J Mol Neurosci DOI 10.1007/s12031-014-0259-x
Association Between VKORC1 Gene Polymorphisms and Ischemic Cerebrovascular Disease in Chinese Han Population Hua Zhang & Linyan Yang & Qingchuan Feng & Yujia Fan & Hong Zheng & Ying He
Received: 28 November 2013 / Accepted: 6 February 2014 # Springer Science+Business Media New York 2014
Abstract The vitamin K epoxide reductase subunit 1 gene (VKORC1) plays a key role in vitamin K recycling, and there is a close association between VKORC1 gene singlenucleotide polymorphisms (SNPs) and the required dose of warfarin, an anticoagulant. However, the association between VKORC1 SNPs and ischemic cerebrovascular disease (ICVD) has not been defined. This case–control study involved 370 patients with ICVD and 408 healthy individuals (controls) from Chinese Han population. Two VKORC1 gene SNPs (1639A/G and 1173T/C) were genotyped by PCR-RFLP method. The G allele frequencies of the 1639A/G locus and C allele frequencies of the 1173T/C locus were higher in the ICVD group than in the control group (p=0.014 and p=0.008, respectively). Haplotype analysis showed that 1639G-1173C was associated with an increased risk of ICVD (odds ratio (OR)=1.163, 95 % confidence interval (CI)=1.137~2.288), while 1639A-1173T was associated with decreased risk of ICVD (OR=0.620, 95 % CI=0.437~0.880). Our findings suggested that individuals carrying the 1639G or 1173C allele might be at increased risk for ICVD. Furthermore, the 1639G1173C haplotype was a risk factor for ICVD, and 1639A1173T was a protective factor in Chinese Han population. Keywords VKORC1 . Polymorphism . Genetics . ICVD
Introduction Vitamin K epoxide reductase complex subunit 1 (VKORC1), a rate-limiting enzyme involved in the process of clotting factor H. Zhang : Q. Feng : Y. Fan : H. Zheng (*) : Y. He (*) Department of Cell Biology & Medical Genetics, Basic Medical College of Zhengzhou University, Zhengzhou 450052, China e-mail: [email protected] e-mail: [email protected] L. Yang Frontage China Clinical Research Center, Zhengzhou 450052, China
generation, plays an essential role in the carboxylation of vitamin K-dependent proteins (Yuan et al. 2005). These proteins include not only procoagulant proteins (factors II, VII, IX, and X) but also anticoagulant proteins (proteins C, S, and Z), which impact on the activation of the coagulation system, maintenance of blood flow, and vascular integrity. Thereby, VKORC1 is believed to be associated with arteriosclerosis and vascular diseases. Recently, Wang et al. first reported that the 2255C allele of the VKORC1 gene might increase the risk of cardiovascular disease in a Chinese Han population (Wang et al. 2006). Subsequently, several studies have tried to replicate this association in different populations. Lacut et al. discovered that the 1173T/C polymorphism of the VKORC1 gene was associated with deep vein thrombosis in a French cohort (Lacut et al. 2007). Hindorff et al. revealed that three SNPs, rs2359612C/T, rs17708427G/A, and rs7294G/A were not relevant to arterial thrombosis (Hindorff et al. 2007). However, to date, the association between VKORC1 gene polymorphisms and ischemic cerebrovascular disease (ICVD) remains undefined. Therefore, to investigate the contribution of variations in the VKORC1 gene to ICVD in Chinese Han population, a case–control association study was designed to clarify the effect of 1639A/G and 1173T/C polymorphisms of VKORC1 gene.
