|Year : 2020 | Volume
| Issue : 1 | Page : 8-15
Genetic variants of vascular endothelial growth factor gene polymorphism affect the risk and severity of ischemic stroke: a case–control study
Muhammad T Abdel Ghafar1, Osama Ragab2, Ayman M Al-Malt2, Radwa A Eissa3, Sara Mina Samy3, Sarah R Abd El-Khalik4, Hanem Rabahd4
1 Department of Clinical Pathology, Faculty of Medicine, Tanta University, Tanta, Egypt
2 Department of Neuropsychiatry, Faculty of Medicine, Tanta University, Tanta, Egypt
3 Department of Microbiology & Immunology, Faculty of Medicine, Tanta University, Tanta, Egypt
4 Department of Medical Biochemistry, Faculty of Medicine, Tanta University, Tanta, Egypt
|Date of Submission||27-Aug-2020|
|Date of Decision||12-Oct-2020|
|Date of Acceptance||01-Dec-2020|
|Date of Web Publication||21-Jul-2021|
Muhammad T Abdel Ghafar
Department of Clinical Pathology, Faculty of Medicine, Medical Campus, Tanta University, Tanta
Source of Support: None, Conflict of Interest: None
Background and objectives
Vascular endothelial growth factor (VEGF) is the key mediator of angiogenesis and atherosclerosis. Hence, it may play a major role in the pathogenic mechanisms underlying ischemic stroke (IS) progression. Thus, we conducted this case–control study to explore the genetic association of a single gene polymorphism of VEGF + 936C/T (rs3025039) with the risk and severity of IS and its relation with serum VEGF level.
Patients and methods
This study included a case series of 49 patients with IS with 41 controls. VEGF + 936C/T (rs3025039) variants were determined via Taqman allelic discrimination PCR. Serum VEGF level was estimated using enzyme-linked immunosorbent assay. The frequency of VEGF rs3025039 genotypes and alleles was calculated manually in both cases and controls. The risk ratio was estimated and represented by odds ratio (OR) and 95% confidence interval (CI) adjusted to the confounding variables via multinomial logistic regressions. The stroke severity was assessed by National Institute of Health Stroke Scale. Moreover, serum VEGF levels were compared between the studied groups and among different genotypes and alleles of IS cases.
Our study detected significantly increased frequencies of TT genotype (P = 0.030) and T allele (P = 0.045) in IS cases when compared with the controls. TT genotype represents an increased adjusted risk for IS progression by 9.94 folds (OR: 9.938, 95% CI: 1.235–79.97) and T allele by 2.19 folds (OR: 2.191, 95% CI: 1.004–4.782) over the CC genotypes and C allele, respectively. Moreover, TT genotypes are associated with higher National Institute of Health Stroke Scale. The serum VEGF level was significantly lower in IS cases than controls, and a more decreased level was associated with the T allele carriers in IS cases groups.
VEGF rs3025039 gene polymorphism is associated with increased risk and severity of IS and decreased VEGF expression, which may play a major role in the pathogenesis of IS progression.
Keywords: genotypes, ischemic stroke, polymorphism, risk, vascular endothelial growth factor
|How to cite this article:|
Abdel Ghafar MT, Ragab O, Al-Malt AM, Eissa RA, Samy SM, Abd El-Khalik SR, Rabahd H. Genetic variants of vascular endothelial growth factor gene polymorphism affect the risk and severity of ischemic stroke: a case–control study. Egypt J Lab Med 2020;32:8-15
|How to cite this URL:|
Abdel Ghafar MT, Ragab O, Al-Malt AM, Eissa RA, Samy SM, Abd El-Khalik SR, Rabahd H. Genetic variants of vascular endothelial growth factor gene polymorphism affect the risk and severity of ischemic stroke: a case–control study. Egypt J Lab Med [serial online] 2020 [cited 2022 Dec 3];32:8-15. Available from: http://www.ejlm.eg.net/text.asp?2020/32/1/8/321899
| Introduction|| |
Stroke is a highly prevalent health condition, presenting with the second highest mortality rate and the third highest disability rate after coronary heart disease worldwide . Ischemic stroke (IS) occupies ∼ 80% of this condition, which occurs as an end result of occlusion of the cerebral vessels . It is a complex disorder with involvement of many etiological factors, including demographic, environmental, and morbidity conditions . The demographic factors include age, sex, and race, whereas environmental factors involve smoking and alcohol consumption. The underlying morbidity conditions such as diabetes, hypertension, obesity, cardiovascular disorders, and dyslipidemia are also found to predispose to IS . All of these risk factors cannot completely explain the high incidence of cerebral stroke in different populations. Thus, genetic background was found to influence the risk for IS progression . Polymorphic variants affecting multiple genes such as apolipoprotein ε4 , nitric oxide synthase , and endothelin 1 are suggested to be associated with IS risk in different ethnic groups. Hence, investigating the genetic role in the pathogenesis of IS is required to improve the preventive and therapeutic measures for this disease.
