Saudi Journal of Kidney Diseases and Transplantation

ORIGINAL ARTICLE
Year
: 2016  |  Volume : 27  |  Issue : 5  |  Page : 908--915

VKORC1 gene (vitamin K epoxide reductase) polymorphisms are associated with cardiovascular disease in chronic kidney disease patients on hemodialysis


Noha A Osman1, Nevine El-Abd2, Mohamed Nasrallah1,  
1 Department of Nephrology, Kasr Al Ainy School of Medicine, Cairo University, Cairo, Egypt
2 Department of Clinical Pathology, Kasr Al Ainy School of Medicine, Cairo University, Cairo, Egypt

Correspondence Address:
Mohamed Nasrallah
Department of Nephrology, Kasr Al Ainy School of Medicine, Cairo University, Cairo
Egypt

Abstract

Vitamin K is necessary for the carboxylation of clotting factors and matrix Gla protein (MGP). Vitamin K epoxide reductase (VKOR) is the enzyme responsible for recirculation of Vitamin K increasing its tissue availability. Polymorphisms of VKOR may alter the function of MGP, thereby influencing vascular calcification. We conducted this study to investigate the relationship of VKORC1 gene single nucleotide polymorphisms (SNPSQs) to vascular calcification and clinically overt cardiovascular disease in chronic kidney disease (CKD) patients on hemodialysis (HD). The study included 54 CKD patients on HD. We excluded those with diabetes or on anticoagulant therapy. Vascular calcifications were measured using computerized tomography scans and roentgenograms. Prevalent clinically overt cardiovascular disease was reported based on the evidence of documented preexisting major cardiovascular events. Genotype detection for the gene VKORC1 C1173T and G-1639A polymorphisms was carried out by polymerase chain reaction. We found a significant association between C1173T polymorphisms and vascular calcification (odds ratio [OR] = 43, P = 0.001). The mutant T allele was also linked with higher odds of vascular calcification (OR = 8.880, 95% confidence interval [CI] = 3.1-25.4, P = 0.001) and clinically overt cardiovascular disease (OR = 4.7, 95% CI = 1.5-14.7, P = 0.005). VKORC1 G-1639A polymorphisms were not associated with vascular calcification and had lower prevalence of clinically overt cardiovascular disease (OR = 0.07, 95% CI = 0.01-0.4, P = 0.001). In patients with CKD on HD, we found that VKORC1 gene polymorphisms did have an association with prevalent cardiovascular calcification and clinically overt cardiovascular disease, C1173T polymorphisms with higher risk for disease, and G-1639A with lower risk.



How to cite this article:
Osman NA, El-Abd N, Nasrallah M. VKORC1 gene (vitamin K epoxide reductase) polymorphisms are associated with cardiovascular disease in chronic kidney disease patients on hemodialysis.Saudi J Kidney Dis Transpl 2016;27:908-915


How to cite this URL:
Osman NA, El-Abd N, Nasrallah M. VKORC1 gene (vitamin K epoxide reductase) polymorphisms are associated with cardiovascular disease in chronic kidney disease patients on hemodialysis. Saudi J Kidney Dis Transpl [serial online] 2016 [cited 2021 Oct 27 ];27:908-915
Available from: https://www.sjkdt.org/text.asp?2016/27/5/908/190782


Full Text

 Introduction



Vitamin K is a cofactor for the enzymatic conversion of glutamic acid (Glu) residues to gamma-carboxyglutamicacid (Gla) in Vitamin K-dependent proteins via Vitamin K-dependent gamma-glutamyl carboxylase. [1] This carboxylation reaction is responsible for the activation of several proteins. [2]

Vitamin K requirements in humans are very low because the vitamin is recycled by Vitamin K epoxide reductase (VKOR). [3] Inactivation of this enzyme increases the Vitamin K requirements to values above those present in the average diet resulting in functional insufficiency of the vitamin. [3] VKORC1 has been identified some years ago as the gene encoding VKOR. Polymorphisms of this gene have been associated with variations related to the availability of active Vitamin K for carboxylation of coagulation factors, particularly coumarin resistance. [4] Increased levels of coagulation factors associated with these polymorphisms may be associated with vascular events related to hypercoagulability. [5]

