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The Egyptian Journal of Neurology, Psychiatry and Neurosurgery
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Role of osteoprotegerin rs3102735 gene polymorphism in acute ischemic stroke patients

The Egyptian Journal of Neurology, Psychiatry and Neurosurgery volume 59, Article number: 49 (2023) Cite this article

Abstract

Background

Ischemic stroke ranks third among leading causes of death and disability. Both endothelial and vascular smooth muscle cells generate osteoprotegerin (OPG). Ischemic stroke and its severity may be enhanced by the OPG rs3102735 gene polymorphism. Our research aims to investigate OPG rs3102735 gene polymorphism role in ischemic stroke risk and to assess its association with stroke severity at presentation and degree of vascular stenosis and evaluate its potential as a predictor of stroke severity. Fifty people with acute ischemic stroke as well as fifty controls were studied. The NIHSS and ASPECTS were utilized to evaluate stroke severity and the infarction size, respectively. All subjects underwent extracranial carotid duplex study and molecular assessment for genotyping of OPG rs3102735) gene polymorphism.

Results

Stroke patients had markedly higher concentrations of OPG in the plasma than controls (311.60 ± 109.48 versus 240.20 ± 75.96 mmol/ml, p = 0.001). The optimal plasma OPG cutoff value for the predicting the occurrence of stroke was determined to be > 250 mmol/ml, the 95% confidence interval (CI) was (0.625–0.843), sensitivity was 68% and specificity was 72%. Ischemic stroke had a significantly different genotype distribution for the OPG rs3102735 gene polymorphism than did controls (36 CC, 13 CT, and 1 TT) versus (28 CC, 15 CT, and 7 TT) respectively. Stroke patients had a significantly greater CC + CT genotype than controls did (P = 0.041), also they had a higher propensity for carrying the C allele than the T allele (P = 0.017). Carotid intima medium thickness and the NIHSS both had positive correlations with OPG serum level (r = 0.39, p = 0.02 and r = 0.4, p = 0.02, respectively), whereas ASPECTS had an inversed correlation (r = − 0.65, p = 0.001).

Conclusions

The current study shows that as an independent risk factor, increased plasma OPG level, may participate in the atherothrombotic ischemic stroke pathophysiology, in addition, genetic variants in the OPG gene (rs3102735) are a separate risk factor for large artery atherosclerosis and plasma OPG level can serve as a biomarker to determine the severity of a stroke.

Background

Ischemic stroke ranks third among leading causes of death and disability. Multiple variables, including genetics, chronic inflammation, and risky behaviors, combine to produce this condition [1, 2]. Osteoprotegerin (OPG) is a receptor for tumor necrosis, specifically a member of the 11B subfamily [3]. The soluble glycoprotein OPG serves as a trap receptor by attaching to and disabling the nuclear factor-B receptor activator and the TNF-related apoptosis-inducing ligand [4]. The primary producers of the OPG are endothelial cells and vascular smooth muscle cells [5]. OPG abnormalities are linked to cardiovascular and cerebrovascular illness, according to numerous studies [6]. With more cerebral arteries affected by atherosclerosis, the plasma levels of OPG considerably increased, and this was thought to be a biomarker for cerebral atherosclerosis [7, 8]. Additionally, it has been determined that ischemic stroke frequency and severity are associated with the OPG rs3102735 gene polymorphism [9, 10]. Our research aims to investigate OPG rs3102735 gene polymorphism role in ischemic stroke risk and to assess its association with stroke severity at presentation and degree of vascular stenosis and evaluate its potential as a predictor of stroke severity.

Methods

The Neurology and Clinical Pathology departments collaborated to carry out this case–control study between November 2017 to January 2021. Fifty adults with ischemic stroke of acute presentation of both sexes, categorized in accordance with the Trial of Org 10172 in Acute Stroke Therapy (TOAST) [11] as a large artery atherosclerosis (LAA) or a small vessel disease (SVD) were included. Patients with other subtypes of stroke, major cardiac, hepatic, renal, endocrinal disorders, malignant diseases, trauma, surgery, or chronic inflammatory illnesses, acute inflammatory or infectious problems or a history of fever lasting at least 15 days before the study, acute organ ischemia during the previous 3 months were excluded. Fifty matched controls without ischemic stroke history were included from the hospital’s outpatient clinic. The study was authorized by the faculty of medicine’s ethical committee and all participants completed informed consent forms.

