Study the Influence of Matrix Metalloproteinase-2 (MMP2) Gene Polymorphisms on Brain Ischemic Stroke in Iraqi Patients
Zahraa Muhammed Jabur 1, Dr. Rand Muhammed Abdul-Hussein Al-Husseini 2
1 Faculty of Science, University of Kufa, IRAQ Email: [email protected]
2 Professor, Faculty of Science, University of Kufa, IRAQ Email: [email protected]
ABSTRACT
Ischemic stroke is a kind of neurologic impairment which characterized by blockage of an arterial vessel supplies blood to the brain, resulting in a neuronal cell death and neurological disabilities. Stroke is one of the leading causes of death worldwide. Matrix Metalloproteinase-2 (MMP2) is an important member in MMPs family encoded by MMP2 gene and plays a critical role in the formation of lesion in blood-brain barriers, cerebral ischemia reperfusion, and neuron apoptosis through destroying the extracellular matrix which may be related with the incidence and development of stroke. This study explored the possible role of MMP2 gene polymorphisms in Iraqi ischemic stroke patients. The study population was included 100 ischemic stroke patients and 50 healthy subjects. The estimated incidence ischemic stroke increased in the age group 60-69 years, with a significant difference (p<0.05) in comparison with the other age groups. There were significant differences (p<0.05) in body mass index (BMI) between ischemic stroke patients and healthy subjects. Tetra- ARMS PCR technique was performed for detection 1306 C/T MMP2 gene polymorphism in ischemic stroke patients and control group. The genotype distribution results of the 1306 C/T SNP of MMP2 gene have shown significant differences (p<0.05) between controls (CC: n=49, 98 %; CT: n=1, 2%; TT: n=0) and patients (CC:
n=40, 40 %; CT: n=28, 28%; TT: n=32, 32 %). The current study showed an increase in CC genotype and C allele of 1306 C/T in ischemic stroke patients than controls. Finally, these results indicated a possible role for the 1306 C/T SNP in MMP2 gene as risk factors in pathogenesis of ischemic stroke in Iraqi population.
Keywords
MMP2 gene; ischemic stroke; polymorphism; matrix metalloproteinase
Introduction
Cerebral ischemic disease, a common form of stroke, remains as one of the leading causes of morbidity and mortality worldwide. Cerebral ischemia is the fifth leading cause of death and disability impacting one million Americans every year [1]. Ischemic stroke (IS) is a severe neurological disorder in humans that results from an interruption of the blood supply to the brain caused by a vascular occlusion or known ashemorrhagic stroke (HS) when stroke results from a blood vessel rupture or leakage. Within seconds, the insufficient blood supply as result leads to a oxygen and glucose deprivation of the cerebral tissue, which caused a serial of pathophysiological events leading to cell death and neurological deterioration [2]. There are many causes of ischemic stroke, such as arteriosclerosis, clotting disorders, hypertension, various forms of vasculitis, and heart emboli which have formed as a result of atrial fibrillation (AF) or rheumatic disease of heart [3]. Epidemiology of stroke in Asia has been recorded high incidence, where ischemic stroke occurs more frequently than hemorrhagic stroke [4, 5]. In the Western world, stroke is themajor cause of disability and death in many countries and it is second cause of death after heart disease. There are some races and ethnic origins that are more prone to stroke than others. People of Asian, African and African Caribbean origin have a much higher risk for stroke [6].
In the early 1960s, the metalloproteinases (MMPs) became known as the enzymes that
responsible for degeneration of the extracellular matrix of cells [7].The MMPs are secreted by vascular smooth muscle (VSM), fibroblasts, and leukocytes cells. MMPs are regulated at the level of mRNA expression and through activation after cleavage of the propeptide domain from their zymogen form or its inactive precursor [8].The single nucleotide polymorphisms in MMP genes may influence the susceptibility of presentation of IS and may be influenced by race/ethnicity [9].Role of polymorphism (rs243865) in the promoter region of MMP2 gene with different population groups has been studied in many malignancies including lung cancer [10]
colon cancer [11], prostate cancer and breast cancer [12]. Currently, some studies reveal that MMP2 polymorphism is correlated with an elevated risk of IS, and most study focuses on the rs243849, rs243865, and rs243864 SNPs [13, 14, 15].
The main aim of this study was to investigate the relationship between 1306 C/T MMP2 gene polymorphism and ischemic stroke in Iraqi patients.
Materials and Methods
This study was carried out in Alssader Teaching Hospital/ Neurosciences Center in Al-Najaf governorate and laboratory of molecular biology in the Department of Biology / Faculty of Science – University of Kufa, Iraq; during the period from September 2020 through April 2021.
