0.229

                    

                  AC + CC

                  64

                  42

                  46

                  30

                  18

                  12

                    

                  Recessive model

                  CC

                  15

                  5

                  0.112

                  9

                  4

                  0.563

                  6

                  1

                  0.063

                    

                  AC + AA

                  286

                  215

                  193

                  122

                  93

                  93

                   

                  Allele

                   

                  A

                  523

                  393

                  0.233

                  349

                  218

                  0.965

                  174

                  175

                  0.082

                     

                  C

                  79

                  47

                  55

                  34

                  24

                  13

                  rs1934967

                  Genotype

                   

                  C/C

                  209

                  152

                  0.126

                  144

                  79

                  0.203

                  65

                  73

                  0.161

                  (SNP4)

                    

                  C/T

                  55

                  61

                  49

                  42

                  6

                  19

                     

                  T/T

                  11

                  7

                  9

                  5

                  2

                  2

                    

                  Dominant model

                  CC

                  209

                  152

                  0.933

                  144

                  79

                  0.105

                  65

                  73

                  0.065

                    

                  CT + TT

                  92

                  68

                  58

                  47

                  34

                  21

                    

                  Recessive model

                  TT

                  11

                  7

                  0.77

                  9

                  5

                  0.832

                  2

                  2

                  0.958

                    

                  CT + CC

                  290

                  213

                  193

                  121

                  97

                  92

                   

                  Allele

                   

                  C

                  499

                  365

                  0.978

                  337

                  200

                  0.19

                  162

                  165

                  0.105

                     

                  T

                  103

                  75

                   

                  67

                  52

                   

                  36

                  23

                   

                  CAD, coronary artery disease.

                  The P value of genotype was calculated by Fisher's exact test.*P>0.05.

                  Table 3 showed that multiple logistic regression analyses were done with or without EH, DM, and smoking. The significant difference of the dominant model (CC vs CT + TT) was retained after adjustment for covariates in women, but not in men (for women, OR: 2.427, 95% confidence interval [CI]: 1.305-4.510, p = 0.005; and for men, OR: 1.372, 95% CI: 0.861-2.186, p = 0.184).
                  Table 3

                  Results of Logistic analysis for the dominant model (CC vs CT + TT) of SNP2

                    

                  Total

                    

                  Men

                    

                  Women

                   
                   

                  OR

                  95% CI

                  p

                  OR

                  95% CI

                  p

                  OR

                  95% CI

                  p

                  CC vs CT + TT

                  1.299

                  0.901-1.873

                  0.161

                  1.372

                  0.861-2.186

                  0.184

                  2.427

                  1.305-4.510

                  0.005*

                  EH

                  1.133

                  0.798-1.609

                  0.486

                  0.981

                  0.627-1.533

                  0.932

                  1.543

                  0.855-2.785

                  0.150

                  DM

                  1.149

                  0.671-1.968

                  0.614

                  1.095

                  0.574-2.089

                  0.783

                  1.186

                  0.425-3.313

                  0.745

                  Somke

                  1.307

                  0.917-1.862

                  0.139

                  0.952

                  0.592-1.530

                  0.839

                  2.905

                  0.536-15.741

                  0.216

                  EH, essential hypertension; DM, diabetes mellitus; CAD, coronary artery disease. * p>0.05.

                  Table 4 showed patterns of linkage disequilibrium in the CYP2C9 gene, with their |D′| and r2 values. |D′| values from 0.7 to 1 indicate strong LD between a pair of SNPs. |D′| values from 0.25 to 0.7 indicate moderate LD and |D′| values of 0–0.25 indicate low LD. In our study, two strong LD patterns were observed between SNP1 and SNP2 (|D′| = 0.998), SNP2 and SNP3 (|D′| = 0.999). Three moderate LD patterns (|D′| values from 0.25 to 0.7) were observed between SNP1 and SNP3 (|D′| = 0.593), SNP1 and SNP4 (|D′| = 0.311), SNP2 and SNP4 (|D′| = 0.392). In addition, a low LD pattern (|D′| < 0.25) was observed between SNP3 and SNP4 (|D′| = 0.032) (Figure 2). Although LD pattern between SNP3 and SNP4 was low, there were linkage disequilibrium between SNP3 and the two SNPs (SNP1, SNP2), the same as SNP4. We can consider that all four SNPs were located in one haplotype block. R2 values of the four SNPs were all <0.5, it means the four SNPs can not replace each other [21, 22]. Then, we use the four SNPs to establish haplotype by the order of SNP1-SNP2-SNP3-SNP4 for all groups.
                  Table 4