Every individual was subjected to a thorough medical history and physical examination. To grade the severity of stroke, the National Institutes of Health Stroke Scale (NIHSS) [12] was utilized. For grading the infarction size in the anterior and posterior circulations, Alberta Stroke Program Early CT Score (ASPECTS) [13] and the posterior circulation ASPECTS (pc-ASPECTS) [14] were applied respectively. Clinically, the ischemic stroke was categorized total and partial anterior (TACI, PACI), lacunar (LACI), and posterior circulation (POCI) infarcts by the Bamford classification [15] are. At presentation, all patients had a non-contrast CT brain to rule out hemorrhagic stroke, followed by a brain magnetic resonance imaging (MRI). TOAST classification [11] was applied to categorize ischemic stroke into: LAA, SVD, cardioembolic (CE), and other determined and undetermined stroke, dependent on electrocardiography, Holter monitoring, transthoracic echocardiography, MRI of the brain and extracranial carotid duplex.

The studied individuals were subjected to the following:

Using the method described by [16, 17], an extracranial carotid duplex investigation was performed using a linear probe B-mode ultrasonography with great resolution (Philips HD11) (7 MHz).

A full blood count, glucose levels (both fasting and post-meal), lipids, C-reactive protein, coagulation profile, serum uric acid, and kidney and liver function tests.

Measurement of plasma OPG concentrations: a commercially available reagent (OPG DuoSet; RandD Systems, Abingdon, UK) was used as an enzyme-linked immunosorbent test according to the outlined procedure. Venous samples obtained in EDTA tubes at the time of admission, plasma is produced by centrifuging blood for 15 min at 1900×g at 4 °C, filtered plasma kept at 80 °C till analysis. This test had a detection range of 31.25–4000 pg/ml.

Molecular assessment for genotyping of osteoprotegerin (OPG rs3102735) gene polymorphism: genomic DNA was isolated from blood leukocytes utilizing Qiagen’s QIA amp® DNA Mini Kit provided by (Qiagen, Germany).

To analyze genetic polymorphism, polymerase chain reaction (PCR) was employed. GenBank sequences were utilized as a reference. Using the following oligonucleotide primers, PCR was used to amplify the OPG rs3102735 promoter sequence: 5′-CTGGAGACATATAACTTGAACA-3′ and 5′-CCATC ATCAAAGGGCTATTGGT-3′. The following were utilized in a 25 µl reaction volume for PCR amplifications: forward and reverse primer (10 mol/l) of equal volumes (0.6 µl each), double distilled water (2.3 µl), LA Taq DNA polymerase (1 µl), 2 buffer solution (12.5 µl), genomic DNA (4 µl). The steps taken to complete the procedure were as follows: a 5-min denaturation at 94 °C is followed by 30 s cycles at 94 °C, 56 °C, and 72 °C, and then a 7-min extension at 72 °C. Following PCR amplification, AseI (New England Biolabs, Beijing, China) as a restriction enzyme was utilized to digest 4 µl of the product for an hour at 37 °C. The product was then examined using 2% agarose gel electrophoresis. The electrophoretic pattern visualized under ultraviolet light showed different lanes as follow (Fig. 1):

Fig. 1
figure 1

OPG rs3102735 polymorphism agarose gel electrophoresis. 1: CT; 2: CC; 3: TT; M: DNA marker

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Statistical analysis

Data analysis was carried out on Windows with SPSS 22.0. In order to make comparisons, the average and standard deviation for continuous data and frequency (%) for categorical variables. When comparing quantitative and qualitative aspects, we employed the Student’s t (t′) and the Chi-square tests. Fisher’s exact and Pearson’s 2 tests were used to compare genotypes and alleles. The cutoff for a significant difference, as determined by the P-value, was set at 0.05.

Results

Patients and controls demographics and clinical characteristics (Table 1). Bamford classification of strokes shows more affection of anterior circulation 43 (86%) than posterior circulation 4 (8.0%) and lacunar stroke 3 (6.0%) and according to the TOAST classification, 29 patients (58%) had large artery atherosclerosis and 21 patients (42%), small vessel disease. Stroke severity was recorded as follows: 44 (88%) moderate stroke, 6 (12%) moderate to severe stroke (Table 2).

Table 1 Patients and controls demographics and clinical characteristics
Full size table
Table 2 Clinical and radiological classification and stroke severity among patients
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Stroke patients had markedly higher concentrations of OPG in the plasma than controls (311.60 ± 109.48 versus 240.20 ± 75.96 mmol/ml, p = 0.001) (Table 3).