Other proteins that require Vitamin Kdependent carboxylation includes matrix Glaprotein (MGP), osteocalcin, and Gla-rich proteins. These proteins are protective for the vasculature and crucial for the prevention of vascular calcification. Impaired expression of gamma-carboxylase is involved in the development of arterial calcification in diabetes possibly due to an inadequate recycling of Vitamin K by VKOR. [6]

Vascular calcification and cardiovascular morbidity/mortality are highly prevalent in patients with chronic kidney disease (CKD) and is not fully explained. [7],[8] We designed this study to investigate the association of VKORC gene polymorphisms with cardiovascular disease in CKD patients on hemodialysis (HD) as denoted by the presence of clinically overt cardiovascular disease and/or vascular calcification.

 Subjects and Methods



Study population

Fifty-four participants attending our university hospital dialysis unit were included in the study. They were 24 males with a mean age of 43.54 ± 12.85 years and 30 females with a mean age of 36.83 ± 11.49 years. Patients on anticoagulation therapy were excluded from the study. All patients were diagnosed with CKD Stage 5 and were receiving 4 h HD sessions, three times weekly, using low flux polysulfone dialyzers and with dialysate calcium of 1.5 mmol/L for at least six months. The study was approved by the Ethical Committee at our university hospital.

Assessment of cardiovascular disease and vascular calcification

Prevalent clinically overt cardiovascular disease was reported based on the presence of documented preexisting major cardiovascular events since the start of HD treatment, namely, myocardial infarction, heart failure, episodes of acute coronary syndrome, nonhemorrhagic cerebrovascular disease, and/or significant peripheral vascular disease.

Vascular calcification was measured in the aorta using computerized tomography (CT) scans of the abdominal aorta and lateral lumbar roentgenograms. CT scans were used to calculate the aortic calcification index after recording the calcified segments present in the abdominal aorta as described in detail elsewhere. [7],[8] Lateral lumbar X-rays were used to view linear calcifications in the abdominal aorta opposite the lumbar vertebrae and calculate the Kauppila score as detailed by Kauppila et al. [9] Peripheral vascular calcification was assessed using plain roentgenograms of the pelvis, forearms, hands, and upper thighs using a simple scoring system described by Adragão et al. [10] Results were reported qualitatively as positive or negative.

Laboratory parameters

Specimen collection: blood samples were obtained immediately before mid-week dialysis sessions. Two milliliters of venous blood was put into a sterile ethylenediaminetetraacetic acid (EDTA) vacutainer tube and stored at −20°C to be used for the genotyping technique.

Laboratory methods: all routine laboratory tests were performed on automated Beckman Coulter AU480 analyzer (Beckman Coulter, Inc., 250 S. Kraemer Blvd., Brea, CA 92821, USA). PTH was done on Cobas-e 411 (Roche Diagnostics GmbH Strasse116, D-68305 Mannheim). Hemoglobin was done on Beckman Coulter LH 750 analyzer.

Genotyping of Vitamin K epoxide reductase complex, subunit 1

DNA was extracted from 200 μL EDTA blood and eluted in 100 μL elution buffer according to the manufacturer's protocol with a MagNA Pure Compact LC (Roche Diagnostics) using MagNA Pure LC DNA High-Performance isolations kit (Roche Diagnostics). The Light Mix® kit for the detection of human VKORC1 C1173T and VKORC1 G-1639A DNA were used; it was tested with the Roche Diagnostics "LightCycler® Fast Start DNA Master Hybridization Probe" ready-to-use reaction mix in the LightCycler 2.0 Instrument.

