CLINICAL PERSPECTIVE

This Article Abstract Full Text (PDF) All Versions of this Article: 113/12/1615    most recent CIRCULATIONAHA.105.580167v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Wang, Y. Articles by Hui, R. Search for Related Content PubMed PubMed Citation Articles by Wang, Y. Articles by Hui, R. Related Collections Clinical genetics Epidemiology

(Circulation. 2006;113:1615-1621.)
© 2006 American Heart Association, Inc.

Vascular Medicine

VKORC1 Haplotypes Are Associated With Arterial Vascular Diseases (Stroke, Coronary Heart Disease, and Aortic Dissection)

Yibo Wang, BS; Weili Zhang, PhD; Yuhui Zhang, MD; Yuejin Yang, MD, PhD; Lizhong Sun, MD; Shengshou Hu, MD; Jilin Chen, MD; Channa Zhang; Yi Zheng, MD; Yisong Zhen, PhD; Kai Sun, PhD; Chunyan Fu, MD; Tao Yang, BS; Jianwei Wang, BS; Jing Sun, BS; Haiying Wu, MD; Wayne C. Glasgow, PhD; Rutai Hui, MD, PhD

From Sino-German Laboratory for Molecular Medicine (Y.W., W.Z., C.Z., Y. Zheng, Y. Zhen, K.S., C.F., T.Y., J.W., J.S., R.H.), Hypertension Division (Y. Zhang, H.W., R.H.), Department of Cardiology (Y.Y., J.C., R.H.), and Department of Cardiovascular Surgery (L.S., S.H), Fuwai Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College and National Genome Center (Beijing); and Division of Basic Medical Sciences (W.C.G.), Mercer University School of Medicine, Macon, Ga.

Reprint requests to Rutai Hui, MD, PhD, Cardiovascular Institute & Fuwai Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, 167 Beilishilu, Beijing 100037, People’s Republic of China. E-mail huirutai{at}sglab.org

Received August 1, 2005; revision received January 18, 2006; accepted January 20, 2006.

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Background— The haplotypes in the gene vitamin K epoxide reductase complex subunit 1 (VKORC1) have been found to affect warfarin dose response through effects on the formation of reduced-form vitamin K, a cofactor for -carboxylation of vitamin K–dependent proteins, which is involved in the coagulation cascade and has a potential impact on atherosclerosis. We hypothesized that VKORC1-dependent effects on the coagulation cascade and atherosclerosis would contribute to susceptibility for vascular diseases.

Methods and Results— To test the hypothesis, we studied the association of polymorphisms of VKORC1 with stroke (1811 patients), coronary heart disease (740 patients), and aortic dissection (253 patients) compared with matched controls (n=1811, 740, and 416, respectively). Five common noncoding single-nucleotide polymorphisms of VKORC1 were identified in a natural haplotype block with strong linkage disequilibrium (D'>0.9, r²>0.9), then single-nucleotide polymorphism (SNP) +2255 in the block was selected for the association study. We found that the presence of the C allele of the +2255 locus conferred almost twice the risk of vascular disease (odds ratio [OR] 1.95, 95% confidence interval [CI] .58 to 2.41, P<0.001 for stroke; OR 1.72, 95% CI 1.24 to 2.38, P<0.01 for coronary heart disease; and OR 1.90, 95% CI 1.04 to 3.48, P<0.05 for aortic dissection). We also observed that subjects with the CC and CT genotypes had lower levels of undercarboxylated osteocalcin (a regulator for the bone), probably vascular calcification, and lower levels of protein induced in vitamin K absence or antagonism II (PIVKA-II, a des--carboxy prothrombin) than those with TT genotypes.

Conclusions— The haplotype of VKORC1 may serve as a novel genetic marker for the risk of stroke, coronary heart disease, and aortic dissection.

Key Words: vitamin K epoxide reductase • haplotypes • stroke • coronary disease • aortic dissection

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Stroke and coronary heart disease (CHD) are the leading causes of morbidity and mortality in China.¹ Each year, more than 2.5 million Chinese have strokes, 1 million have heart attacks, and 2 million die of stroke- and CHD-related causes. Furthermore, 7 million patients have survived a stroke and are disabled (http://www.moh.gov.cn). Elucidation of the pathogenesis of these diseases and identification of subjects at risk for these events are major challenges to medical society. A high degree of comorbidity and common risk factors have been observed among myocardial infarction (MI), stroke, and aortic dissection. Most of these cases result from atherosclerotic disease that is characterized by lesions in large and medium-sized elastic and muscular arteries.^2–4

Editorial p 1550

Clinical Perspective p 1621

Platelet adhesion and mural thrombosis are ubiquitous in the initiation and generation of lesions of atherosclerosis in animals and in humans.^5,6 Plaque adhesion and thrombosis formation result from activation of the coagulation cascade, initiated by atherosclerotic plaque erosion or rupture contributing to propagation of thrombosis.^7–9

Vitamin K–dependent proteins play very important roles in activation of the coagulation cascade and in maintaining blood flow and integrity of the vasculature.^10–13 The biological activity of all known vitamin K–dependent proteins is highly dependent on correct -carboxylation.¹⁴ Vitamin K hydroquinone, a cofactor for -carboxylation, is converted to vitamin K epoxide, which in turn is recycled to vitamin K hydroquinone by vitamin K epoxide reductase.^15,16