Sampling of Cases
Study group consists from 100 patients. They were admitted to Alsader Specialized Center of Neurosciences. All the patients selected for the present study were having ischemic stroke.
Control group consists from 50 healthy individuals; all were without any inflammatory disorders or clinical manifestation of any disease. Data from participants were collected using a questionnaire which included their gender, age, body height and weight.
Body mass index
The BMI value included the ratio of weight and height squared (in kilogram and meter respectively) for each patient, which calculated according to the following universal formula:
BMI=Wt (kg) / Ht (m) 2. The range of BMI for an adult was as follows: 18.5-24.9 kg/m2 was considered as healthy or normal BMI, 25-29.9 kg/m2 was considered as overweight, and higher than 30 kg/m2 was considered as obese.
Determination of MMP2 1306 C/T polymorphism
Genomic DNA was isolated from blood using protocol from Genomic DNA Kit (Geneaid Biotech Ltd., Taiwan) designed specifically for purifying DNA from blood.
Tetra-Primer Amplification refractory mutation system (ARMS PCR) method was performed for detection and genotyping MMP2 single-nucleotide polymorphism in blood samples. The reaction was conducted in accordance with the temperature profile: initial denaturation 94°C for 5 minutes; 35 cycles: 94°C for 45 seconds, 58°C for 45 seconds, 72°C for 45 seconds in; final extension 72°C for 5 minutes.
DNA from each sample was amplified with primer-pairs specific for 1306 C/T SNP of MMP2 gene (table 1).The sequences of primers were published previously [16, 17]. The primers were synthesized by Microgen, Inc. Korea. All these primers were reached as lyophilized form with different concentration. Primer containers were first centrifuged at 13,000 rpm for 3 minutes, and then reconstituted with appropriate volume of Tris-EDTA buffer (TE buffer) for each one (according to the manufacturer) in order to get 100 pmole/µl (stock solutions). Working solution (10 pmol/µl) was prepared from stock solutions.
Group1 Group2 Group3 Group4 Group5 Total patients 10.00% 12.00% 15.00% 38.00% 25.00% 100%
0.00%
20.00%
40.00%
60.00%
80.00%
100.00%
120.00%
Percentages of Patients
Age Groups
Table 1. Primers sequences for MMP2 1306 C/T polymorphism
Primers sequences PCR product
Outer forward 5′-ACCAGACAAGCCTGAACTTGTCTGA-3′
542 bp, 379 bp, 211bp
Outer reverse 5′-TGTGACAACCGTCTCTGAGGAATG-3′
Inner forward 5′- ATATTCCCCACCCAGCACGCT-3′
Inner reverse 5′-GCTGAGACCTGAAGAGCTAAAGAGTTG-3′
The amplification products of the polymorphic MMP2 gene fragments were separated in a 2%
agarose gel dyed with DSRed Nucleic Acid Stain (Dongsheng Biotech Co., Ltd. China). Based on the amplification products obtained, three possible allele arrangements were identified: CC (542 bp and 379 bp), CT (542 bp, 379 bp and 211 bp) and TT (542 bp and 211 bp).
Results
Characteristics of the study population
The clinical assessment of ischemic stroke patients revealed that the frequency of distribution of patients according to gender were 54 (54%) females and 46 (46%) males.
The differences of gender were statistically not significant. Assessment of age presentation of patients at diagnosis revealed that 10(10%) in age group 30-39, 12 (12%) in age group 40-49, 15 (15%) in age group 50-59, 38 (38%) in age group 60-69, and 25(25%) in age group 70-79 (Figure 1). Their ages ranged from 30 to 79 years, with a mean age of 62.322 years.The patients and the controls were not statistically (P> 0.05) different with regard to gender and age. The estimated incidence of ischemic stroke increased in the fourth age group (60-69), with a significant difference (p<0.05) in comparison with the other groups.
Figure 1: Age distribution of patients presented with ischemic stroke (Group1: 30-39 years, Group2: 40-49 years, Group3: 50-59 years, Group4: 60-69 years, Group5: 70-79 years).
The results in table 2 describe the characteristic of ischemic stroke patients according to the BMI.
Twenty five (25%) ischemic stroke patients had a normal body mass index; overweight was found in 45 (45%) ischemic stroke patients while obesity was found in 30 (30%) ischemic stroke patients.There were significant differences (p< 0.05) in BMI between normal BMI and overweight in ischemic stroke patients.
Table 2. Body mass index in ischemic Stroke patients and controls
BMI (Range) Ischemic stroke patients
Normal weight (18.5-24.9 kg/m2 ) 25(25%)
Overweight (25-29.9 9 kg/m2 ) 45(45%)*
Obese (≥30kg/m2 ) 30(30%)
Results values were expressed as mean ±SE (results also represented as a percentage (%)).