A 176 bp fragment and a 289 bp fragment of the human VKORC1 gene were amplified with specific primers. Human VKORC1 C1173T gene was analyzed with a simple probe and human VKORC1 G-1639A gene was analyzed with LightCycler red labeled hybridization probes. The primers and probes were designed and custom-made by TIB MOLBIOL (Berlin, Germany). For mutation detection with the LightCycler 2.0, a 20 μL reaction was performed. Polymerase chain reaction cycling conditions were: 120 s at 95°C for DNA denaturation, 50 cycles, 5 s at 95°C (denaturation), 8 s at 55°C (annealing), and 10 s at 72°C (extension). The genotypes were identified by running a melting curve with specific melting points (Tm). The wild type VKORC1 C1173 exhibits a Tm of 51.8°C in channel 530 and the wild-type VKORC1 G-1639 a Tm of 52.6°C in channel 640. The allele variant VKORC1 C-1173T exhibits a Tm of 58.1°C in channel 530, and the allele variant VKORC1 G-1639A exhibits a Tm of 61.3°C in channel 640. A color compensation file was generated with the TIB MOLBIOL "Light Mix Color Compensation" 530/640 for identifying the genotypes in the corresponding channel (melting curves of heterozygous genotype of the studied single nucleotide polymorphisms (SNPs) are illustrated in [Figure 1] and [Figure 2].{Figure 1}{Figure 2}

The investigators who examined the radiological studies were blinded to the clinical and laboratory data of the study participants and vice versa.

 Statistical Analysis



Statistical Package for Social Analysis (SPSS) version 22 (IBM SPSS Statistics for Windows, Version 22.0. Armonk, NY: IBM Corp.) was used for data analysis. Data were summarized as percentage, mean, and standard deviation (SD). Based on the sample distribution and the relationship between different samples, comparisons were performed using Mann-Whitney U-test. Pearson's Chi-squared test was used for categorical data and Fisher's exact test for cells with expected counts of <6. Logistic regression analysis was used to verify the independence of the reported associations. P ≤0.05 was considered statistically significant.

 Results



General characteristics of the studied patients are shown in [Table 1].{Table 1}

VKORC1 C1173T Polymorphisms

We found a significant positive association between C1173T SNP's (homozygous and heterozygous compared to homozygous CC wild-type) and vascular calcification [Table 2]. This association remained valid after correction for age, diabetes mellitus (DM), body mass index (BMI), duration on HD, and systolic blood pressure using logistic regression analysis (P = 0.001). Moreover, the mutant T allele was also linked with higher odds ratio (OR) of vascular calcification (OR = 8.9, 95% confidence interval [CI] = 3.1-25.4, P = 0.001).{Table 2}

There was no association between the presence of preexisting cardiovascular disease and C1173T polymorphism [Table 3]. However, the mutant allele (T) was associated with higher OR of having clinically overt cardiovascular disease (OR = 4.7, 95% CI = 1.5-14.7, P = 0.005).{Table 3}

VKORC1 G-1639A polymorphisms

VKORC1 G-1639A polymorphisms (homozygous and heterozygous compared to homozygous GG wild-type) were not associated with vascular calcification [Table 2]. However, we found the mutant allele was significantly associated with lower OR of vascular calcification (OR = 0.12, 95% CI = 0.03-0.4, P = 0.001).

Patients carrying the genotypes with the mutant allele had a significantly lower prevalence of clinically overt cardiovascular disease [Table 3]. This association was confirmed by logistic regression corrected for age, BMI, DM, smoking, and duration on CKD and vascular calcification, P = 0.02. Furthermore, the mutant allele (A) was linked to lower OR for the presence of overt cardiovascular disease compared to the wild allele (G) (OR = 0.07, 95% CI= 0.02-0.3, P <0.001).

 Discussion



VKORC1 gene polymorphisms were associated with prevalent cardiovascular calcification, and clinically overt cardiovascular disease in patients with CKD on HD. C1173T polymorphism was associated with higher odds of vascular calcification and clinical cardiovascular disease, whereas VKORC1 G-1639A polymorphism was associated with lower odds.