Undercarboxylation of vitamin K–dependent proteins in patients with a specific mutation in the -carboxylase results in bleeding disorders.^17,18 Common polymorphisms in the gene vitamin K epoxide reductase complex subunit 1 (VKORC1) affect warfarin dose response and blood clotting through effects on the formation of the reduced form of vitamin K, which subsequently alters carboxylation of vitamin K–dependent hemostatic and nonhemostatic proteins.^19–25 Numerous studies indicate that vitamin K–dependent proteins have additional activities that extend their roles beyond hemostatic and bone metabolism, perhaps in vascular calcification and atherosclerotic complications. Price et al²⁶ showed that inhibition of VKORC1 by warfarin results in undercarboxylation of matrix Gla protein (MGP) and subsequent medial calcification of the arterial vessel wall. Gene-deletion studies in mice have shown that MGP is an inhibitor of calcification.²⁷

Arterial calcification has been correlated with an increased probability of dissection and a higher incidence of future ischemic episodes in patients undergoing angioplasty.²⁸ Intimal calcification of atherosclerotic plaques correlates with plaque burden and high risk of cardiovascular events.²⁹ Medial calcification has been shown to be associated with diabetes mellitus and end-stage renal disease and as a prognostic marker for cardiovascular mortality in patients requiring hemodialysis.³⁰ Despite the fact that VKORC1 mediates vitamin K–dependent -carboxylation, which is involved in calcification, it remains unknown whether this could translate into higher risk for arterial disease. We hypothesized that VKORC1–dependent effects on the coagulation cascade and vascular calcification would contribute to susceptibility to vascular diseases. To test the hypothesis, we investigated the association of VKORC1 polymorphisms with stroke, CHD, or aortic dissection.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
The study was approved by both the ethics committee of Fuwai Hospital and the local ethics committee of the collaborative hospitals. All subjects who participated in the study provided written informed consent and reported themselves to be of Han nationality.

Stroke Cohort
The primary study population has previously been used to investigate risk factors of stroke.^31,32 Briefly, case and control subjects were recruited from 7 clinical centers located in 7 provinces from the same demographic area and at the same time from November 2000 to November 2001. Only 3 subtypes of stroke—cerebral atherosclerosis, lacunar infarction, and intracerebral hemorrhage—were included. Diagnosis of stroke was based on strict neurological examination, computed tomography, or MRI according to the International Classification of Diseases, 9th revision. Controls were selected from inpatients (21.5%) with minor illness from the departments of ophthalmology, gastroenterology, otorhinolaryngology, and orthopedics and from community-based inhabitants (78.5%) free of neurological diseases, following the same exclusion criteria as case subjects. In each local community, both men and women 35 to 74 years of age in the range of selection were grouped by age (5-year range for each group), and the control subject was randomly selected from the corresponding group.

CHD and Aortic Dissection Cohorts
Patients for the 2 studies were consecutive patients admitted to Fuwai Hospital from December 2001 to December 2002. The second population comprised 740 unrelated patients with CHD aged from 30 to 75 years and 740 control subjects. Inclusion criteria were 70% narrowing of the lumina of at least 1 major coronary artery. A detailed history of angina or MI was obtained. Of the patients, 57.8% had MI judged by typical ECG change (Minnesota Code 1.1 or 1.2 in ECG) and by changes in serum enzymes (troponin T, troponin I, creatine kinase-MB, aspartate aminotransferase, and glutamic pyruvic transaminase). The 740 control subjects were selected from hospital inpatients whose major coronary arteries had no more than 20% stenosis and who did not have any vascular disease.

The third study sample comprised 253 unrelated cases with aortic dissection who were 20 to 75 years old and 416 control subjects. Aortic dissection was diagnosed on the basis of symptoms, signs, ECG, cardiac enzymes, coronary angiography, magnetic resonance angiography, and/or aortic angiography; 70% of the patients underwent aortography, aortic surgery, and pathological examination. One hundred sixty-three patients were excluded because of known causes of disease, including aortic disease caused by coarctation of aorta, congenital aortic valve disease, inflammation arteritis, and trauma during cardiovascular procedure or glucocorticoid treatment. The diagnosis of Marfan syndrome (11.5% in these patients with aortic dissection) was based on revised clinical criteria of the Gent nosology.³³ None of the control subjects had a history or symptoms of cardiovascular disease.

Biological Variable Determination and Clinical Data Collection
Blood samples were collected after a 12-hour overnight fast before cardiovascular procedures. In subjects with an acute event, the drawing of blood was delayed for at least 6 weeks. The plasma and cell buffet coat were kept at –70°C. Genomic DNA was extracted, and biological variables were determined within 3 months. A complete clinical history was obtained from all subjects. In addition to neurological history and family history of hypertension, CHD, and diabetes mellitus (DM), the following vascular risk factors were also recorded: history of vascular disease, cigarette smoking, alcohol consumption, body mass index, systolic blood pressure (SBP), diastolic blood pressure (DBP), blood glucose, HDL cholesterol (HDL-C), non–HDL-C lipids, total plasma cholesterol (TC), and triglycerides (TG). Plasma biochemical parameters were assayed by an automatic analyzer (Hitachi 7060, Hitachi, Tokyo, Japan). Non-HDL-C was calculated by the Friedewald formula. Hypertension was defined as a mean of 3 independent measures of blood pressure >140/90 mm Hg or the use of antihypertensive drugs.³⁴ DM was diagnosed when the subject had a fasting glucose level >7.8 mmol/L, >11.1 mmol/L at 2 hours after oral glucose challenge, or both. All lipids were determined in a Centers for Disease Control and Prevention (CDC)–qualified laboratory in Fuwai hospital.