Up to now, cerebral atherosclerosis is considered one of the major causes for IS with the proliferation and formation of new blood vessels in atheromatous plaque leading to microthrombi formation, as well as atheromatous plaque instability or rupture .
Vascular endothelial growth factor (VEGF) is the main mediator of angiogenesis . However, its role in the pathogenesis of atherosclerosis is contradictory and needs to be further clarified . It is encoded by a gene present on chromosome 6 (6p21.3) and is composed of 14-kb coding region with eight exons and seven introns . The VEGF family is a group of seven proteins: six VEGF (A–F) and placental growth factor. Its action is mediated via interaction with high-affinity tyrosine kinases receptor VEGFR2 . It is distributed mainly in the vascular endothelial cells, smooth muscle cells, and macrophages. However, it is also expressed in some malignant cells.
More than 30 VEGF gene single nucleotide polymorphisms (SNPs) have been identified and found to be associated with multiple disorders such as osteosarcoma, renal cell carcinoma, diabetic retinopathy, and autoimmune disorders [14–17]. Among these polymorphisms, three common polymorphisms were extensively studied and found to affect the VEGF expression level or signaling activity, such as +936C/T (rs3025039), −2578C/A (rs699947), and − 1154G/A (rs1570360) . Some of these VEGF gene SNPs had been suggested to be related with IS risk in several studies . Thus, we conduct this case–control study to explore the possible association of a single nucleotide polymorphism +936C/T (rs3025039) with genetic susceptibility and severity of IS and to assess its relation to VEGF expression via estimation of its serum level.
| Patients and methods|| |
This case–control study was performed on 49 cases (group I) with IS who were admitted to the ICU of Neuropsychiatry Department at Tanta University Hospitals and were diagnosed clinically by a neurologist based on the presence of clinical signs of acute neurological deficit that were continued for more than 1 day and confirmed by either brain MRI and/or cranial computed tomography (CT) scan according to the updated definition of stroke of American Stroke Association . The different pathological types of IS were determined according to the Trial of Org 10172 in Acute Stroke Treatment (TOAST) criteria and cases with cardio-embolic, lacunar subtypes were excluded. We excluded also all other cases suspected to have cranial hemorrhage, coronary artery disease, arteritis, tumors, and head trauma. All patients presented beyond the thrombolytic therapy time window, so they had not received recombinant tissue plasminogen activator or mechanical thrombectomy. Patients with IS were treated according to American Stroke Association guideline for management of IS .
Furthermore, we recruited 41 participants from the Outpatient Clinic of Neuropsychiatry Department at Tanta University Hospitals experiencing neurological disorders other than stroke with no previous history of any types of cerebrovascular diseases, tumors, and coronary artery diseases. They served as a study control (group II). The recruitment started in June 2018 till May 2019. All participants were asked to sign an informed consent. The study protocol was approved by Ethics Committee of Faculty of Medicine, Tanta University, and the study was performed in accordance with Helsinki Declaration.
Data collection and assessment
Full medical data were obtained from all participants. A questionnaire concerning the family history of IS and history of smoking, diabetes, or hypertensive disorder was applied. A complete clinical examination with measurement of blood pressure and calculation of BMI was performed. All participants were examined by ultrasonography to evaluate the carotid system and measure the intima-media thickness (IMT) of common carotid artery, using Ultrasound Philips HD (22100 Bothell Everett Hwy, Bothell, WA 98021, USA) 11 linear array transducer of multifrequency (3–12 MHz), in real time and in sagittal, coronal, and axial views. IMT considered abnormal if more 0.9 mm and indicate the presence of atherosclerosis . Echocardiography to exclude cardio-embolic causes of stroke, lipid profile, fasting blood glucose (FBS), and glycated hemoglobin (HbA1C) were done. Stroke severity was assessed by the validated Arabic version of the National Institute of Health Stroke Scale (NIHSS) on admission and 3 months after stroke onset .
