|Year : 2018 | Volume
| Issue : 4 | Page : 206-211
von Willbrand factor Thr789Ala gene variant determines the type of myocardial ischemia in Egyptian patients
Nahla F Osman1, Marwa A Younes1, Reehab I Yaseen2, Amro Fathy1
1 Department of Clinical Pathology, Menoufia University, Shebin Elkom, Egypt
2 Department of Cardiology, Menoufia University, Shebin Elkom, Egypt
|Date of Submission||11-Feb-2017|
|Date of Acceptance||16-Feb-2017|
|Date of Web Publication||10-Apr-2019|
Nahla F Osman
Department of Clinical Pathology, Faculty of Medicine, Menoufia University, Shebin Elkom
Source of Support: None, Conflict of Interest: None
Objective von Willbrand factor antigen level (vWF: Ag) was shown to contribute to the risk of cardiovascular disease. vWF Thr789Ala single nucleotide polymorphism was thought to affect the factor level and function. This study aimed to investigate the impact of genetic variants at that position on the risk of acute coronary syndrome (ACS).
Patients and methods The study included 112 patients of ACS; 31 with unstable angina (UA) and 81 with myocardial infarction (MI) as well as 118 healthy controls. vWF: Ag level was measured by enzyme-linked immunosorbent assay. The gene analysis was carried out by PCR using restriction fragment length polymorphism principles.
Results vWF: Ag levels were significantly higher in patients (111.29±24.43 IU/dl) compared to the controls (71.13±13.72 IU/dl, P<0.001). The majority of patients with UA (80.6%) were Ala789 homozygous, 6.5% were Thr789Ala heterozygous, and 12.9% were Thr789 homozygous. With respect to the MI group, Ala789 genotype was present in 34.6% of the patients, Thr789Ala genotype was the predominant genotype and was seen in 48.1% of the patients, and Thr789 homozygous was present in 17.3% of the patients. The genotype frequency in the control group was as follow; 47.4% were Ala789 homozygous, 33.1% were heterozygous, and 19.5% were Thr789 homozygous. The difference in genotype distribution was significantly different among the three groups (P<0.001), and between the groups with UA and MI (P<0.001). Ala789 homozygous genotype was an independent risk factor for UA while the Thr789Ala genotype was shown as an independent risk factor of MI.
Conclusion vWF Thr789Ala polymorphism is an independent risk factor for ACS and has significant impact on the type of myocardial ischemia. It should be incorporated in the risk assessment model to identify and guide the management in these patients.
Keywords: acute coronary syndrome, gene polymorphism, genotype, risk, vWF
|How to cite this article:|
Osman NF, Younes MA, Yaseen RI, Fathy A. von Willbrand factor Thr789Ala gene variant determines the type of myocardial ischemia in Egyptian patients. Egypt J Haematol 2018;43:206-11
|How to cite this URL:|
Osman NF, Younes MA, Yaseen RI, Fathy A. von Willbrand factor Thr789Ala gene variant determines the type of myocardial ischemia in Egyptian patients. Egypt J Haematol [serial online] 2018 [cited 2020 Mar 31];43:206-11. Available from: http://www.ehj.eg.net/text.asp?2018/43/4/206/255878
| Introduction|| |
Cardiovascular disease is the leading cause of death worldwide accounting for 17.3 million deaths per year. The global burden of cardiovascular mortality for the next decades is expected to predominantly occur among developing countries ,.
Interaction of genetic and acquired factors is known to lead to coronary atherosclerosis and/or thrombosis. Risk stratification in individual patient is crucial in optimizing the treatment plan and prevention of the development or recurrence of ischemic attacks and hence of myocardial damage .
von Willbrand factor (vWF) is synthesized and stored by vascular endothelium. It is involved in thrombus formation at the site of vascular injury through promotion of platelet adhesion and is considered as a key player in arterial thrombosis .