Screening for Single-Nucleotide Polymorphisms
The entire gene region of VKORC1 and the 2-kilobase (kb) 5' upstream promoter region and 2-kb 3' downstream region were screened by sequencing (ABI Prism 377, Perkin-Elmer Applied Biosystems, Foster City, Calif) in 50 stroke patients and 50 control subjects. No polymorphism was found in the entire coding sequence. The alleles with frequencies <0.05 were excluded; 5 common noncoding polymorphisms were identified in the VKORC1 gene, including –1639A/G (rs9923231) in the promoter region, +1173T/C (rs9934438) in the first intron, +1542C/G (rs8050894) and +2255T/C (rs2359612) in the second intron, and +3730G/A (rs7294) in the 3' downstream region (defined by the nucleotide position from the translational start site). These single-nucleotide polymorphisms (SNPs) are located at positions 3673, 6484, 6853, 7566, and 9041 of the VKORC1 reference sequence (GenBank accession number AY587020). We calculated linkage disequilibrium (LD) between pairs of SNPs using the standard definition of D' and r² and found all 5 SNPs were in strong LD, with D'>0.9 and r²>0.9, which indicates that any 1 of the 5 SNPs could reflect the natural haplotype block of VKORC1. SNP +2255 was selected for genotyping in all studied subjects because its frequency was slightly higher than the other 4 SNPs, and there is a natural digestion site of NcoI in the SNP +2255 fragment.

Genotyping of SNP +2255
The polymorphism +2255 was analyzed by amplification of a 198-bp sequence with the use of the following primers: 5'-TCTGAACCATGTGTCAGCCAGGACC-3' and 5'-GAACAG-AGAGAGGAACCAAGGGAGTGGA-3'. The resultant polymerase chain reaction products were digested with NcoI (New England Biolabs, Beverly, Mass), which yielded 2 DNA fragments of 26 and 172 bp for the T allele on 4% agarose gel and only 1 band for the C allele. Reproducibility of genotyping was confirmed by bidirectional sequencing in 500 samples, and the reproducibility was 100%.

Testing of Genetic Stratification of the Populations
The possible unequal genetic admixture or population subdivision in the control and patient populations could have resulted in a spurious association between a marker and disease. We additionally typed 7 unlinked microsatellite markers: TNNT2 (1q32; D1S2622), MYL3 (3p21.3-p21.2; D3S3560), NEXILIN (1p31.1; D1S2876), MYH7 (14q12; D14S990), TPM1 (15q22.1, D15S993), PRKG2 (7q35-q36; D7S483), and TNNI3 (19q13.4; D19S927). The allele frequencies at those markers were tested for association with phenotypes. The primer sequences were obtained from the Human Genome Database (http://www.gdb.org/), and the primers were synthesized and fluorescently labeled commercially. Genotype results were analyzed with GeneScan and Genotyper software (Applied Biosystems), with the positive control DNA 1347-02 (Centre d’Etude du Polymorphisme Humain [CEPH]) and the negative control. ² Tests were used to compare allele frequencies of the microsatellite loci in patients and controls.³⁵ Alleles with frequencies <0.05 were combined at a given microsatellite locus in both groups.

Undercarboxylated Osteocalcin and Protein Induced in Vitamin K Absence or Antagonism II Measurements
To investigate the impact of the gene polymorphisms on -carboxylation of vitamin K–dependent proteins, we determined the levels of undercarboxylated osteocalcin and PIVKA-II (protein induced in vitamin K absence or antagonism II) antigen in the serum of 49 subjects aged 45 to 65 years. To avoid potential confounding effects of risk factors on the serum levels of undercarboxylated osteocalcin and PIVKA-II, subjects without conventional risk factors were selected, including 26 with homozygotes, 18 with a heterozygote for the T allele, and 5 with a homozygote for the C allele of SNP +2255. The assay was performed with ELISA kits, undercarboxylated osteocalcin from Takara Shuzo (Tokyo, Japan), and PIVKA-II from Diagnostica Stago (Asnieres Sur Seine, France).

Statistical Analysis
The distribution of quantitative variables was tested for normality by use of a 1-sample Kolmogorov-Smirnov test. Because the TG level was highly skewed, we compared the difference between cases and controls with a Mann-Whitney nonparametric test. Quantitative variables, including age, body mass index, SBP and DBP, glucose, HDL-C, non-HDL-C, and TC, were compared with the 1-way ANOVA test. A ² test was used to test for qualitative variables, genotype/allele frequencies, and Hardy-Weinberg equilibrium of the polymorphisms. Association of SNP +2255 with vascular disease was analyzed by multivariable logistic regression adjusted by age, sex, body mass index, blood pressure, smoking, alcohol consumption, glucose, HDL-C, non-HDL-C, TC, and TG. The association was expressed as an odds ratio. The Student t test and Spearman correlation were used to assess the difference and correlation between serum levels of undercarboxylated osteocalcin and PIVKA-II in 3 genotypes of SNP +2255. A 2-tailed probability value of 0.05 was considered significant. Analyses were performed with SPSS 11.0 (SPSS Inc, Chicago, Ill) for Windows (Microsoft Corp, Redmond, Wash). The r² measurement was used to determine LD with the software EMLD (http://request.mdacc.tmc.edu/qhuang/Software/pub.htm). The D' and r² were used to indicate the strength of LD.