For the laboratory analysis, all participants were asked for a venous blood sample. Overall, 7–10 ml was withdrawn by a sterile syringe and evacuated into one plain vacutainer tube, and then into two K3 EDTA vaccutainer tubes. The plain tubes were centrifuged for 15 min at 3000 rpm, and the serum was separated into two aliquots, where one was used for immediate biochemical analysis for FBS and lipids, and the other aliquot was stored at − 20°C for estimation of VEGF serum level. One of the EDTA tubes was used for HBA1C determination, and the other was stored at − 20°C for VEGF rs3025039 (+936C/T) genotyping.
Routine biochemical analysis
FBS, total cholesterol, triglyceride, high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol were measured on automated chemistry analyzer, Konelab-60I (Thermo Scientific, Vantaa, Finland), using commercial kits provided by Thermo-Fisher Scientific (Waltham, Massachusetts, USA). The HbA1C was measured by immune-turbidimetric method using commercial HbA1C kits on Twin A1c semi-automated analyzer, provided by Spectrum Diagnostics (Obour City, Egypt).
Serum vascular endothelial growth factor estimation
The quantitative estimation of VEGF was performed via enzyme immunoassay sandwich technique (enzyme-linked immunosorbent assay) for all of the study participants in the serum sample using DRG Human VEGF ELISA Kit, USA, catalog no EIA-4819 (304 Route 22 West, Springfield, NJ 07081, USA). The instructions for reagent preparation and assay procedure were obtained from the package insert, and accordingly, the standard provided was reconstituted in 1-ml sample diluent to obtain a 4200 pg/ml concentrate, and then diluted serially to obtain seven concentrations starting from 0 to 1200 pg/ml. In each well of 96 precoated microtiter plate with monoclonal antibody specific for human VEGF, 50 μl of each standard and samples was added. After washing, biotin-conjugated VEGF antibody and HRP-streptavidin were added sequentially. After repeated washing, the enzyme detection was performed via adding the substrate (TMB), and then the reaction was stopped by acidification. The color developed was measured as absorbance on Tecan Spectra II micro-plate reader (Switzerland) at 450 nm. The standard curve was displayed in logit-log manner with standard concentrations versus their absorbance values, and the sample concentration was calculated from the curve. The interassay and intraassay CVs were less than 10.0 and less than 6.0%, respectively.
Determination of vascular endothelial growth factor rs3025039 (+936C/T) polymorphism
The genomic DNA was isolated from the peripheral blood leukocytes using human DNA extraction kit, GeneJET genomic DNA purification kit (Thermo-Fisher Scientific), according to the manufacturer's protocol. The DNA yield was quantified on the ultraviolet spectrophotometer (Jenway, Staffordshire, UK), and the purity was determined as 260/280 ratio. The DNA yield was used as a template for amplification and determination of VEGF rs3025039 (+936C/T) via Taqman SNP real-time PCR allele discrimination method. The primers used for the amplification was provided in the following sequences (For. 5′-AAGGAAGA GGAGACTCTGCGCAGAGC-3′ and Rev.5′- CCTGTAGACACACCCAC CCACATACATACATTTA-3′). The reaction was prepared in 25-μl mixture comprising the following: 2 × Taqman genotyping master mix, Taqman SNP genotyping assay (Thermo-Fisher Scientific); 20 ng of the extracted genomic DNA; and nuclease-free water. The amplification protocol was an initial degeneration (95°C/10 min) and then 40 cycles (95°C/15 s, 60°C/1 min). In all samples, cycle threshold was detected for each genotype, and the results were displayed on a multicomponent plot using Applied Biosystem (1241 E Hillsdale Blvd #270, Foster City, CA 94404, USA), step I version software analysis modules.
