Ismail Kara1, Tammam Sipahi1, Aslı Sert Sunal2, Mustafa Yıldız1, Babürhan Güldiken2

1Trakya University Faculty of Medicine, Department of Biophysics, Edirne, Türkiye
2Trakya University Faculty of Medicine, Department of Neurology, Edirne, Türkiye

Keywords: Matrix metalloproteinase, stroke, MMP9, C-1562T, rs3918242


Objective: Matrix metalloproteinases (MMPs) are calcium-dependent zinc-containing proteases containing more than 28 enzymes that cause the degrading of the extracellular matrix. Although MMPs play key roles in many biological processes, they influence some pathological processes such as ischemic stroke (IS). Among MMPs, the enzyme most associated with IS is the MMP9 enzyme. This study aimed to evaluate the effect of the C-1562T (rs3918242) gene polymorphism of MMP9 on the development of IS in Turkish patients living in the Trakya Region.

Materials and Methods: Our study involved 60 patients with IS and 60 controls. The patients with IS were categorized according to the Trial of ORG 10172 in Acute Stroke Treatment classification of stroke subtypes, and MMP9 C-1652T gene polymorphism was identified using polymerase chain reaction, followed by restriction fragment length polymorphism using the Pael (SphI) restriction enzyme.

Results: Genotypes were defined as CC, CT, and TT according to the presence of C and T alleles. No significant differences were identified in the genotype distribution and allele frequency of MMP9 C-1562T gene polymorphism between the patients with IS and controls.

Conclusion: Our findings suggest that MMP9 C-1562T gene polymorphism is not associated with the risk of IS in Turkish patients living in the Trakya Region.


Stroke is the second leading cause of death and the most common cause of disability globally (1). A considerable amount of research has been conducted to clarify the etiological and pathological mechanisms of this multifactorial disease. The etiology and pathogenesis of ischemic stroke (IS) are diverse. Environmental factors (such as diabetes, hypertension, alcohol intake, and smoking) have been confirmed as key risk factors of IS (2). In addition, because not all individuals have been exposed to the aforementioned environmental factors, genetic factors have been correlated with the occurrence and progress of IS (3,4).

The activation and altered expression of matrix metalloproteinases (MMPs) are thought to play a role in the development of IS. The levels and activity of MMP9 (gelatinase B), which has a molecular weight of 92 kDa and is also known as type IV collagenase, increase in the acute phase of stroke and remain high for months after the incident (5). Studies have demonstrated that MMP9 activity is controlled by a functional substitution of the C and T (C/T) alleles at the −1562 position in the promoter region of the human MMP9 gene, which is located on chromosome 20q12.2-13.1. These studies have revealed that the T-1562 allele has higher promoter activity in driving gene expression than the C-1562 allele (6,7). In particular, relatively high expression levels of MMP9 were identified in the human brain tissue of patients with IS, indicating that MMP9 may contribute to ischemic brain injury (8).

Some meta-analyses have been conducted to assess the association of MMP9 gene polymorphism with IS susceptibility, but no association has been detected (9,10). In recent years, studies have associated MMP9 variants with the risk of IS. Although studies have reported an association between MMP9 variants and IS susceptibility, the results remain controversial (11). In our study, we aimed to investigate the role of MMP9 C-1562T gene polymorphism in the development of IS in Turkish patients living in the Trakya Region.

Material and Methods


The patient group consisted of 60 Turkish adult patients with IS, who were categorized according to the Trial of ORG 10172 in Acute Stroke Treatment classifications for stroke subtypes into large and small vessel stroke (12). All the patients with IS were enrolled in the study when they were hospitalized during acute stroke or when they came to the neurology department for further examinations in the chronic phase of stroke. Stroke was diagnosed through clinical findings after neurologic examination and imaging data obtained from either cerebral magnetic resonance imaging or computed tomography. Patients diagnosed with cerebral hemorrhage, transient ischemic attack, cardioembolic stroke, or recent myocardial infarction were excluded from the study. The control group consisted of 60 Turkish adults who were treated at the hospital’s department of physical therapy and rehabilitation without any clinical evidence of cerebrovascular disease. Both the controls and patients were recruited from the province of Edirne and its surrounding areas. All participants gave informed consent, and the study was approved by Trakya University Ethics Committee of Scientific Research in Edirne (protocol no: TÜTFBAEK 2016/176, decision no: 13/20, date: 20.07.2016).