Several studies demonstrated an association between plasma vWF antigen (vWF: Ag) levels, its activity and ischemic heart disease. Genetic and nongenetic factors are known to affect plasma vWF ,.
Single nucleotide polymorphism (SNP) at codon 789 of vWF gene (rs1063856) lies in exon 18 that encodes part of the D’ domain. This SNP results in substitution of the amino acid threonine (Thr) by alanine (Ala) at that position. Three genotypes are recognized, Thr789 homozygous (T/T, wild type), Ala789 homozygote (A/A), and the Thr789Ala heterozygous (T/A) ,.
D’ domain is involved in vWF multimerization, its interaction with endothelium and platelets as well as factor VIII binding; hence, structural change might have significant impact on vWF function as a coagulation protein. In addition, this polymorphism is thought to affect plasma vWF levels and vWF ristocetin cofactor activity (vWF: Ri-Cof) and subsequently might contribute to the genetic susceptibility to cardiovascular disease ,,.
The aim of this study was to explore the association of Thr789Ala SNP of vWF gene and the risk of acute coronary syndrome (ACS).
| Patients and methods|| |
In a single center case–control study, carried out in Menoufia University Hospitals, Egypt, we recruited 112 patients, 35 females and 77 males, with diagnosis of ACS (aged between 38 and 66 years). Of the ACS patients; 31 were diagnosed with unstable angina (UA) and 81 with myocardial infarction (MI). Within the MI group; 20 and 61 patients had the diagnosis of ST-elevation MI and non-ST elevation MI, respectively. The study also included 118 sex- matched healthy controls (40 females and 78 males and aged from 48 to 63 years). The study was approved by the local ethics committee for postgraduate studies and research and an informed consent was obtained from involved subjects.
Diagnosis of MI required the presence of typical chest pain with elevated cardiac enzymes with or without the characteristic ECG changes, whereas diagnosis of UA was made in patients with typical chest pain at rest and confirmed by the standard ECG changes with normal levels of cardiac markers.
Under standardized conditions, blood was collected into citrate containing vacutainer tubes. Plasma was separated and stored at −80°C for subsequent measurement of vWF antigen levels.
For DNA isolation and SNP study, blood was collected into EDTA containing vacutainer tubes. DNA was extracted and stored at −20°C for subsequent genetic analysis.
vWF: Ag plasma levels
Plasma vWF: Ag level was measured by REAADS vWF Antigen ELISA kit (catalog #: K034-001) supplied by Diapharma, West Chester, USA.
- DNA extraction from whole blood was carried out by spin-column technique using Gene Jet Genomic DNA Purification Kit, supplied by ThermoFisher Scientific (Waltham, MA, USA).
- vWF Thr789Ala genotype was determined by restriction fragment length polymorphism–PCR. The sense and antisense primers (supplied by Biosearchtech, Petaluma, CA, USA) were 5′-TGG GCA ACT CTG AGT CTC TT-3′ and 5′-AGA AAA CTG AAG GGC AGG CA-3′ 
- DNA amplification was performed in a reaction mixture volume of 50 µl and comprised of; 1 µl of each of the primers, 1 µl of template DNA and 25 µl of master mix (MyTaq Red Mix Kit, catalog number: BIO-25043; supplied by Bioline, Toronto, Canada) and nuclease-free water.
- After an initial denaturation step of 5 min at 95°C, 35 repeated cycles consisting of, denaturation at 95°C for 30 s, annealing at 60°C and extension at 72°C each for 60 s followed by a final extension step of 10 min at 72°C.