The genotype and allele frequency of VEGF gene SNP (rs3025039) were calculated by manual counting. The genotype distribution was analyzed according to the Hardy–Weinberg equilibrium in the control group. The relative risk of VEGF gene SNP (rs3025039) for IS was expressed as odds ratio (OR) with the corresponding 95% confidence interval (95% CI). Multiple logistic regression analysis was used for adjustment of the risk ratio for the confounding variables such as age, sex, BMI, family history, smoking, history of diabetes mellitus and hypertension, and IMT. Other results of this study were statistically analyzed using Student t test for normally distributed variables, Mann–Whitney U test for non-normally distributed variables, and χ2 for nominal variables. Analysis of variance test was used for comparing the basic characteristics of IS cases among the different genotypes. Two-sided P value was employed, with value less than 0.05 as significant. The statistical analysis was performed using SSPS software, version 22, and the graph was designed using graphpad software (IBM Corp., version 22.0, Armonk, NY, USA).
| Results|| |
Basic features of the study population
In this case–control study, 49 cases with IS were included in addition to 41 participants as controls. The IS cases were aged 57.1 ± 5.52 years, with 28 males and 21 females. They were matched with the control group regarding the age and sex distribution. Overall, 57.1% of the IS cases were diabetic and 49.0% were hypertensive. NIHSS of IS cases was 15.29 ± 4.6. The case group had a significantly increased BMI, systolic blood pressure, diastolic blood pressure, IMT, FBS, HbA1C, and serum lipids when compared with the control group [Table 1].
Distribution of vascular endothelial growth factor rs3025039 (+936C/T) genotypes between the studied groups
We detected that the dominant TT genotype is significantly higher than the CC genotype in the IS cases when compared with the controls (OR: 4.800, 95% CI: 1.137–20.27). Moreover, the T allele presents 1.83-fold increased risk for IS over the C allele (OR: 1.828, 95% CI: 1.009–3.313). After adjustment for the confounding variables such as age, sex, BMI, family history, smoking, history of diabetes mellitus and hypertension, and IMT, this association increases to 9.94 folds for TT genotypes and to 2.19 folds for T allele when compared with CC genotype and C allele, respectively [Table 2].
|Table 2: Distribution of vascular endothelial growth factor rs3025039 (+936C/T) genotypes and alleles between the studied groups|
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Vascular endothelial growth factor rs3025039 (+936C/T) genotypes and the characteristics of the ischemic stroke case group
In this study, a higher percentage of VEGF rs3025039 TT genotype was observed in patients with IS who were hypertensive, smoker, or had a positive family history for IS. Furthermore, TT genotype was associated with a significantly higher NIHSS (P < 0.001), IMT (P = 0.007), FBS (P = 0.005), HbA1C (P = 0.007), total cholesterol (P = 0.009), and low-density lipoprotein cholesterol (P = 0.004) levels and lower high-density lipoprotein cholesterol (P = 0.008) level than the CC genotype in the IS cases [Table 3].
|Table 3: Distribution of the basic characteristics of the ischemic stroke cases among the different vascular endothelial growth factor rs3025039 (+936C/T) genotypes|
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Vascular endothelial growth factor rs3025039 (+936C/T) polymorphism and stroke severity
NIHSS has been used for assessment of the stroke severity among the case group at admission (baseline) and after 3 months following treatment (follow-up), and the difference was expressed as a percent of change. Among the different VEGF rs3025039 (+936C/T) genotypes, TT genotype carriers were associated with a higher NIHSS at admission and after 3 months following treatment when compared with CC genotype carriers. The percent of improvement in NIHSS was significantly lower in TT genotypes than CC genotypes [Table 4].
|Table 4: Distribution of NHISS in ischemic stroke cases among the different vascular endothelial growth factor rs3025039 (+936C/T) genotypes|
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Vascular endothelial growth factor rs3025039 (+936C/T) polymorphism and serum vascular endothelial growth factor level
The median serum VEGF level in IS cases was 126.0 pg/ml, with a range of 40.0–421.0, and in the control group was 348.0 pg/ml, with a range of 77.0–751.0. The median serum VEGF level in the IS group was significantly lower than that in the control group (P < 0.001) [Figure 1]. No significant differences were detected in serum VEGF levels among the three VEGF rs3025039 (+936C/T) genotypes in IS cases; however, patients with T allele had a significantly lower serum VEGF level than C allele (P = 0.021) [Table 5].