Materials and Methods Study Subjects We recruited 370 inpatients (age, mean ± SD 65.7 ± 11.21 years; male, 59.7 %) with a clinical diagnosis of ICVD from the Neurology Department of Henan Provincial Hospital during 2010–2012. ICVD was defined as patients with an acute or sudden focal neurological defect lasting for >24 h and positive brain image lesions by computed tomography, magnetic resonance imaging, or magnetic resonance
J Mol Neurosci
angiography examination (Chen et al. 2006). The type of ICVD was clinically categorized according to the Classification of Cerebrovascular Disease III revised by the National Institute of Neurological Disorders and Stroke (Chen et al. 2006). Brain hemorrhage and cardiogenic brain infarction were excluded from the study. Also excluded were patients with arterial fibrillation, peripheral vascular diseases, or kidney diseases. The control group consisted of 408 unrelated subjects (age, mean±SD 64.68±12.01 years; male, 65.4 %) who were recruited from the same demographic area and were matched to cases on the basis of age, gender, and residency. All controls were unrelated native Henan Han individuals without cerebrovascular and cardiovascular diseases, cancer, and hepatic or renal diseases. The study protocol was reviewed and approved by the Zhengzhou University Ethics Committee on Human Research. Informed consent was obtained from all participants. DNA Extraction and Genotyping Genomic DNA was extracted from peripheral white blood cells using the phenol/chloroform method (Sangon, Shanghai, China). We selected 1639A/G and 1173T/C for genotyping with PCR-RFLP method. For the 1639A/G polymorphism, the primers were forward (F), 5′-GCCAGCAGGAGAGGGA AATA-3′ and reverse (R), 5′-AGTTTGGACTACAGGTGC CT-3′ (Sangon, Shanghai, China). For the 1173T/C polymorphism, the primers were F, 5′-AATCCTGACGTGGCCAAA3′ and R, 5′-CAGCACATGCTCCACCAG-3′ (Sangon, Shanghai, China). PCR was carried out in 25-μl reactions containing 2.0 μl genomic DNA, 3.0 μl dNTPs (TaKaRa, Dalian, China), 2.5 μl 10× buffer (TakaRa, Dalian, China), 1 U/0.3 μl Taq DNA polymerase (TaKaRa, Dalian, China), 2 μl of each primer and 13.2 μl ddH2O. For the 1639A/G polymorphism, the PCR (Biometra, Germany) involved initial denaturation at 95 °C for 5 min, followed by 45 cycles at 94 °C for 30 s, 63 °C for 30 s, 72 °C for 60 s, and final extension at 72 °C for 7 min. For the 1173T/C polymorphism, the PCR was as follows: initial denaturation at 95 °C for 15 min, followed by 40 cycles at 95 °C for 30 s, 56 °C for 30 s, 72 °C for 50 s, and final extension at 72 °C for 5 min. The amplified products were digested by MspI (Sangon, Shanghai, China) and StyI (Sangon, Shanghai, China), respectively. For quality control, 10 % of the samples underwent repeat PCR and genotyping, and no discrepancies were detected. Moreover, 5 % of the samples of three different genotypes in each SNP were randomly selected for DNA sequencing (100 % concordance) (Sangon, Shanghai, China). Statistical Analysis Continuous data are presented as the mean±SD or number (percentage). Hardy–Weinberg equilibrium (HWE) testing
was carried out using SHEsis software (http://analysis.bio-x. cn) (Shi and He 2005). The chi-square test or Fisher’s exact test was used to compare the genotype and allele distributions of the two SNPs between case and control. The multivariate logistic regression model was used to exclude the effects of possible confounding factors including sex, age, smoking, hypertension, and diabetes. In addition, Bonferroni’s adjustment was used for multiple comparisons. The adjusted p value for significance was set at 0.05/2=0.025. All statistical tests were performed with SPSS version 17.0.