- A final amplification product of 322bp was obtained ([Figure 1]a).
|Figure 1 Agarose gel electrophoresis shows (a) amplification product (b) RSaI restriction digestion of PCR product. (a) PCR amplification; Lane 1: DNA ladder Lanes 2-8: amplification bands of 322bp. (b) products of DNA restriction, Lane 1: DNA ladder, Lanes 3 and 7: Ala789 homozygous, Lanes 2 and 4: Thr789 homozygous, Lanes 5 and 6: Thr789Ala heterozygous and Lane 8: negative control.|
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- DNA restriction was done using RsaI endonuclease (#FD1124; ThermoFisher Scientific, Fermentas, Canada). The digestion mixture contained: 10 μl PCR product, 2 μl restriction enzyme buffer, 1 μl restriction enzyme Rsa1 and finally 17 μl nuclease-free water to make a final volume of 30 μl. Gentle mix was applied and the mixture was incubated for 2 h at 37°C. DNA restriction gave two bands; 200 and 122bp and indicates the presence of the mutant allele (A), whereas the 322bp band in [Figure 1]b.
- The DNA product was visualized on 1.5% agarose-gel electrophoresis using UV illuminator.
Results were collected, tabulated, and statistically analyzed by an IBM compatible personal computer with SPSS statistical package version 20 (IBM SPSS Statistics for Mac, Released 2011; IBM Corp., Armonk, New York, USA; SPSS Inc., Chicago, Illinois, USA). Descriptive statistics were expressed as number, percentage, mean, and SD. Qualitative data were analyzed by χ2. Quantitative data were analyzed by t-test for comparison of two parametric groups. For comparison of more than two parametric groups, one-way analysis of variance was used and post-hoc test was applied to show which group is statistically different. Multivariate analysis was used to identify the independent risk factors in ACS patients. P <0.05 was considered statistically significant.
| Results|| |
Characteristics of involved subjects are presented in [Table 1]. Of the patients, 31 had UA and 81 were diagnosed as MI (20 ST-elevation MI and 61 non-ST-elevation MI). The study also included 118 healthy controls. Patients were significantly younger (52.5±6.65 years) than controls (55.25±6.58 years, P=0.002).
Study of vWF Thr789Ala polymorphism in the involved subjects revealed that 17.8% (n=41) were Thr789 homozygous, 34.8% (n=80) carried the Thr789Ala genotype and 47.4% (n=109) were Ala789 homozygous. Allele frequencies were 64.7 and 35.3% for the A and T alleles respectively. Allele frequencies in subjects from different ethnic groups and in those involved in our study are shown in [Figure 2]a and b, respectively.
|Figure 2 Frequency of the vWF Thr789Ala alleles in different races; quoted from Ahmed et al.  (a) and in Egyptian participants (b). vWF, von Willbrand factor.|
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Plasma vWF levels were measured in 85 participants (55 patients and 30 controls). The factor level was 107.9±28.4 IU/dl in those who were homozygous for the wild allele (T/T, n=15), 115.2±18.6 IU/dl in heterozygous (T/A, n=30), and 112.6±24.4 IU/dl in the A/A homozygous (n=40). There was no significant difference in antigen levels among different genotypes (P=0.599; [Table 2]).
|Table 2 Comparison of von Willbrand factor levels in different genotypes|
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vWF: Ag levels were significantly higher in MI (111.68±24.77 IU/dl) and UA (110.27±23.44 IU/ml) patients compared to healthy controls (71.13±13.72 IU/dl), P less than 0.001 for both groups, with no significant difference between the two patient groups (P=0.9; [Table 3]).
|Table 3 Comparison of von Willbrand factor levels among patients with different types of ischemia and controls|
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Comparison of the genotype distribution among the groups with UA, MI, and controls showed that the majority of patients with UA (80.6%) were A/A, whereas T/A genotype was seen in 6.5% and T/T in 12.9%. With respect to the MI group, A/A genotype was present in 34.6%, T/A was the predominant genotype and was seen in 48.1% of the patients and T/T was present in 17.3% of the patients. The genotype distribution in the control group was as follow; 47.4% were A/A, 33.1% were T/A, and 19.5% were T/T. The genotype distribution was significantly different among the three groups (P<0.001); and between the groups with UA and MI (P<0.001; [Table 4]).
|Table 4 Comparison of von Willbrand factor genotype and allele frequency in different studied groups|
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There was also statistically significant difference in allele frequency among patients with MI, UA, and control groups with the A allele being more frequent in UA group (83.9%) ([Table 4]).