Table 3 Patients’ and controls’ serum levels of osteoprotegerin (OPG)
Full size table

Ischemic stroke had a significantly different genotype distribution for the OPG rs3102735 gene polymorphism than did controls (36 CC, 13 CT, and 1 TT) versus (28 CC, 15 CT, and 7 TT) respectively. Stroke patients had a significantly greater CC + CT genotype than controls did (P = 0.041), also they had a higher propensity for carrying the C allele than the T allele (P = 0.017) (Table 4).

Table 4 Patients’ and controls’ different types of osteoprotegerin (OPG) rs3102735 polymorphism
Full size table

The optimal plasma OPG cutoff value for the predicting the occurrence of stroke was determined to be > 250 mmol/ml, the 95% confidence interval (CI) was (0.625–0.843), sensitivity was 68% and specificity was 72%, according to analysis of ROC curve (Table 5 and Fig. 2).

Table 5 Area under the curve for analysis of serum osteoprotegerin (OPG) for differentiating stroke event from controls
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Fig. 2
figure 2

ROC analysis based on plasma OPG and the occurrence of stroke

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Patients with posterior circulation syndrome (POCS) stroke had substantially higher plasma OPG values than those with other types (432.50 ± 51.23, p = 0.010), in large artery atherosclerosis stroke than in small vessel disease stroke (332.80 ± 116.56, p = 0.049) and in moderate to severe stroke patients than in those with moderate strokes (410.0 ± 70.0 versus 305.3 ± 109.0 mmol/ml, p = 0.048) (Table 6). Patient OPG rs3102735 polymorphism was not associated with Bamford classification, TOAST classification, or stroke severity (P value > 0.05). (Table 7).

Table 6 Relationship between serum osteoprotegerin (OPG) level and Bamford and TOAST classifications and stroke severity among patients
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Table 7 Relation between Bamford and TOAST classifications and stroke severity and osteoprotegerin (OPG) rs3102735 polymorphism
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When compared to controls, stroke patients’ carotid intima media thickness (IMT) (1.26) was statistically substantially larger than that of controls (0.91) (P value < 0.001) (Table 8). Carotid intima medium thickness and the NIHSS both had positive correlations with OPG serum level (r = 0.39, p = 0.02 and r = 0.4, p = 0.02, respectively), whereas ASPECTS had an inversed correlation (r = − 0.65, p = 0.001) signifying that larger infarctions are associated with higher serum OPG levels (Table 9).

Table 8 Patients’ and controls’ carotid artery evaluation
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Table 9 Correlation analysis between serum osteoprotegerin (OPG) level and carotid intima-media thickness, NIHSS and ASPECT score among patients
Full size table

Discussion

Our research aims to investigate OPG rs3102735 gene polymorphism role in ischemic stroke risk and to assess its association with stroke severity at presentation and degree of vascular stenosis and evaluate its potential as a predictor of stroke severity.

Our research showed that acute ischemic stroke patients’ plasma OPG levels were much greater than those of controls. Previous research has shown that having a high OPG level is strongly linked to increased ischemic stroke risk [7, 18, 19].

According to the findings of this study, a patient’s risk of suffering an ischemic stroke may increase if they have the CC genotype of OPG rs3102735. Additionally, the C allele constituted a separate risk factor. Previous research demonstrated a strong connection between the with the OPG rs3102735 CC + CT genotypes and ischemic stroke [9, 10].

According to the findings of this study, the higher plasma OPG levels the more severe stroke and larger infarction size at presentation. Similar outcomes were noted in earlier research [18, 20, 21]. However, no correlation between the incidence of ischemic stroke and OPG levels was discovered in one investigation [22].

Our research showed that plasma OPG level could serve as a biomarker to determine the severity of a stroke. The ideal plasma OPG level cutoff value was > 250 based on our AUC study. At this cutoff point, the specificity was 72%, while the sensitivity was 68%.

The assessment of stroke severity is the most typical clinical application of NIHSS scores. These patients’ OPG plasma levels may provide a critical objective indicator for assessing the severity of a stroke. Consequently, a potential method of assessing the severity of a stroke might be using plasma OPG levels. Additionally, some stroke patients’ NIHSS scores may be lower when they are admitted, but they increase as soon as their impairment gets worse in the first few days after admission. OPG levels in plasma may be an important indicator of how these people’s strokes are progressing [21]. However, more research is necessary to determine whether OPG is a reliable indicator of stroke progression a few days after the stroke’s onset.

Our results showed a significant association between the OPG rs3102735 CC + CT genotype and LAA ischemic stroke, as in other previous studies [9, 10].