|Figure 1: Serum VEGF level between the studied groups. VEGF, vascular endothelial growth factor.|
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|Table 5: Serum vascular endothelial growth factor level distribution among different vascular endothelial growth factor rs3025039 genotypes and alleles in ischemic stroke cases|
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| Discussion|| |
The pathogenesis of acute IS is heterogeneous with involvement of multiple factors. The genetic epistasis may play a role in the predisposition for IS, which is different in various ethnic groups. Recognition of these genetic factors is important for early detection of IS in the risky persons, and hence, effective preventative and curative measures could be applied. As the vascular dysfunction is the main mechanism involved in development of the cerebrovascular disorders, VEGF with its genetic variants had been suggested to contribute for the enhanced IS risk. In the hypoxic state, VEGF enhances the angiogenesis and neovascularization with increased vascular permeability. Thus, it is suggested to have a role in the pathogenesis of many ischemic disorders, specifically the cerebrovascular diseases. Several polymorphisms had been described for VEGF gene, with + 936C/T (rs3025039) being commonly detected to be associated with the susceptibility of various disorders, such as malignancies ,, endometriosis , and other vascular disorders ,.
In this study, we aimed to explore the association of VEGF single gene polymorphism + 936C/T (rs3025039) with the risk and severity of IS and to assess its relation to VEGF expression via estimation of its serum level.
We detected that the frequency of VEGF rs3025039 TT genotype was significantly higher in IS cases when compared with CC genotypes. Moreover, the carriers of T allele had higher risk for IS by 2.19 folds more than the C allele carriers. These results were consistent with the results of other studies previously conducted and found that the VEGF rs3025039 TT genotype is associated with IS ,. The same findings were detected collectively in a meta-analysis performed by Xu et al. on 3747 IS cases and 2868 controls, particularly in the Asian population after stratification to the ethnic variables. Furthermore, the frequency of T allele was elevated and associated with hypertensive cerebellar hemorrhage in the findings of He et al. study. Contradictory to our study findings, no association was detected between VEGF rs3025039 polymorphism and either the risk of IS in Chinese population or aneurysmal subarachnoid hemorrhage in Italian population .
Our study detected that the VEGF rs3025039 TT genotype carriers were significantly associated with a higher IS severity and a lower percent of improvement as assessed by NHISS than CC genotype carriers. These findings agree with Zhao et al. , who detected that TT and CT genotypes were associated with increased IS severity and poor outcome after adjustment for the confounding risk factors.
Several studies had assessed the role of VEGF in IS and have found that it may be under debate whether it is predisposing or protective . VEGF can enhance the progression of atheroma via stimulation of endothelial cells migration, smooth muscle cell proliferation, plaque neovascularization, and increasing the vascular permeability that predispose to brain edema and IS ,. On the contrary, it has an anti-atherosclerotic effect through enhancing the re-endothelialization, reducing intimal thickening, and inhibiting thrombus formation ,. Moreover, it may promote the resolution of the cerebral infarction region via stimulation of neovascularization with restoration of adequate blood supply in the ischemic areas .
In our study, the VEGF rs3025039 TT genotype was significantly associated with a higher IMT when compared with CC genotype. Moreover, the serum VEGF was significantly lower in IS cases than controls. Thus, we suggested that decreased VEGF level may be a risk factor for the progression of IS. Moreover, we detected that the T allele is associated with lower VEGF serum level when compared with C allele in IS cases. The same association was detected in two previous studies ,, which suggest the possible influence of this polymorphism on VEGF either at transcriptional or post-transcriptional level. This can be explained by the presence of this polymorphism in the 3'UTR which subsequently interferes with the binding of AP-4 , or hypoxia-induced protein, resulting in a marked reduction of the VEGF mRNA half-life .
These findings may support the contradictory role of VEGF in the pathogenesis of IS with more acceptance for its anti-atherosclerotic and angiogenesis role over its effect in the propagation of the atheromatous plaque. Our findings were also confirmed by a previous study, which found an improved neurological outcomes of patients with IS following recombinant human VEGF administration .
| Conclusion|| |
In conclusion, VEGF rs3025039 gene polymorphism is associated with increased susceptibility and severity of IS and decreased VEGF expression, which may play a major role in the pathogenesis of IS. There are some imitations in this study. First, the included participants were from a single center, arising a selection bias; however, the distribution of genotypes were in accordance with Hardy–Weinberg equilibrium that reflects a sufficient presentation. Second, the number of included participants was small; therefore, we recommend further studies to be carried out on larger or different populations to support our results.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]