                  Pairwise linkage disequilibrium (| D'| above diagonal and r 2 below diagonal) for the four SNPs

                    

                  | D'|

                   

                  SNP

                  SNP1

                  SNP2

                  SNP3

                  SNP4

                  r 2

                  SNP1

                   

                  0.998

                  0.593

                  0.311

                   

                  SNP2

                  0.101

                   

                  0.999

                  0.393

                   

                  SNP3

                  0.255

                  0.127

                   

                  0.032

                   

                  SNP4

                  0.069

                  0.238

                  0.001

                   

                  | D'|

                  http://static-content.springer.com/image/art%3A10.1186%2F1476-511X-13-143/MediaObjects/12944_2014_Article_1157_Fig2_HTML.jpg
                  Figure 2

                  Pairwise estimates of linkage disequilibrium (LD) between each CYP2C9 polymorphism is plotted for Han population using SHEsis platform. Each polymorphism is numbered according to its position in the CYP2C9 gene as presented in Figure 1. (a) showed | D'| and different colors represent different degree of linkage disequilibrium. The darker the color,wasthe stronger the degree of linkage disequilibrium was (b) showed r2.

                  Table 5 showed the distribution of haplotypes in CAD patient and control participants. There were twelve haplotypes established in all subjects. The overall distribution of the haplotypes were significantly different between the CAD patients and the control subjects (all p < 0.0001). The most frequence haplotype in this study was 0100 (C-T-A-C) haplotype. For women, the frequency of C-T-A-C was significantly lower in the CAD patients than in the control subjects (nominal p = 0.0032, adjusted p* = 0.016). In addition, the frequency of the 0001 (C-C-A-T) haplotype was higher in the CAD patients than in the control subjects in women (nominal p = 0.0016, adjusted p* = 0.016). For total and men, the frequency of haplotypes was no diffenerce between the CAD patients and the control subjects.
                  Table 5

                  The distubution of haplotype in CAD patient and control participants

                   

                  Total

                  Men

                  Women

                   

                  CAD (%)

                  Control (%)

                  Nominal p

                  Adjusted p

                  CAD (%)

                  Control (%)

                  Nominal p

                  Adjusted p

                  CAD (%)

                  Control (%)