Results Characteristics of the Subjects The characteristics of the study population were described in Table 1. The percentages of hypertension, diabetes, smokers, and alcohol cases were higher than those of control groups and showed statistical significance (p0.025). The distribution of genotype frequencies of the two SNPs in three genetic models between the case and control groups were described in Table 2. For the 1639A/G polymorphism, the G allele frequency (11.4 %) in ICVD group was significantly higher than that (7.7 %) in control group (p=0.014, OR=1.530, 95 % CI=1.086–2.157). There was a significant difference in the dominant model (combined heterozygote and variant homozygote group versus wild-type homozygotes) between the case and control groups (p=0.012). Using the Table 1 The characteristics of the study population Characteristics
Cases (n=370)
Controls (n=408)
p value
Age (years) Hypertension (n, %) Diabetes (n, %) Smokers (n, %) Alcohol (n, %) Cholesterol (mg/dl) Total triglyceride (mmol/L)
65.7±11.21 219 (59.2) 80 (21.6) 111 (30) 76 (20.5) 5.22±1.85 1.63±0.92
64.68±12.01 134 (32.8) 42 (10.3) 48 (11.8) 53 (13) 5.06±0.92 1.52±0.74
0.272 0.000* 0.000* 0.000* 0.000* 0.055 0.097
*p
Association Between VKORC1 Gene Polymorphisms and Ischemic Cerebrovascular Disease in Chinese Han Population Hua Zhang & Linyan Yang & Qingchuan Feng & Yujia Fan & Hong Zheng & Ying He
Received: 28 November 2013 / Accepted: 6 February 2014 # Springer Science+Business Media New York 2014
Abstract The vitamin K epoxide reductase subunit 1 gene (VKORC1) plays a key role in vitamin K recycling, and there is a close association between VKORC1 gene singlenucleotide polymorphisms (SNPs) and the required dose of warfarin, an anticoagulant. However, the association between VKORC1 SNPs and ischemic cerebrovascular disease (ICVD) has not been defined. This case–control study involved 370 patients with ICVD and 408 healthy individuals (controls) from Chinese Han population. Two VKORC1 gene SNPs (1639A/G and 1173T/C) were genotyped by PCR-RFLP method. The G allele frequencies of the 1639A/G locus and C allele frequencies of the 1173T/C locus were higher in the ICVD group than in the control group (p=0.014 and p=0.008, respectively). Haplotype analysis showed that 1639G-1173C was associated with an increased risk of ICVD (odds ratio (OR)=1.163, 95 % confidence interval (CI)=1.137~2.288), while 1639A-1173T was associated with decreased risk of ICVD (OR=0.620, 95 % CI=0.437~0.880). Our findings suggested that individuals carrying the 1639G or 1173C allele might be at increased risk for ICVD. Furthermore, the 1639G1173C haplotype was a risk factor for ICVD, and 1639A1173T was a protective factor in Chinese Han population. Keywords VKORC1 . Polymorphism . Genetics . ICVD
Introduction Vitamin K epoxide reductase complex subunit 1 (VKORC1), a rate-limiting enzyme involved in the process of clotting factor H. Zhang : Q. Feng : Y. Fan : H. Zheng (*) : Y. He (*) Department of Cell Biology & Medical Genetics, Basic Medical College of Zhengzhou University, Zhengzhou 450052, China e-mail: [email protected] e-mail: [email protected] L. Yang Frontage China Clinical Research Center, Zhengzhou 450052, China
generation, plays an essential role in the carboxylation of vitamin K-dependent proteins (Yuan et al. 2005). These proteins include not only procoagulant proteins (factors II, VII, IX, and X) but also anticoagulant proteins (proteins C, S, and Z), which impact on the activation of the coagulation system, maintenance of blood flow, and vascular integrity. Thereby, VKORC1 is believed to be associated with arteriosclerosis and vascular diseases. Recently, Wang et al. first reported that the 2255C allele of the VKORC1 gene might increase the risk of cardiovascular disease in a Chinese Han population (Wang et al. 2006). Subsequently, several studies have tried to replicate this association in different populations. Lacut et al. discovered that the 1173T/C polymorphism of the VKORC1 gene was associated with deep vein thrombosis in a French cohort (Lacut et al. 2007). Hindorff et al. revealed that three SNPs, rs2359612C/T, rs17708427G/A, and rs7294G/A were not relevant to arterial thrombosis (Hindorff et al. 2007). However, to date, the association between VKORC1 gene polymorphisms and ischemic cerebrovascular disease (ICVD) remains undefined. Therefore, to investigate the contribution of variations in the VKORC1 gene to ICVD in Chinese Han population, a case–control association study was designed to clarify the effect of 1639A/G and 1173T/C polymorphisms of VKORC1 gene.