Multivariate regression analysis showed that the A/A genotype is an independent risk factor for UA and is associated with an estimated 6.87 fold increase in the risk of UA [95% confidence interval (CI): 2.448–19.284 and P<0.001]. In addition, T/A genotype was associated with 13.46 folds estimated increase in the risk of MI (95% CI: 3.01–60.20 and P<0.001). vWF antigen level was also identified as independent risk factor for UA (odds ratio=1.43, 95% CI: 1.25–1.64, P<0.001) and MI (odds ratio=1.19, 95% CI: 1.15–1.24, P<0.001).
| Discussion|| |
vWF has been blamed for being a risk factor for arterial thrombosis for decades due to its established role in clot formation. Genetic variations in vWF genes and its relation to the factor level and consequently to the risk of ischemic heart disease are still not clear.
This study, the first report from the region to the best of our knowledge; aimed to address the association of vWF789 genetic variants and ACS.
The current study, in agreement with others , demonstrated significantly higher vWF antigen levels in patients with ACS compared to the control group. This cannot be explained, in our cohort, by the influence of Thr789Ala genetic variants as we did not demonstrate significant relation between vWF789 gene variants and the plasma antigen levels. In fact, increased vWF, in our patients, can be merely a ‘marker’ of endothelial damage rather than a primary event as previous studies reported an association between vWF release and vascular–endothelial damage. However, high vWF level can be a risk for ACS regardless to its cause .
The lack of relation between vWF789 genotype and its plasma level was contradicted by Klemm et al. , whose study was carried out on Caucasians, as they found higher plasma vWF: Ag levels in those with heterozygous genotype (T/A). Lacquemant et al. . also reported an association between vWF antigen levels and Thr789Ala polymorphism; however, unlike Klemm et al. , higher levels were seen in those with A/A genotype.
Our study has elucidated an association between vWF Thr789Ala genetic variants and the type of myocardial ischemia as Ala789 homozygous, and hence the A allele, was associated with increased risk of UA, whereas the Thr789Ala genotype was more prevalent in the MI group. It seems likely that the heterozygous genotype, which was reported previously to be associated with higher (Ri-Cof) activity , have led to an additional event, that is, thrombus formation in predisposed subjects, for example, those with underlying atherosclerosis.
In addition, D’ domain is crucial for a number of vWF procoagulant activities as well as factor VIII binding and hence nonsynonymous polymorphism at that region can alter those functions which in turn can affect clotting mechanisms .
A number of studies investigated the impact of vWF Thr789Ala polymorphism on the risk of coronary heart disease (CHD).
Van Loon et al.  did not find correlation between Thr789Ala variants and the risk of CHD, which was defined as history of MI, percutaneous coronary intervention, or coronary artery bypass grafting. Lack of correlation in their study was explained by involvement of elderly patients (mean age 71.7±6.7) in whom the effect of genetic factors is superseded by environmental and the lifestyle factors .
Lacquemant et al. , in agreement with our findings, have reported an association between vWF Thr789Ala polymorphism and the risk of CHD with increased frequency of Ala789Ala genotype (and hence A allele) and decreased frequency of Thr789Thr genotype in CHD subjects with type 1 diabetes. In addition, Klemm et al.  have identified Ala789 homozygous to be more frequent in nondiabetic patients with CHD. However, they did not demonstrate direct effect of vWF Thr789Ala variant on the development of CHD in patients with type 2 diabetes.
The discrepancy in the results among different studies addressing the relation between vWF789 polymorphism with the antigen level or with risk of CHD is likely due to the diversity in the ethnic background of the studied population, which is known to be associated with significant variation in allele frequency and linkage disequilibrium pattern .
This is supported by data published by Keightly et al.  who suggested that circulating levels of vWF is determined, at least partially, by polymorphic variation in the promoter region, which can be in linkage disequilibrium with other polymorphisms, through affecting gene expression.