All this information encourages the idea that stroke risk could be related to genetic variations in OPG. Previous research suggested that the OPG serum level and the CC/CT genotype of rs3102735 were positively correlated [23]. Many studies have linked inflammation to an increased risk of LAA stroke [1]. According to some research, increased plasma OPG levels enhance the inflammatory response, increase the likelihood of ischemic stroke, and may aggravate stroke severity, elevated plasma OPG levels could amplify the inflammatory response, increase the threat of ischemic stroke, and worsen its severity [24]. The exact way of involvement of OPG in these vascular processes is not well understood. OPG may participate in ischemic stroke occurrence by increasing dendritic cells and T lymphocytes invasion into atheromatous plaque, stimulating angiopoietin-2 synthesis, and encouraging the adherence of endothelial cells [25, 26].

In our study the plasma OPG levels were also positively correlated to CIMT values. Previous studies also demonstrated that OPG levels were linked to the severity and development of carotid artery disease [20]. OPG has also been shown to be crucial in the calcification of atherosclerotic plaque and endothelial dysfunction [27]. According to Ohmori et al. [28], there was no difference in the vascular stenosis grade depending on the OPG rs2073617 gene polymorphism variant [28]. In contrast, vascular stenosis was associated with a substantial increase in plasma OPG levels [7]. The vascular stenosis grade depends on OPG plasma levels and gene variation. These variations may exist because different OPG gene loci may influence plasma OPG levels depending on the vascular stenosis degree [9].

There are various limitations in this study, we focused on the polymorphism at the OPG rs3102735 gene locus, which may possibly not fully reflect the gene. Furthermore, the study’s sample size is small. A more sample size and more comprehensive research on this issue are required across more gene loci and to make sure of the results. The long-term outcome of the stroke was not investigated because we did not follow the patients.

Conclusions

The current study shows that as an independent risk factor, increased plasma OPG level, may participate in the atherothrombotic ischemic stroke pathophysiology, in addition, genetic variants in the OPG gene (rs3102735) are a separate risk factor for large artery atherosclerosis and plasma OPG level can serve as a biomarker to determine the severity of a stroke.

Availability of data and materials

The data can be publicly available at the Faculty of Medicine, Suez Canal University.

Abbreviations

ASPECTS:

Alberta stroke program early CT score

AUC:

Area under the curve

C:

Cytosine nucleotide

CE:

Cardioembolic

CIMT:

Carotid intima-media thickness

LAA:

Large artery atherosclerosis

LACS:

Lacunar syndrome

NIHSS:

National Institutes of Health Stroke Scale

OPG:

Osteoprotegerin

PACS:

Partial anterior circulation stroke

pc-ASPECTS:

Posterior circulation Alberta stroke program early CT score

PCR:

Polymerase chain reaction

POCS:

Posterior circulation syndrome

SVD:

Small vessel disease

T:

Thiamine nucleotide

TACS:

Total anterior circulation stroke

TOAST:

Trial of Org 10172 in Acute Stroke Treatment

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Acknowledgements

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Funding

The authors were responsible for the cost of this study including the design of the study and collection, analysis, and interpretation of data and in writing the manuscript and no funding.

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Authors and Affiliations

  1. Department of Neurology, Faculty of Medicine, Suez Canal University, Ismailia, Egypt

    Dina Monir, Ahmed Osama, Mohamed Negm & Reda Abd El-Razek

  2. Department of Clinical Pathology, Faculty of Medicine, Suez Canal University, Ismailia, Egypt

    Alaa Eldin Saad

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  2. Ahmed Osama
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Contributions

MD carried out the Study conception and design, participated by acquisition of data and performed the statistical analysis and drafted the manuscript. OA carried out the design and conception of the study, the analysis and interpretation of data and helped to draft the manuscript. SA carried out the design and conception of the study, participated in the sequence alignment, laboratory data collection interpretation of data and drafting of manuscript. NM carried out the design and conception of the study, the analysis and interpretation of data and helped to draft the manuscript. AR carried out the study conception and design, participated in its design, and drafted the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Reda Abd El-Razek.

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Ethics approval and consent to participate

The study was approved by the Ethics committee of Suez Canal Faculty of medicine on November 21, 2017. Committee Number: 3294. An informed written consent was taken from all the participants in the study.

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The authors declare that they have no competing interests (financial or non-financial).

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Monir, D., Osama, A., Saad, A.E. et al. Role of osteoprotegerin rs3102735 gene polymorphism in acute ischemic stroke patients. Egypt J Neurol Psychiatry Neurosurg 59, 49 (2023). https://doi.org/10.1186/s41983-023-00652-4

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