Materials and Methods Study Subjects We recruited 370 inpatients (age, mean ± SD 65.7 ± 11.21 years; male, 59.7 %) with a clinical diagnosis of ICVD from the Neurology Department of Henan Provincial Hospital during 2010–2012. ICVD was defined as patients with an acute or sudden focal neurological defect lasting for >24 h and positive brain image lesions by computed tomography, magnetic resonance imaging, or magnetic resonance
J Mol Neurosci
angiography examination (Chen et al. 2006). The type of ICVD was clinically categorized according to the Classification of Cerebrovascular Disease III revised by the National Institute of Neurological Disorders and Stroke (Chen et al. 2006). Brain hemorrhage and cardiogenic brain infarction were excluded from the study. Also excluded were patients with arterial fibrillation, peripheral vascular diseases, or kidney diseases. The control group consisted of 408 unrelated subjects (age, mean±SD 64.68±12.01 years; male, 65.4 %) who were recruited from the same demographic area and were matched to cases on the basis of age, gender, and residency. All controls were unrelated native Henan Han individuals without cerebrovascular and cardiovascular diseases, cancer, and hepatic or renal diseases. The study protocol was reviewed and approved by the Zhengzhou University Ethics Committee on Human Research. Informed consent was obtained from all participants. DNA Extraction and Genotyping Genomic DNA was extracted from peripheral white blood cells using the phenol/chloroform method (Sangon, Shanghai, China). We selected 1639A/G and 1173T/C for genotyping with PCR-RFLP method. For the 1639A/G polymorphism, the primers were forward (F), 5′-GCCAGCAGGAGAGGGA AATA-3′ and reverse (R), 5′-AGTTTGGACTACAGGTGC CT-3′ (Sangon, Shanghai, China). For the 1173T/C polymorphism, the primers were F, 5′-AATCCTGACGTGGCCAAA3′ and R, 5′-CAGCACATGCTCCACCAG-3′ (Sangon, Shanghai, China). PCR was carried out in 25-μl reactions containing 2.0 μl genomic DNA, 3.0 μl dNTPs (TaKaRa, Dalian, China), 2.5 μl 10× buffer (TakaRa, Dalian, China), 1 U/0.3 μl Taq DNA polymerase (TaKaRa, Dalian, China), 2 μl of each primer and 13.2 μl ddH2O. For the 1639A/G polymorphism, the PCR (Biometra, Germany) involved initial denaturation at 95 °C for 5 min, followed by 45 cycles at 94 °C for 30 s, 63 °C for 30 s, 72 °C for 60 s, and final extension at 72 °C for 7 min. For the 1173T/C polymorphism, the PCR was as follows: initial denaturation at 95 °C for 15 min, followed by 40 cycles at 95 °C for 30 s, 56 °C for 30 s, 72 °C for 50 s, and final extension at 72 °C for 5 min. The amplified products were digested by MspI (Sangon, Shanghai, China) and StyI (Sangon, Shanghai, China), respectively. For quality control, 10 % of the samples underwent repeat PCR and genotyping, and no discrepancies were detected. Moreover, 5 % of the samples of three different genotypes in each SNP were randomly selected for DNA sequencing (100 % concordance) (Sangon, Shanghai, China). Statistical Analysis Continuous data are presented as the mean±SD or number (percentage). Hardy–Weinberg equilibrium (HWE) testing
was carried out using SHEsis software (http://analysis.bio-x. cn) (Shi and He 2005). The chi-square test or Fisher’s exact test was used to compare the genotype and allele distributions of the two SNPs between case and control. The multivariate logistic regression model was used to exclude the effects of possible confounding factors including sex, age, smoking, hypertension, and diabetes. In addition, Bonferroni’s adjustment was used for multiple comparisons. The adjusted p value for significance was set at 0.05/2=0.025. All statistical tests were performed with SPSS version 17.0.
Results Characteristics of the Subjects The characteristics of the study population were described in Table 1. The percentages of hypertension, diabetes, smokers, and alcohol cases were higher than those of control groups and showed statistical significance (p0.025). The distribution of genotype frequencies of the two SNPs in three genetic models between the case and control groups were described in Table 2. For the 1639A/G polymorphism, the G allele frequency (11.4 %) in ICVD group was significantly higher than that (7.7 %) in control group (p=0.014, OR=1.530, 95 % CI=1.086–2.157). There was a significant difference in the dominant model (combined heterozygote and variant homozygote group versus wild-type homozygotes) between the case and control groups (p=0.012). Using the Table 1 The characteristics of the study population Characteristics
Cases (n=370)
Controls (n=408)
p value
Age (years) Hypertension (n, %) Diabetes (n, %) Smokers (n, %) Alcohol (n, %) Cholesterol (mg/dl) Total triglyceride (mmol/L)
65.7±11.21 219 (59.2) 80 (21.6) 111 (30) 76 (20.5) 5.22±1.85 1.63±0.92
64.68±12.01 134 (32.8) 42 (10.3) 48 (11.8) 53 (13) 5.06±0.92 1.52±0.74
0.272 0.000* 0.000* 0.000* 0.000* 0.055 0.097
*p