The authors had full access to the data and take full responsibility for its integrity. All authors have read and agree to the manuscript as written.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Characteristics of the Subjects
Clinical characteristics of the subjects in the present study are shown in Table 1. In the stroke study, of the 4000 subjects initially recruited for that study, 189 cases and 189 controls were excluded because of lack of definite diagnosis, insufficient DNA, or failure of genotyping. Significantly higher levels of SBP and DBP and a higher incidence of cigarette smoking and hypertension were found in cases than in controls (all P<0.01). In the CHD study, 740 cases and 740 controls were enrolled; higher frequencies of DM, hypertension, and cigarette smoking were found in cases than in controls (all P<0.01). In the aortic dissection study, 253 cases and 416 controls were recruited. Significantly higher levels of SBP and HDL-C, lower levels of non-HDL-C, TC, and TG, and a lower drinking frequency were found in patients than in controls (all P<0.05).

View this table: [in this window] [in a new window]   TABLE 1. Clinical Characteristics

The C Allele of +2255 Was Associated With Stroke, CHD, and Aortic Dissection
The distribution of VKORC1 genotypes of +2255 is shown in Tables 2, 3, and 4, respectively, and fulfilled expectations of Hardy-Weinberg equilibrium in both cases and controls. The estimated risk of vascular disease for subjects with the CT genotype (1 copy of the risk allele C) was significantly higher than for those with the TT genotype (no copy of the risk allele C) but was comparable to those with the CC genotype, which indicates a dominant effect of the at-risk allele C. We used the dominant model to analyze the association of the SNP with vascular disease. The frequency of the CC+CT genotype was significantly higher in patients than in controls (stroke 19.7% versus controls 11.7%, P<0.001; CHD 16.9% versus controls 11.2%, P<0.01; aortic dissection 17.0% versus controls 9.9%, P<0.05). The association remained after adjustment for age, sex, and other conventional risk factors with multiple logistic regression analysis; the OR was 1.95 (95% CI 1.58 to 2.41) for stroke (P<0.001), 1.72 (95% CI 1.24 to 2.38) for CHD (P<0.01), and 1.90 (95% CI 1.04 to 3.48) for aortic dissection (P<0.05). In subtypes of stroke patients, the frequencies of CC+CT were 18.4% for thrombosis (OR 1.75, 95% CI 1.34 to 2.29, P<0.001), 23.2% for lacunar stroke (OR 2.33, 95% CI 1.09 to 2.16, P<0.05), and 18.2% for hemorrhage (OR 1.53, 95% CI 1.76 to 3.06, P<0.001). The presence of the C allele of the +2255 locus conferred an almost 2-fold increase in vascular disease risk within these studied populations. Because the C allele of +2255 can reflect the G-C-G-C-A (3673-6484-6853-7566-9041) haplotype of VKORC1 gene, one can reasonably expect that the G-C-G-C-A haplotype of VKORC1 is also associated with a high risk of stroke, CHD, and aortic dissection.

View this table: [in this window] [in a new window]   TABLE 2. Association of SNP+2255 in VKORC1 Gene With Stroke

View this table: [in this window] [in a new window]   TABLE 3. Association of SNP+2255 in VKORC1 Gene With CHD

View this table: [in this window] [in a new window]   TABLE 4. Association of SNP+2255 in VKORC1 Gene With Aortic Dissection

No Evidence of Strong Population Stratification
Among the 7 highly polymorphic markers that we genotyped, no significant allele-frequency differences were detected between controls and patients within each clinic center and among these centers, which indicates that there was no obvious evidence for genetic stratification in the cohort. The serum levels of undercarboxylated osteocalcin and PIVKA-II were lower in the subjects carrying the C allele of the +2255 locus. The levels of both undercarboxylated osteocalcin and PIVKA-II antigen were lower in carriers of the C allele than in T allele carriers (Figure). The Spearman coefficient for the existing correlation was r=–0.219 for undercarboxylated osteocalcin and –0.567 for PIVKA-II with genotype, respectively. The differences were also significant in undercarboxylated osteocalcin and in PIVKA-II between TT and CC carriers (P=0.021 and P<0.001, respectively), which suggests that the C allele contributes to a higher functional efficiency of the VKORC1 complex.

View larger version (34K): [in this window] [in a new window]   Undercarboxylated osteocalcin and PIVKA-II antigen serum levels in carriers of different genotypes of the VKORC1 gene SNP +2255. Levels of the 2 proteins are presented as means; T bars represent SDs. PIVKA-II antigen serum levels were significantly correlated with SNP +2255 (P<0.001). Five samples in the CC group, 18 in the CT group, and 26 in the TT group were analyzed. *P<0.05, ***P<0.001 for the comparison between genotype TT and CC groups.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The present study is the first clinical investigation of the significance of the polymorphisms of VKORC1 in vascular disease, including stroke, CHD, and aortic dissection. We found that the variants of VKORC1 are associated with almost double the risk of stroke, CHD, and aortic dissection. Next, we examined whether the disease-associated allele was related to specific vascular risk factors, such as hypertension, diabetes, age, and sex. No significant association was observed between the +2255 allele and these risk factors, which suggests that the contribution of SNP +2255 to the risk of vascular disease is independent of those conventional vascular risk factors. We examined the population stratification effect by genotyping 7 unlinked highly polymorphic microsatellite markers in the case subjects and in control subjects, and no association was found between these markers and vascular disease.