                  Nominal p

                  Adjusted p

                  1

                  0100

                  CTAC

                  34.02

                  37.16

                  0.2699

                  0.6747

                  38.09

                  34.7

                  0.2299

                  0.3831

                  26.02

                  41.38

                  0.0032*

                  0.016*

                  2

                  0000

                  CCAC

                  34.47

                  35.16

                  0.7979

                  0.9973

                  31.87

                  31.41

                  0.3916

                  0.3916

                  39

                  40.04

                  0.3360

                  0.4200

                  3

                  1000

                  TCAC

                  2.5

                  2.48

                  0.9727

                  0.9727

                  1.55

                  3.04

                  0.1537

                  0.3074

                  4.43

                  1.73

                  0.0761

                  0.1900

                  4

                  1100

                  TTAC

                  1.22

                  ˉ

                  0.0221

                  0.2210

                  1.06

                  ˉ

                  0.1472

                  0.3680

                  1.55

                  ˉ

                  0.0902

                  0.1503

                  5

                  0010

                  CCCC

                  5.4

                  4.3

                  0.4255

                  0.8510

                  5.83

                  6.08

                  0.3707

                  0.4119

                  4.41

                  1.41

                  0.0634

                  0.1133

                  6

                  1010

                  TCCC

                  2.27

                  2.34

                  0.9226

                  1.0000

                  2.28

                  2.89

                  0.3394

                  0.4243

                  2.34

                  2.02

                  0.3537

                  0.3537

                  7

                  0110

                  CTCC

                  3.9

                  1.52

                  0.0300

                  0.1500

                  3.75

                  1.25

                  0.1020

                  0.5100

                  4.06

                  1.18

                  0.0877

                  0.1754

                  8

                  0001

                  CCAT

                  10.05

                  9.18

                  0.5749

                  0.8212

                  8.16

                  13.3

                  0.1000

                  1.0000

                  13.15

                  3.04

                  0.0016*

                  0.016*

                  9

                  1001

                  TCAT

                  2.49

                  3.12

                  0.5333

                  0.8888

                  3.88

                  2.7

                  0.2834

                  0.4049

                  0

                  3.72

                  -

                  -

                  10

                  0101

                  CTAT

                  0

                  2.23

                  -

                  -

                  0

                  1.36

                  -

                  -

                  1.74

                  3.18

                  0.1573

                  0.2242

                  11

                  1101

                  TTAT

                  2.25

                  0

                  -

                  -

                  2.08

                  0

                  -

                  -

                  1.98

                  0

                  -

                  -

                  12

                  0011

                  CCCT

                  1.43

                  2.52

                  0.1746

                   

                  1.47

                  3.27

                  0.1230

                  0.4100

                  1.31

                  1.05

                  0.3476

                  0.3862

                  13

                  1011

                  TCCT

                  -

                  -

                  -

                  -

                  -

                  -

                  -

                  -

                  0

                  1.26

                  -

                  -

                  The p value of haplotype was calculated by Fisher's exact test, and revised by False discovery rate. * p>0.05;

                  ‘0 represents major allele’ and 1 represents minor allele’.

                  “0100” refers respectively the major allele of the SNP1,minor allele of the SNP2,major allele of the SNP3,major allele of the SNP4.

                  The p value of each haplotype by the order of SNP1-SNP2-SNP3-SNP4 is relative to the other haplotypes as a group (overall p <0.0001).

                  Table 6 showed the distribution of diplotypes in CAD patients and control participants. For women, the two diplotypes (CTAC/CTAC, CTAC/CCAC) in CYP2C9 gene were significantly lower in the CAD patients than in the control subjects (nominal p = 0.004, and p = 0.016 respectively), while after revised by False discovery rate, the diplotypes (CTAC/CCAC) was no difference between CAD patients than the control subjects (adjusted p = 0.072). The homozygous diplotype (CTAC/CTAC) was associated with decreased risk of CAD in women. For total and men, the frequencies of diplotypes were no diffenerce between the CAD patients and the control subjects.
                  Table 6

                  The distubution of diplotype of CYP2C9 in CAD patient and control participants

                   

                  Total

                  Men

                  Women

                   