Determination of the Matrix Metalloproteinase-9 Genotype

Peripheral venous blood of both patient and control groups was collected in ethylenediamine tetraacetic acid vials, and DNA was extracted from whole blood using blood DNA kits (Thermo Fisher Purelink® Genomic DNA Mini Kit). The DNA purity and quantity were assessed using a spectrophotometer and checked using 0.8% concentration agarose gel electrophoresis stained with 2 µl of ethidium bromide. Polymerase chain reaction (PCR) followed by restriction fragment length polymorphism using the Pael (SphI) restriction enzyme was used to identify C-1562T (rs3918242) gene polymorphism in the MMP9 gene. Subsequently, C-1562T polymorphism in the MMP9 gene was analyzed through the amplification of a 436 bp (Figure 1) fragment using PCR (6,13). The following primers were used for the amplification reaction: forward primer 5’-GCC TGG CAC ATA GTA GGC CC-3’; reverse primer 5’-CTT CCT AGC CAG CCG GCA TC-3’ (Figure 1). The PCR reaction mixture (25 µl) contained 200 ng of DNA, deoxynucleotide triphosphates (0.1 mM of each), primers (200 pmol of each), 1X Taq buffer with ammonium sulfate [(NH4 ) 2 SO4 ; 75 mM of Tris hydrochloride buffer (pH 8.8), 20 mM of (NH4 ) 2 SO4 , 0.01% Tween 20], 2.5 mM of magnesium chloride, and 1.25 U of Taq DNA polymerase (Thermo Fisher Scientific). This mixture was amplified using a Techne DNA Thermal Cycler (TechGene) with 6 min of denaturation at 95 °C, followed by 35 cycles with denaturation for 30 s at 94 °C, annealing for 30 s at 61.5 °C, and extension for 1 min at 72 °C, followed by 7 min of extension at 72 °C. The PCR products were checked using 2% concentration agarose gel electrophoresis stained with 2 µl of ethidium bromide (Figure 2). The Pael (SphI) restriction enzyme then digested 10 µl of the PCR products. Fragments were separated on 2% concentration agarose gel electrophoresis stained with 2 µl of ethidium bromide (Figure 3).

Statistical Analysis

The statistical analysis was performed using TURCOSA (Turcosa Analytics, Kayseri, Türkiye) statistical software. Allele frequencies were calculated from the genotypes of all participants. The Hardy–Weinberg equilibrium was evaluated using a chisquared test with 1 degree of freedom. Genotype distribution and allele frequencies were compared between the patient and control groups using Pearson’s chi-squared test of independence with 2 × 2 contingency and z-statistics. The relationship between the genotypes of MMP9 C-1562T gene polymorphism and development of IS was assessed through logistic linear regression analysis after adjusting for the confounding covariates. Age, hypertension, diabetes mellitus, smoking, and alcohol intake were included as the covariates in the regression model. Statistical significance was set at P < 0.05.


In the present study, −1562 C/T (rs3918242) gene polymorphism in the MMP9 gene was investigated in 60 patients and 60 controls. The demographic and clinical characteristics of all research participants are summarized in Table 1. In the comparison between patients and controls, no significant statistical differences were identified in terms of age (P = 0.52), sex (P = 0.28), diabetes mellitus (P = 0.67), alcohol intake (P = 0.82), or smoking (P = 0.08), but significant differences were determined for hypertension (P = 0.01).

All genotype distributions were in Hardy–Weinberg equilibrium (X2 : 4.69, P = 0.09). The genotype distribution of MMP9 C-1562T gene polymorphism and allele frequency for the C and T alleles in the patient and control groups are presented in Table 2. No significant statistical differences were identified in either allele frequency or genotype distribution between the two groups. The genotype distribution among patient subgroups is summarized in Table 3. No significant statistical differences were observed in the genotype distribution of patient subtypes.


MMPs, produced by endothelial cells, microglia, astrocytes, and neurons, are involved in the progress of atherosclerosis by activating the migration and proliferation of smooth muscle cells and inducing the destabilization of atherosclerotic plaque. An imbalance in MMP activity is associated with cardiovascular and cerebrovascular diseases (14), and some MMP9 gene polymorphisms regulate MMP9 activity. Studies have identified MMP9 C-1562T polymorphism within the promoter of the MMP9 gene (15). The transition of the T-1562 allele to the C-1562 allele increases the promoter activity of MMP9 (16). Because MMP9 exhibits relatively high expression levels in the human brain tissue of patients with IS, it may contribute to ischemic brain injury (17).