Stroke, CHD, and aortic dissection are all vascular diseases. The pathologies of these diseases are quite different. Atherothrombotic stroke mainly results from large-artery atherosclerosis, intracerebral hemorrhages may be due to microaneurysms in intracerebral arteries (whether this is the main cause is still in debate), and lacunar infarction is usually caused by lipohyalinosis or microatheromata with thrombosis of the vascular lumen (30 to 100 µm). Although stroke results from a number of different pathological processes, predisposing factors for each stroke subtype are surprisingly similar, such as increased age, hypertension, smoking, and DM. These are risk factors for CHD and aortic dissection as well. Some of the same factors are manifested in Marfan syndrome, which is caused primarily by mutations in the FBN1 gene. However, other factors are clearly important in the expression of Marfan syndrome; relatives who share the same mutations show dramatically different phenotypes.³⁶ Therefore, all mechanisms that weaken the aorta’s medial layers will contribute to higher aortic wall stress. This can induce aortic dilation and aneurysm formation and eventually cause intramural hemorrhage, aortic dissection, or rupture.

VKORC1 variation could serve as a common genetic risk factor for all vascular diseases. It involves mediation of -carboxylation of hemostatic and nonhemostatic proteins by control of the vitamin K cycle. Polymorphisms of VKORC1 have been shown to affect the expression and activity of VKORC1 and thus warfarin dose response and blood clotting.^23,24 Vitamin K–dependent nonhemostatic proteins mediate calcification. Vessel calcification decreases vessel elasticity and increases shear stress and is associated with the increased morbidity and mortality of arterial disease.^30,37–39

D’Andrea et al¹⁹ recently found that patients with the TT genotype of SNP rs9934438 require a lower dose of warfarin, which indicates a lower activity of the coagulation system in these patients, due at least in part to a lower activity of VKORC1. This leads to less conversion of vitamin K epoxide back to its reduced form, which results in less carboxylation of vitamin K–dependent proteins.¹⁹ The T allele at rs9934438 is on the same haplotype as the T allele of SNP +2255. Rieder et al²⁴ reported that mRNA levels in the group with the G-C-G-C-A (3673-6484-6853-7566-9041) haplotype were 3 times as high as those in the wild-type group. Yuan et al²³ reported that the VKORC1 promoter with the G allele yielded a 44% increase of activity compared with the A allele of –1639. Consistent with these results, we found that subjects with the TT genotype of +2255 had higher levels of undercarboxylated vitamin K–dependent proteins, osteocalcin and PIVKA-II. We proposed that the VKORC1 haplotype G-C-G-C-A (3673-6484-6853-7566-9041) would increase the promoter activity and higher expression of VKORC1 mRNA and protein. This would result in increased -carboxylation, as indicated by the decreased levels of the undercarboxylated vitamin K–dependent proteins osteocalcin and PIVKA-II and a potential need for increased warfarin dosage.

The biological activity of all known vitamin K–dependent proteins is highly dependent on correct -carboxylation, which affects not only procoagulatory (factor II, VII, IX, and X) but also anticoagulatory (protein C, S, and Z) clotting factors and osteocalcin in the same way as MGP.³⁹ The greater functional activity of the C allele may also lead to higher levels of MGP and less vascular calcification. Therefore, the observed association may not be related to the effect on the -carboxylation of the vitamin K–dependent proteins but may be caused by another yet-unknown function of VKORC1.

One possibility is that VKORC1 haplotypes are only markers for LD, and some genes in the linkage region independent of or in conjunction with VKORC1 confer susceptibility to arterial disease. At least 6 other genes are found in the natural haplotype block over the 68 000 bases around VKORC1⁴⁰: ZNF668 (zinc finger protein), ZNF646, BCKDK (branched chain ketoacid dehydrogenase kinase), MYST1 (histone acetyltransferase1), PRSS8 (protease serine 8), and PRSS36. No evidence through a search of the PubMed database shows that these genes have any definite biological function. Previously, we found that VKORC1 is involved in angiogenesis¹⁰; others have shown that polymorphisms of VKORC1 are associated with differences in dose requirements in warfarin sensitivity among patients of different ancestries. In the present report, lower levels of undercarboxylated osteocalcin and PIVKA-II antigen were associated with the C-allele of SNP +2255 of the VKORC1, which supports the idea that the association between the haplotype of the VKORC1 block and vascular disease is attributable to VKORC1.

Furthermore, vascular calcification is more complex in humans than in rodents. In women, progression of atherosclerotic calcification is associated with increased bone loss during menopause.⁴¹ In patients with mutations in the MGP gene, arterial calcification is not a common feature.⁴² In severely atherosclerotic patients, circulating MGP is significantly increased, as is osteocalcin in women with aortic atherosclerosis.⁴³ These have been proposed as feedback mechanism to attempt calcium clearance. Thus far, the pathophysiology behind this exciting finding is not clear and needs to be addressed by further studies.

In conclusion, the prevalence of the haplotype G-C-G-C-A of VKORC1 was significantly more frequent in patients with vascular disease than in controls. The haplotype may serve as a novel genetic marker for the risk of stroke, CHD, and aortic dissection.

	Acknowledgments

 
We thank Dr Thomas Eling (NIH/NIEHS) for his text editing. The study was supported by the Ministry of Science and Technology of China with grant 2002BA711A05 and National 973 Basic Research Project (973 G2000056901) to Dr Rutai. The authors also acknowledge the International HapMap Consortium for the data of LD around VKORC1.

Dislosures

None.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. World Health Organization. World Health Statistics Annual, 1993. Geneva, Switzerland: World Health Organization; 1994.
   