                  CHD

                  Control

                  OR

                  95% CI

                  Nominal p

                  Adjusted p

                  CHD

                  Control

                  OR

                  95% CI

                  Nominal p

                  Adjusted p

                  CHD

                  Control

                  OR

                  95% CI

                  Nominal p

                  Adjusted p

                  1

                  1/1

                  0100/0100

                  CTAC/CTAC

                  47

                  52

                  1.511

                  0.893-2.557

                  0.122

                  1

                  40

                  28

                  0.824

                  0.421-1.611

                  0.334

                  0.334

                  7

                  24

                  0.251

                  0.105-0.876

                  0.004*

                  0.036*

                  2

                  1/2

                  0100/0000

                  CTAC/CCAC

                  72

                  59

                  0.858

                  0.576-1.278

                  0.451

                  1

                  53

                  25

                  1.437

                  0.839-2.462

                  0.149

                  0.745

                  19

                  34

                  0.419

                  0.218-0.806

                  0.016*

                  0.072

                  3

                  2/2

                  0000/0000

                  CCAC/CCAC

                  39

                  24

                  1.216

                  0.708-2.088

                  0.479

                  0.958

                  32

                  10

                  2.184

                  1.033-4.615

                  0.074

                  0.740

                  7

                  14

                  0.435

                  0.167-1.130

                  0.108

                  0.324

                  4

                  1/3

                  0100/0001

                  CTAC/CCAT

                  19

                  18

                  0.756

                  0.387-1.477

                  0.412

                  1

                  12

                  13

                  0.549

                  0.242-1.245

                  0.195

                  0.488

                  7

                  5

                  1.354

                  0.414-4.425

                  0.351

                  0.526

                  5

                  1/4

                  0100/0010

                  CTAC/CCCC

                  12

                  8

                  1.100

                  0.442-2.739

                  0.837

                  1

                  9

                  6

                  0.933

                  0.324-2.686

                  0.326

                  0.408

                  3

                  2

                  0.438

                  0.235-8.800

                  0.308

                  0.554

                  6

                  1/5

                  0100/1001

                  CTAC/TCAT

                  10

                  5

                  1.478

                  0.498-4.386

                  0.479

                  1

                  9

                  3

                  1.912

                  0.508-7.201

                  0.220

                  0.440

                  1

                  2

                  0.469

                  0.042-5.264

                  0.424

                  0.424

                  7

                  2/5

                  0000/1001

                  CCAC/TCAT

                  5

                  4

                  0.912

                  0.242-3.437

                  0.900

                  0.981

                  5

                  0

                  -

                  -

                  -

                   

                  0

                  4

                  -

                  -

                  -

                   

                  8

                  5/5

                  1001/1001

                  TCAT/TCAT

                  3

                  1

                  2.205

                  0.228-21.338

                  0.484

                  0.830

                  1

                  1

                  0.622

                  0.039-10.032

                  0.294

                  0.49

                  2

                  0

                  -

                  -

                  -

                   

                  9

                  1/7

                  0100/1000

                  CTAC/TCAC

                  6

                  4

                  1.098

                  0.306-3.939

                  0.891

                  1

                  1

                  2

                  0.308

                  0.028-3.437

                  0.321

                  0.467

                  5

                  2

                  2.477

                  0.463-12.931

                  0.278

                  0.626

                  10

                  1/8

                  0100/1010

                  CTAC/TCCC

                  3

                  3

                  0.728

                  0.146-3.642

                  0.698

                  1

                  1

                  1

                  0.622

                  0.039-10.032

                  0.327

                  0.363

                  2

                  2

                  0.948

                  0.131-6.874

                  0.383

                  0.431

                  11

                  2/8

                  0000/1010

                  CCAC/TCCC

                  7

                  5

                  1.024

                  0.321-3.269

                  0.968

                  0.968

                  0

                  5

                  -

                  -

                  -

                   

                  7

                  0

                  -

                  -

                  -

                   

                  12

                  1/9

                  0100/1011

                  CTAC/TCCT

                  5

                  8

                  0.448

                  0.144-1.387

                  0.153

                  0.918

                  4

                  6

                  0.404

                  0.112-1.461

                  0.154

                  0.513

                  1

                  2

                  0.469

                  0.042-5.264

                  0.353

                  0.454

                  The p value of diplotype was calculated by Fisher's exact test, and revised by False discovery rate.*p>0.05; The odds ratio (OR) and 95 % confidence interval (CI) of each diplotype are relative to the other diplotype as a group. The total diplotypes with very rare count (<3) are not shown. 0 represents major allele and 1 represents minor allele.

                  Discussion

                  Endogenous CYP metabolites such as epoxyeicosatrienoic acids (EETs), hydroxyeicosatetraenoic acids, prostacyclin (PGI2), aldosterone, and sex hormones have been demonstrated to be involved in coronary artery disease, stroke, hypertension, and other cardiovascular diseases [11]. Arachidonic acid can be metabolized by the CYP2C9 subfamily to EETs, which has been established to have five physiological functions. First, EETs produces vasodilation in a number of vascular beds by activating the smooth muscle large conductance Ca2+-activated K+ channels (BKCa) [6, 14]. Second, EETs may act as endothelium-derived hyperpolarizing factors (EDHF) [16], particularly in the coronary circulation. EDHF possess potent vasodilating effects hyperpolarize vascular smooth muscle cells (VSMCs) by activating K-Ca[6, 23, 24]. Third, EETs inhabit inflammation responses by decreasing the cytokine-induced endothelial expression of vascular cell adhesion molecule-1 (VCAM-1) and to decrease leukocytes adhesion to the vascular wall by inhibiting nuclear factor κB (NF-κB) and IκB kinase [25]. Fourth, EETs have antithrombotic effects by inhibiting platelet adhesion to endothelial cells, inhibiting platelet aggregation, and enhancing the expression and activity of tissue plasminogen activator [26]. Fifth, in the kidney, EETs are important regulators of glomerular filtration by activating Na+/H+ exchanger and mediate pressure natriuresis and long-term control of blood pressure [27, 28]. CYP2C9 polymorphisms might affect the biosynthesis and activity of EETs, which determines susceptibility to the development of CAD. In this study, we hypothesized that variability in CYP2C9 gene might affect the risk of CAD. We genotyped four SNPs of the gene in a Han population, and assessed the association between the CYP2C9 gene and CAD using diplotype-based case–control analyses.