In addition, the difference in the characteristics of the involved participants, such as age, medications, and associated comorbidities should have contributed .
This study identified the A/A homozygous as an independent risk factor for UA, whereas the heterozygous genotype was an independent risk factor for MI.
In conclusion, the Thr789Ala polymorphism has an influence on the type of ACS; by a mechanism not involving vWF: Ag levels as the Ala789 homozygous and the Thr789Ala heterozygous were independent risk factors for UA and MI, respectively.
On the basis of our results, vWF Thr789Ala genetic variants and vWF: Ag levels should be incorporated in a risk assessment model to identify precisely the risk and the likely type of ischemia in individual patient. This should allow efficient use of therapeutic options and thus improve the outcome.
This research was supported by Menoufia University Research Grant, Egypt.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Laslett L, Alagona P, Clark B, Drozda J, Saldivar F, Wilson S et al.
The worldwide environment of cardiovascular disease: prevalence, diagnosis, therapy, and policy issues: a report from the American College of Cardiology. J Am Coll Cardiol
Oliveira G, Avezum A, Roever L. Cardiovascular disease burden: evolving knowledge of risk factors in myocardial infarction and stroke through population-based research and perspectives in global prevention. Front Cardiovasc Med
Van Schie M, de Maat M, Issacs A, van Duijin C, Deckers J, Dippel D, Leebeek W. Variation in von Willbrand factor gene is associated with von Willebrand factor levels and with risk of cardiovascular disease. Blood
Xu A, Xu R, Lu C, Y M, Zhao W, Fu X et al.
Correlation of von Willbrand factor gene polymorphism and coronary heart disease. Mol Med Rep
Van Schie MC, van Loon J, de Maat M, Leebeek F. Genetic determinants of von Willbrand factor levels and activity in relation to the risk of cardiovascular disease: a review. J Thromb Haemost
Van Loon J, Kavousi M, Leebeek F, Felix J, Hofman A, Witeman J, Maat M. von Willbrand factor plasma levels, genetic variations and coronary heart disease in older population. J Thromb Haemos
Ahmad F, Kannan M, Biswas A, Saxena R. Impact of 789Ala/Ala genotype on quantitative type of von Willebrand disease. Ann Hematol
Klemm T, Mehnert A, Siegemund A, Wiesner TD, Gelbrich G, Blüher M, Paschke R. Impact of the Thr789Ala variant of the von Willebrand factor levels, on ristocetin co-factor and collagen binding capacity and its association with coronary heart disease in patients with diabetes rtmellitus type 2. Exp Clin Endocrinol Diabetes
Lenting PJ, Christophe OD, Dennis CV. Von willbrand factor biosynthesis, secretionand clearance. connecting the far ends. Blood
Lenting PJ, Casari C, Christophe OD, Denis CV. von Willbrand factor: the old, the new and the unknown. J Thromb Haemos
Kunkel G, Graham J, Fowlkes D, Lord S. Rsa1 polymorphism in von Willbrand factor (vWF) at codon 789. Nucleic Acids Res
Whincup PH, Danesh J, Walker M, Lennon L, Thomson A, Appelby P et al.
von Willebrand factor and coronary heart disease: prospective study and meta-analysis. Eur Heart J
Vischer UM. Von Willbrand factor, endothelial dysfunction and cardiovascular disease. J Thromb Haemos
Lacquemant C, Gaucher C, Delorme C, Chatellier G, Gallois Y, Rodier M et al.
Association between high von Willebrand factor levels and the Thr789Ala vWF gene polymorphism but not with nephropathy in type I diabetes. Kidney Int
Keightley AM, Lam YM, Brady JN, Cameron CL, Lillicrap D. Variation at the von Willebrand factor (vWF) gene locus is associated with plasma vWF:Ag levels: identification of three novel single nucleotide polymorphisms in vWF gene promoter. Blood
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4]