2. Stary HC, Chandler AB, Glagov S, Guyton JR, Insull W Jr, Rosenfeld ME, Schaffer SA, Schwartz CJ, Wagner WD, Wissler RW. A definition of initial, fatty streak, and intermediate lesions of atherosclerosis: a report from the Committee on Vascular Lesions of the Council on Arteriosclerosis, American Heart Association. Circulation. 1994; 89: 2462–2478.[Abstract/Free Full Text]
   
3. Fuster V, Badimon L, Badimon JJ, Chesebro JH. Mechanisms of disease: the pathogenesis of coronary artery disease and the acute coronary syndromes (part one). N Engl J Med. 1992; 326: 242–250.[Medline] [Order article via Infotrieve]
   
4. Falk E. Unstable angina with fatal outcome: dynamic coronary thrombosis leading to infarction and/or sudden death. Circulation. 1985; 71: 699–708.[Abstract/Free Full Text]
   
5. Merten M, Dong JF, Lopez JA, Thiagarajan P. Cholesterol sulfate: a new adhesive molecule for platelets. Circulation. 2001; 103: 2032–2034.[Abstract/Free Full Text]
   
6. Kiechl S, Willeit J; Bruneck Study Group. The natural course of atherosclerosis, part II: vascular remodeling. Arterioscler Thromb Vasc Biol. 1999; 19: 1491–1498.[Abstract/Free Full Text]
   
7. Huo Y, Schober A, Forlow SB, Smith DF, Hyman MC, Jung S, Littman DR, Weber C, Ley K. Circulating activated platelets exacerbate atherosclerosis in mice deficient in apolipoprotein E. Nat Med. 2003; 9: 61–67.[CrossRef][Medline] [Order article via Infotrieve]
   
8. Wagner DD, Burger PC. Platelets in inflammation and thrombosis. Arterioscler Thromb Vasc Biol. 2003; 23: 2131–2137.[Abstract/Free Full Text]
   
9. Tremoli E, Camera M, Toschi V, Colli S. Tissue factor in atherosclerosis. Atherosclerosis. 1999; 144: 273–283.[CrossRef][Medline] [Order article via Infotrieve]
   
10. Wang Y, Zhen Y, Shi Y, Chen J, Zhang C, Wang X, Yang X, Zheng Y, Liu Y, Hui R. Vitamin K epoxide reductase: a protein involved in angiogenesis. Mol Cancer Res. 2005; 3: 317–323.[Abstract/Free Full Text]
    
11. Xu N, Dahlback B, Ohlin AK, Nilsson A. Association of vitamin K–dependent coagulation proteins and C4b binding protein with triglyceride-rich lipoproteins of human plasma. Arterioscler Thromb Vasc Biol. 1998; 18: 33–39.[Abstract/Free Full Text]
    
12. Burns P, Hoffman CJ, Katz JP, Miller RH, Lawson WE, Hultin MB. Vitamin K–dependent clotting factors are elevated in young adults who have close relatives with ischemic heart disease. J Lab Clin Med. 1993; 122: 720–727.[Medline] [Order article via Infotrieve]
    
13. Reitsma PH, Poort SR, Allaart RC, Briet E, Bertina RM. The spectrum of genetic defects in a panel of 40 Dutch families with symptomatic protein C deficiency type I: heterogeneity and founder effects. Blood. 1991; 78: 890–894.[Abstract/Free Full Text]
    
14. Stenflo J, Fernlund P, Egan W, Roepstorff P. Vitamin K dependent modifications of glutamic acid residues in prothrombin. Proc Natl Acad Sci U S A. 1974; 71: 2730–2733.[Abstract/Free Full Text]
    
15. Li T, Chang CY, Jin DY, Lin PJ, Khvorova A, Stafford DW. Identification of the gene for vitamin K epoxide reductase. Nature. 2004; 427: 541–544.[CrossRef][Medline] [Order article via Infotrieve]
    
16. Rost S, Fregin A, Ivaskevicius V, Conzelmann E, Hortnagel K, Pelz HJ, Lappegard K, Seifried E, Scharrer I, Tuddenham EG, Muller CR, Strom TM, Oldenburg J. Mutations in VKORC1 cause warfarin resistance and multiple coagulation factor deficiency type 2. Nature. 2004; 427: 537–541.[CrossRef][Medline] [Order article via Infotrieve]
    
17. Brenner B, Sanchez-Vega B, Wu SM, Lanir N, Stafford DW, Solera J. A missense mutation in gamma-glutamyl carboxylase gene causes combined deficiency of all vitamin K–dependent blood coagulation factors. Blood. 1998; 92: 4554–4559.[Abstract/Free Full Text]
    
18. Spronk HM, Farah RA, Buchanan GR, Vermeer C, Soute BA. Novel mutation in the gamma-glutamyl carboxylase gene resulting in congenital combined deficiency of all vitamin K–dependent blood coagulation factors. Blood. 2000; 96: 3650–3652.[Abstract/Free Full Text]
    
19. D’Andrea G, D’Ambrosio RL, Di Perna P, Chetta M, Santacroce R, Brancaccio V, Grandone E, Margaglione M. A polymorphism in the VKORC1 gene is associated with an interindividual variability in the dose-anticoagulant effect of warfarin. Blood. 2005; 105: 645–649.[Abstract/Free Full Text]
    
20. Harrington DJ, Underwood S, Morse C, Shearer MJ, Tuddenham EG, Mumford AD. Pharmacodynamic resistance to warfarin associated with a Val66Met substitution in vitamin K epoxide reductase complex subunit 1. Thromb Haemost. 2005; 93: 23–26.[Medline] [Order article via Infotrieve]
    
21. Bodin L, Verstuyft C, Tregouet DA, Robert A, Dubert L, Funck-Brentano C, Jaillon P, Beaune P, Laurent-Puig P, Becquemont L, Loriot MA. Cytochrome P450 2C9 (CYP2C9) and vitamin K epoxide reductase (VKORC1) genotypes as determinants of acenocoumarol sensitivity. Blood. 2005; 106: 135–140.[Abstract/Free Full Text]
    