We found a significant association between VKORC1 gene polymorphism C1173T [both homozygous (TT) and heterozygous (CT) in comparison to the homozygous wild-type (CC)] and vascular calcification. Moreover, the mutant T allele was also linked with higher OR of vascular calcification. This could be explained by the role of Vitamin K in the carboxylation of MGP. MGP is expressed in a variety of tissues and undergoes posttranslational modification by glutamate carboxylation. [3],[11] The glutamate carboxylation step is a crucial activation step that paves the way to the binding with the bone morphogenetic protein 2 (BMP2) preventing the transdifferentiation of vascular smooth muscle cells to osteoblast-like cells and hence protecting against vascular calcification. This binding is activated by the carboxylated form of Vitamin K which is provided by the enzyme VKORC1. [3],[11] Individuals with VKORC1 gene polymorphism C1173T have lifelong reduced activity of the enzyme VKORC1 and were shown to require the lower doses of warfarin for anticoagulation due to the inadequate recirculation of Vitamin K, [12],[13] and possibly also produce less active MGP resulting in enhanced vascular calcification. Our results concord with the findings of Teichert et al who found that this polymorphism was associated with increased risk of calcification of the aortic wall in a large population-based cohort without CKD. [14]

We have also shown that the VKORC1 gene SNPC1173T was associated with higher odds of having clinically overt cardiovascular disease which could be easily explained by the role played by vascular calcification in the development of cardiovascular diseases, especially in the studied uremic population. [15] Vascular calcification is highly prevalent among CKD patients [7],[8],[16],[17] and may be responsible for isolated systolic hypertension and is associated with left ventricular hypertrophy and coronary heart disease. [18]

We have also found that VKORC1 G-1639A SNP and the mutant allele A were significantly associated with lower odds of vascular calcification. This is unlike what we anticipated and hypothesized since this SNP is probably associated with lower VKORC1 activity and thus lower Vitamin K carboxylation and thus lower levels of carboxylate MGP. It is noteworthy here to highlight two facts. First, not all studies investigating VKORC1 and cardiovascular disease have yielded uniform results. [14],[19],[20],[21] Second, the only study investigating VKORC1 polymorphisms and cardiovascular risk in CKD (pre-dialysis patients) has revealed findings such as our findings with the G-1639A polymorphism, i.e., that polymorphisms increasing VKOR activity conferred cardiovascular risk unlike what would be expected due to anticipated drop of MGP levels. [19] The authors interpreted this counter intuitive finding by showing that this polymorphism was associated with increased levels of Vitamin K-dependent coagulation factors that could pose an increased cardiovascular risk. [19] It is possible that the uremic milieu, present in the CKD population, may modify the expression of the VKOR gene. This may disrupt the delicate balance needed between protective Vitamin K-dependent factors as MGP and potentially harmful Vitamin K-dependent coagulation factors. However, further evidence is needed to support this hypothesis.

It is noteworthy that our study is the first to investigate these SNP's of the VKORC1 gene in CKD patients on HD and link them to clinically overt cardiovascular disease as well as its surrogate, vascular calcification. The main drawback of the study is the relatively small number of patients included. We have tried to overcome this weakness using a very sensitive technique, namely, aortic calcification index using CT scans to assess vascular calcification. [7],[8],[16],[17] This study raises the possibility of a potentially modifiable risk factor for cardiovascular disease in CKD. The results need to be confirmed in larger studies possibly involving the measurement of carboxylated Vitamin K levels, MGP, and coagulation factors to interpret the results evidently.

Conflict of interest: None declared.