                  EH and DM were both common risk factors for CAD. As expected, in our study, we found that the prevalence of EH and DM were significantly higher for patients with CAD than for control participants for women. The frequency of T allele of SNP2 (rs2475376) was about 0.409, which was slightly higher than the frequency of T allele of the Chinese Han people (about 0.378) in PubMed database. The distribution of genotypes, dominant model and alleles of SNP2 (rs2475376) showed significant difference between the CAD patients and control participants (p = 0.033, P = 0.010 and p = 0.038, respectively). However, the significant difference of the dominant model (CC vs CT + TT) was retained after adjustment for covariates in women, but not in men (for women, OR: 2.427, 95% confidence interval [CI]: 1.305-4.510, p =0.005; and for men, OR: 1.372, 95% CI: 0.861-2.186, p =0.184). We speculated women carrying CC genotype seem to have a lower ability to synthesize EETs. It means CC genotype of rs2475376 may be an increased risk factor of CAD. The frequency of T alleles (rs2475376) was significantly lower for CAD patients than for control subjects. Women carrying mutant T allele seem to have a higher ability to synthesize EETs. It means that T alleles (rs2475376) may be a decreased risk factor of CAD.

                  For women, the frequency of the 0100 (C-T-A-C) haplotype was significantly lower in the CAD patients than in the control subjects, and the frequency of the 0001 (C-C-A-T) haplotype was higher in the CAD patients than in the control subjects. Human being is of homologous chromosomes, therefore, diplotype is more convincing than haplotype. For women, the homozygous diplotype (CTAC/CTAC) was significantly lower in the CAD patients than in the control subjects. It means that C-T-A-C haplotype, the homozygous diplotype (CTAC/CTAC) may be decreased risk factors of CAD. C-C-A-T haplotype may be an increased risk factor of CAD. These results of haplotype and diplotype were consistent with the results of CC genotype and T allele of SNP2 (rs2475376).

                  In our study, we observed a protective effect of the CYP2C9 mutant allele for the development of CAD only in women. For the gender differences, we can explain it by the following reasons. First, this could be attributed to sex hormones. Sex hormones such as estrogens protect against oxidative stress and are known to be vasoprotective [17, 18, 29]. Second, there were some researches [30, 31], which show that estrogens protect the EETs against being hydrolyzed by soluble epoxide hydrolase (sEH). Hence, women carrying mutant T allele seem to have a lower risk for suffering CAD. Third, this may be related to antioxidation of CYP2C9. CYP2C9 has been shown to be a major source of reactive oxygen species (ROS) within coronary artery endothelial cells [32]. Lower formation of oxygen radicals in carriers of mutant alleles might explain our findings.

                  In addition, many previous studies showed the polymorphisms of CYP2C9 gene (rs1057910) associated with the cardiovascular risk. An increased risk of MI was found in association with CYP2C9 variants (rs1057910) among women [17, 18, 33, 34]. In addition, there was the study that showed men carriers of the CYP2C9 mutant genotype (rs1057910) seem to have a lower risk for suffering MI in Austria [35]. There was also the research which suggested CYP2C9 gene interaction with smoking was associated with CAD [36]. In our study, it was not found that rs1057910 of CYP2C9 gene was associated with the risk of CAD. There might be ethnic and geographical environment factors explaining the difference among clinical trials.

                  Conclusion

                  In conclusion, CC genotype of rs2475376 and C-C-A-T haplotype in CYP2C9 may be a risk genetic marker of CAD in women. T allele of rs2475376, the haplotype (C-T-A-C) and the diplotype (CTAC/CTAC) could be protective genetic markers of CAD for women in Han population of China.

                  Declarations

                  Authors’ Affiliations

                  (1)
                  Department of Cardiovascular Medicine, First Affiliated Hospital of Xinjiang Medical University

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                  © Fu et al.; licensee BioMed Central Ltd. 2014

                  This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://​creativecommons.​org/​licenses/​by/​4.​0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://​creativecommons.​org/​publicdomain/​zero/​1.​0/​) applies to the data made available in this article, unless otherwise stated.

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