22. Wadelius M, Chen LY, Downes K, Ghori J, Hunt S, Eriksson N, Wallerman O, Melhus H, Wadelius C, Bentley D, Deloukas P. Common VKORC1 and GGCX polymorphisms associated with warfarin dose. Pharmacogenomics J. 2005; 5: 262–270.[CrossRef][Medline] [Order article via Infotrieve]
    
23. Yuan HY, Chen JJ, Lee MT, Wung JC, Chen YF, Charng MJ, Lu MJ, Hung CR, Wei CY, Chen CH, Wu JY, Chen YT. A novel functional VKORC1 promoter polymorphism is associated with inter-individual and inter-ethnic differences in warfarin sensitivity. Hum Mol Genet. 2005; 14: 1745–1751.[Abstract/Free Full Text]
    
24. Rieder MJ, Reiner AP, Gage BF, Nickerson DA, Eby CS, McLeod HL, Blough DK, Thummel KE, Veenstra DL, Rettie AE. Effect of VKORC1 haplotypes on transcriptional regulation and warfarin dose. N Engl J Med. 2005; 352: 2285–2293.[Abstract/Free Full Text]
    
25. Sconce EA, Khan TI, Wynne HA, Avery P, Monkhouse L, King BP, Wood P, Kesteven P, Daly AK, Kamali F. The impact of CYP2C9 and VKORC1 genetic polymorphism and patient characteristics upon warfarin dose requirements: proposal for a new dosing regimen. Blood. 2005; 106: 2329–2333.[Abstract/Free Full Text]
    
26. Price PA, Faus SA, Williamson MK. Warfarin causes rapid calcification of the elastic lamellae in rat arteries and heart valves. Arterioscler Thromb Vasc Biol. 1998; 18: 1400–1407.[Abstract/Free Full Text]
    
27. Luo G, Ducy P, McKee MD, Pinero GJ, Loyer E, Behringer RR, Karsenty G. Spontaneous calcification of arteries and cartilage in mice lacking matrix GLA protein. Nature. 1997; 386: 78–81.[CrossRef][Medline] [Order article via Infotrieve]
    
28. Fitzgerald PJ, Ports TA, Yock PG. Contribution of localized calcium deposits to dissection after angioplasty: an observational study using intravascular ultrasound. Circulation. 1992; 86: 64–70.[Abstract/Free Full Text]
    
29. Kondos GT, Hoff JA, Sevrukov A, Daviglus ML, Garside DB, Devries SS, Chomka EV, Liu K. Electron-beam tomography coronary artery calcium and cardiac events: a 37-month follow-up of 5635 initially asymptomatic low- to intermediate-risk adults. Circulation. 2003; 107: 2571–2576.[Abstract/Free Full Text]
    
30. London GM, Guerin AP, Marchais SJ, Metivier F, Pannier B, Adda H. Arterial media calcification in end-stage renal disease: impact on all-cause and cardiovascular mortality. Nephrol Dial Transplant. 2003; 18: 1731–1740.[Abstract/Free Full Text]
    
31. Sun L, Li Z, Zhang H, Ma A, Liao Y, Wang D, Zhao B, Zhu Z, Zhao J, Zhang Z, Wang W, Hui R. Pentanucleotide TTTTA repeat polymorphism of apolipoprotein(a) gene and plasma lipoprotein(a) are associated with ischemic and hemorrhagic stroke in Chinese: a multicenter case-control study in China. Stroke. 2003; 34: 1617–1622.[Abstract/Free Full Text]
    
32. Li Z, Sun L, Zhang H, Liao Y, Wang D, Zhao B, Zhu Z, Zhao J, Ma A, Han Y, Wang Y, Shi Y, Ye J, Hui R; Multicenter Case-Control Study in China. Elevated plasma homocysteine was associated with hemorrhagic and ischemic stroke, but methylenetetrahydrofolate reductase gene C677T polymorphism was a risk factor for thrombotic stroke: a Multicenter Case-Control Study in China. Stroke. 2003; 34: 2085–2090.[Abstract/Free Full Text]
    
33. De Paepe A, Devereux RB, Dietz HC, Hennekam RC, Pyeritz RE. Revised diagnostic criteria for the Marfan syndrome. Am J Med Genet. 1996; 62: 417–426.[CrossRef][Medline] [Order article via Infotrieve]
    
34. O’Donnell CJ, Lindpaintner K, Larson MG, Rao VS, Ordovas JM, Schaefer EJ, Myers RH, Levy D. Evidence for association and genetic linkage of the angiotensin-converting enzyme locus with hypertension and blood pressure in men but not women in the Framingham Heart Study. Circulation. 1998; 97: 1766–1772.[Abstract/Free Full Text]
    
35. Pritchard JK, Rosenberg NA. Use of unlinked genetic markers to detect population stratification in association studies. Am J Hum Genet. 1999; 65: 220–228.[CrossRef][Medline] [Order article via Infotrieve]
    
36. Dietz HC, Pyeritz RE, Puffenberger EG, Kendzior RJ Jr, Corson GM, Maslen CL, Sakai LY, Francomano CA, Cutting GR. Marfan phenotype variability in a family segregating a missense mutation in the epidermal growth factor-like motif of the fibrillin gene. J Clin Invest. 1992; 89: 1674–1680.[Medline] [Order article via Infotrieve]
    