When the cells of the central nervous system are injured by ischemia, MMP9 is released (18). The MMP9 gene is located in the chromosome 20q12.2-13.1 region and contains 13 exons and 12 introns (7), and MMP9 is vital for matrix degradation, extracellular matrix remodeling, cellular adhesion, and cell growth (18,19). Additionally, MMP9 is thought to play a role in regulating cell proliferation, differentiation, angiogenesis, apoptosis, and host defense (2,19).

In our study, we did not identify any significant relationship between the C-1562T polymorphism of the MMP9 gene and the development of IS. This is consistent with the results of numerous other studies. A study performed in a Polish population revealed no correlation between MMP9 C-1562T variants and the risk of IS. In this study, no significant difference was determined between CC, CT, or TT genotype ratios or C and T allele ratios in patients with IS compared with controls (20). Wang et al. (21) conducted a study in a Chinese population in Guangxi Province, revealing results consistent with those of Szczudlik and Borratynska (20);

However, significant associations between MMP9 C-1652T variants and an increased risk of IS have been identified in some case-control studies (12,22,23,24,25).

The study performed in a Polish population in 2015 by Buraczynska et al. (13) revealed that the T allele in −1562 C/T polymorphism, compared with that of a control group, might lead to an approximately 1.9-fold increase in the risk of stroke in patients carrying the T allele and a 2.3-fold increase in T allele carriers with diabetes. This finding is inconsistent with a previous study conducted in a Polish population, which demonstrated no association between MMP9 C-1562T polymorphism and an increased risk of IS (20).

Nie et al. (22) conducted a study in 396 Chinese patients with IS and 400 controls, revealing that MMP9 C-1562T genotype and allele frequencies differed between the two groups and the MMP9 C-1562T allele was associated with a 1.5-fold increase in IS incidence.

Zhao et al. (23) demonstrated that C-1562T gene polymorphism and IS pathogenesis are closely related and that this gene polymorphism interacts with body mass index to increase the risk of IS. Moreover, the CC genotype of C-1652T of the MMP9 gene increases the risk of IS compared with the TT genotype in a Chinese population (23).

A similar result was obtained in a study conducted by Hao et al. (24) involving 317 patients with stroke and 317 healthy participants. They identified that individuals carrying the CC genotype and TC + CC genotype were associated with a significantly increased risk of IS compared with those carrying the TT genotype, thus correlating MMP9 C-1652T polymorphism with an elevated risk of IS. The authors also demonstrated that this gene polymorphism has a significant interaction with smoking to increase the risk of IS (24).

Li et al. (25) demonstrated a significant difference in the genotype and allele frequencies of MMP9 C-1652T polymorphism between patients with IS and controls, indicating that the MMP9 C-1562T T allele was a genetically susceptible gene for cerebral infarction and cerebral hemorrhagic stroke.

In a meta-analysis of 13 studies conducted by Jiang et al. (26) involving 3,996 patients and 3,815 controls, a correlation was identified between MMP9 C1562T polymorphism and IS in the Chinese population, with the TT+TC genotype determined to increase the risk of IS.


In this study, no relationship was identified between the C-1562T gene polymorphism of the MMP9 gene and the probability of developing IS in the Turkish population living in the Trakya Region. These findings suggest that individuals with genotype CC, CT, or TT or with allele C or T do not have a relatively high risk of IS compared with the whole Turkish population.

In conclusion, although some research suggests an association between single nucleotide polymorphisms such as MMP9 C-1562T and an increased risk of IS, further investigation based on larger sample sizes utilizing more diverse population selections will help us gain better insight into any potential linkages between genetic variations and diseases such as stroke.

Early diagnosis of IS may be possible if the genes that are effective in the development of IS are identified. The identification and understanding of these genetic factors can help identify individuals at risk of developing this condition, allowing for early intervention and treatment. This improved knowledge could also lead to more successful therapies as well as preventive measures such as lifestyle modifications or medications that target those specific genes associated with this disorder.

Ethics Committee Approval

Trakya University Ethics Committee of Scientific Research in Edirne (protocol no: TÜTFBAEK 2016/176, decision no: 13/20, date: 20.07.2016).

Peer Review

Externally and internally peer-reviewed.

Author Contributions

Surgical and Medical Practices: A.S.S., B.G., Concept: İ.K., T.S., Design: İ.K., T.S., Data Collection or Processing: İ.K., A.S.S., Analysis or Interpretation: İ.K., T.S., B.G., M.Y., Literature Search: İ.K., Writing: İ.K.

Conflict of Interest

No conflict of interest was declared by the authors.

Financial Disclosure

The study was supported by Trakya University Scientific Research Projects Unit with project grant number (2017/55).


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