References

1Vermeer C, De Boer-Van den Berg MA. Vitamin K-dependent carboxylase. Haematologia (Budap) 1985;18:71-97.
2McDonald JF, Shah AM, Schwalbe RA, Kisiel W, Dahlbäck B, Nelsestuen GL. Comparison of naturally occurring Vitamin K-dependent proteins: correlation of amino acid sequences and membrane binding properties suggests a membrane contact site. Biochemistry 1997;36:51207.
3Theuwissen E, Smit E, Vermeer C. The role of Vitamin K in soft-tissue calcification. Adv Nutr 2012;3:166-73.
4Müller E, Keller A, Fregin A, Müller CR, Rost S. Confirmation of warfarin resistance of naturally occurring VKORC1 variants by coexpression with coagulation factor IX and in silico protein modelling. BMC Genet 2014;15:17.
5Leung A, Huang CK, Muto R, Liu Y, Pan Q. CYP2C9 and VKORC1 genetic poly-morphism analysis might be necessary in patients with Factor V Leiden and pro-thrombin gene G2021A mutation(s). Diagn Mol Pathol 2007; 16:184-6.
6Doyon M, Mathieu P, Moreau P. Decreased expression of γ-carboxylase in diabetesassociated arterial stiffness: impact on matrix Gla protein. Cardiovasc Res 2013; 97:331-8.
7Kabaya T, Nitta K, Kimura H, Kawashima A, Narusawa K, Nihei H. Increased aortic calcification index in hemodialysis patients. Nephron 1999;81:354-5.
8NasrAllah M M, Nassef A, Elshaboni T H, Morise F, Osman N A, Sharaf El Din U A. Comparing different calcification scores to detect outcomes in chronic kidney disease patients with vascular calcification. Int J Cardiol 2016; 220: 884-889
9Kauppila LI, Polak JF, Cupples LA, Hannan MT, Kiel DP, Wilson PW. New indices to classify location, severity and progression of calcific lesions in the abdominal aorta: a 25year follow-up study. Atherosclerosis 1997;132: 245-50.
10Adragão T, Pires A, Birne R, et al. A plain Xray vascular calcification score is associated with arterial stiffness and mortality in dialysis patients. Nephrol Dial Transplant 2009;24:9971002.
11Schurgers LJ, Cranenburg EC, Vermeer C. Matrix Gla-protein: the calcification inhibitor in need of Vitamin K. Thromb Haemost 2008;100: 593-603.
12D'Andrea G, D'Ambrosio RL, Di Perna P, et al. A polymorphism in the VKORC1 gene is associated with an interindividual variability in the dose-anticoagulant effect of warfarin. Blood 2005;105:645-9.
13Rieder MJ, Reiner AP, Gage BF, et al. Effect of VKORC1 haplotypes on transcriptional regulation and warfarin dose. N Engl J Med 2005; 352:2285-93.
14Teichert M, Visser LE, van Schaik RH, et al. Vitamin K epoxide reductase complex subunit 1 (VKORC1) polymorphism and aortic calcification: the Rotterdam Study. Arterioscler Thromb Vasc Biol 2008;28: 771-6.
15Sigrist M, Bungay P, Taal MW, McIntyre CW. Vascular calcification and cardiovascular function in chronic kidney disease. Nephrol Dial Transplant 2006;21:707-14.
16Nasrallah MM, El-Shehaby AR, Osman NA, et al. The association between fibroblast growth factor-23 and vascular calcification is mitigated by inflammation markers. Nephron Extra 2013; 3:106-12.
17Nasrallah MM, El-Shehaby AR, Osman NA, Salem MM, Nassef A, El Din UA. Endogenous soluble receptor of advanced glycation endproducts (esRAGE) is negatively associated with vascular calcification in non-diabetic hemodialysis patients. Int Urol Nephrol 2012; 44:1193-9.
18Ketteler M, Biggar PH, Liangos O. FGF23 antagonism: the thin line between adap-tation and maladaptation in chronic kidney disease. Nephrol Dial Transplant 2013;28: 821-5.
19Holden RM, Booth SL, Tuttle A, et al. Sequence variation in Vitamin K epoxide reductase gene is associated with survival and progressive coronary calcification in chronic kidney disease. Arterioscler Thromb Vasc Biol 2014;34:1591-6.
20Wang Y, Zhang W, Zhang Y, et al. VKORC1 haplotypes are associated with arterial vascular diseases (stroke, coronary heart disease, and aortic dissection). Circulation 2006;113:1615-21.
21Ortak H, Sögüt E, Demir H, Ardagil A, Benli I, Sahin S. Predictive value of the Vitamin K epoxide reductase complex subunit 1 G-1639A and C1173T single nucleotide polymorphisms in retinal vein occlusion. Clin Experiment Ophthalmol 2012;40:743-8.