37. Voetsch B, Loscalzo J. Genetic determinants of arterial thrombosis. Arterioscler Thromb Vasc Biol. 2004; 24: 216–229.[Abstract/Free Full Text]
    
38. Huang H, Virmani R, Younis H, Burke AP, Kamm RD, Lee RT. The impact of calcification on the biomechanical stability of atherosclerotic plaques. Circulation. 2001; 103: 1051–1056.[Abstract/Free Full Text]
    
39. Berkner KL, Runge KW. The physiology of vitamin K nutriture and vitamin K–dependent protein function in atherosclerosis. J Thromb Haemost. 2004; 2: 2118–2132.[CrossRef][Medline] [Order article via Infotrieve]
    
40. The International HapMap Consortium. The International HapMap Project. Nature. 2003; 426: 789–796.[CrossRef][Medline] [Order article via Infotrieve]
    
41. Hak AE, Pols HA, van Hemert AM, Hofman A, Witteman JC. Progression of aortic calcification is associated with metacarpal bone loss during menopause: a population-based longitudinal study. Arterioscler Thromb Vasc Biol. 2000; 20: 1926–1931.[Abstract/Free Full Text]
    
42. Taylor BC, Schreiner PJ, Doherty TM, Fornage M, Carr JJ, Sidney S. Matrix Gla protein and osteopontin genetic associations with coronary artery calcification and bone density: the CARDIA study. Hum Genet. 2005; 116: 525–528.[CrossRef][Medline] [Order article via Infotrieve]
    
43. Jie KS, Bots ML, Vermeer C, Witteman JC, Grobbee DE. Vitamin K intake and osteocalcin levels in women with and without aortic atherosclerosis: a population-based study. Atherosclerosis. 1995; 116: 117–123.[CrossRef][Medline] [Order article via Infotrieve]
    

---

 

CLINICAL PERSPECTIVE

Epidemiological data indicate that coronary atherosclerosis is an important cause of morbidity and mortality worldwide. To minimize the devastating consequence of vascular disease, we must reliably distinguish those individuals who will experience an event from those who will not. The haplotypes in VKORC1 affect warfarin dose response through effects on the formation of the reduced form of vitamin K, a cofactor for -carboxylation of vitamin K–dependent proteins. Vitamin K–dependent proteins play a critical role in the coagulation cascade and may potentially impact atherosclerosis. We tested the association of the single-nucleotide polymorphisms of the VKORC1 gene and arterial vascular diseases including coronary heart disease, stroke, and aortic dissection. We observed that VKORC1 variation is significantly associated with each of these diseases. Although the pathophysiology behind the present findings is not entirely clear, confirmation of these observations in additional studies is warranted. Additional research is necessary to elucidate the potential role of VKORC1 and its downstream products in the pathogenesis of vascular disease.

This article has been cited by other articles:

				N. S. Rost, S. M. Greenberg, and J. Rosand The Genetic Architecture of Intracerebral Hemorrhage Stroke, July 1, 2008; 39(7): 2166 - 2173. [Abstract] [Full Text] [PDF]

				M. Teichert, L.E. Visser, R.H.N. van Schaik, A. Hofman, A.G. Uitterlinden, P.A.G. M. De Smet, J.C.M. Witteman, and B.H.Ch. Stricker Vitamin K Epoxide Reductase Complex Subunit 1 (VKORC1) Polymorphism and Aortic Calcification: The Rotterdam Study Arterioscler. Thromb. Vasc. Biol., April 1, 2008; 28(4): 771 - 776. [Abstract] [Full Text] [PDF]

				W. Song, J. Chen, K. Sun, H. Yu, and R. Hui No Association of PLIN Polymorphisms With Hemorrhagic and Ischemic Stroke Stroke, February 1, 2008; 39(2): 470 - 472. [Abstract] [Full Text] [PDF]

				D. A. Pearson Bone Health and Osteoporosis: The Role of Vitamin K and Potential Antagonism by Anticoagulants Nutr Clin Pract, October 1, 2007; 22(5): 517 - 544. [Abstract] [Full Text] [PDF]

				Y. Wang, Y. Zheng, W. Zhang, H. Yu, K. Lou, Y. Zhang, Q. Qin, B. Zhao, Y. Yang, and R. Hui Polymorphisms of KDR Gene Are Associated With Coronary Heart Disease J. Am. Coll. Cardiol., August 21, 2007; 50(8): 760 - 767. [Abstract] [Full Text] [PDF]

				J. Sanz, P. R. Moreno, and V. Fuster The Year in Atherothrombosis J. Am. Coll. Cardiol., April 24, 2007; 49(16): 1740 - 1749. [Full Text] [PDF]

				M. Dichgans and R. A. Hegele Update on the Genetics of Stroke and Cerebrovascular Disease 2006 Stroke, February 1, 2007; 38(2): 216 - 218. [Full Text] [PDF]

				B. F. Gage, E. Birman-Deych, and M. J. Radford The Association of Warfarin Use With Osteoporotic Fracture in Elderly Patients With Atrial Fibrillation--Reply Arch Intern Med, July 24, 2006; 166(14): 1525 - 1525. [Full Text] [PDF]

				H. M.H. Spronk Vitamin K Epoxide Reductase Complex and Vascular Calcification: Is This the Important Link Between Vitamin K and the Arterial Vessel Wall? Circulation, March 28, 2006; 113(12): 1550 - 1552. [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 113/12/1615    most recent CIRCULATIONAHA.105.580167v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Wang, Y. Articles by Hui, R. Search for Related Content PubMed PubMed Citation Articles by Wang, Y. Articles by Hui, R. Related Collections Clinical genetics Epidemiology