Abbreviations: BMI: body mass index, *: P< 0.05 or significant differences between percentages.
MMP2 gene SNP (1306 C/T) and ischemic stroke:The present study found that among the 50 healthy subject; 49 (98 %) had found as homozygous CC alleles, and 1(2%) found as heterozygous genotype with the C and T alleles (CT); (CC: n=49, 98 %; CT: n=1, 2%; TT: n=0).
While in ischemic stroke patients; 40 (40 %) had found as homozygous CC alleles, and 28 (30%) found as heterozygous genotype (with the C and T alleles (CT) and 32 (32 %) had found as homozygous TT alleles; (CC: n=40, 40 %; CT: n=28, 28%; TT: n=32, 32 %) (Table 3).
Table 3. Genotyping frequencies of 1306 C/T SNP at MMP2 gene in ischemic stroke patients and controls
Genotypes Healthy controls (N=50) Ischemic stroke patients
CC 49 (98 %) 40 (40 %)*
CT 1 (2%) 28 (28%)*
TT 0 32 (32%)*
P-value 0.0007*
Alleles frequency N(%) N(%)
C allele 99(99%) 108(54%)
T allele 1 (1 %) 92(46%)
X2 P-value OR(95%CI)
63.114 0.000019*
0.012 (0.002-0.087)
Data were expressed as number of patients (results values also represented as a percentages (%)).Abbreviation:
X2 =chi-square, OR= odds ratio, CI= confidence interval. *P< 0.05 significant differences between values.
Statistical analysis
Statistical analyses of all results were carried out by the help of Statistical Package for the Social Sciences (SPSS) version 23 software statistical package using t-test and Chi-square test (with P value at level of significance less than 0.05) to compare value of results between groups. Result values were expressed as mean ± SE, number of patients, or percentages.
Discussions
In study population, the incidence of ischemic stroke was higher in females than in men. These findings were consistent with the study of Touzé and Rothwell [18] which showed that the heritability of ischemic stroke is higher in women, and that women have a profile of higher baseline risk factors. The study of Goto et al. [19] reported that men and women are not
susceptible to same stroke risk factors. While women will more often have hypertension and intracranial stenosis, other risk factors are more prevalent in men, like peripheral vascular disease, hyperlipidemia, abdominal aneurysm, severe carotid stenosis or aortic atherosclesrosis.
In study of Mozaffarian et al. [20], females have a higher lifetime risk for stroke, they are more likely to have more severe strokes as compared with males, and they more experience recurrent stroke even after surviving a stroke, Also these results agree with the Kapral et al. study [21], they found that the relationship of gender to stroke risk depends on patient age. At young ages, females are more likely to have IS than men, although the relative risk is slightly higher in men of older ages. These results agree with Reeves et al. [22] study, which has showed that more strokes occur in women than men, because of the longer life span of women compared with men.
While these results do not agree with the Miah et al. study [23], which found that stroke occurs more often in male than in female and Stroke rates for men are 1.25 times higher, but because women tend to live longer than men, more women than men die from IS each year. On average women experience more severe strokes and have longer hospital stays [24]. Arboix et al. [25]
study results recorded that females have higher stroke-related mortality.
The prevalence rate of ischemic stroke was higher in the age group 60-69 years, with a significant difference (p<0.05) in comparison with the other age groups. These results agree with other studies [26, 27, 28] that showed age is a non-adjustable risk factor (stroke risk doubles with every decade of life after age 55).These findings are comparable to the findings of Guan et al. study [29] That reported a higher stroke prevalence and IS-related risk factors among adults aged ≥40 years. Another study showed that most strokes occur in people >65 years and the stroke is more related with aging patients [30]. These results also comparable with previous study [31] found the mean age of the patients was 55.1 ± 14.0 years and 65 patients (56.0%) were in age group 45–65 years. These results also agree with the Rosamond and his colleagues study [32], which indicated that 75–89% of strokes occur in individuals aged >65 years.
In an epidemiological study, which reviewed 98 articles haveshown that the mean age of first- ever IS is 72.9 years in women compared with 68.6 years in men [33].
The BMI results revealed that there were significant differences (p<0.05) in BMI between ischemic stroke patients and healthy subjects. 45% of ischemic stroke patients are overweight.
These results were in agreement with another study [34] which has recorded an association between elevated BMI and risk of stroke. Similar results were found in Zhang et al. [35] study found that BMI >27 kg/m2 was associated with increased risk for stroke compared with normal weight subjects.
Whitlock et al. [36] study showed obesity was associated with an increased risk for stroke. Our result also agrees with the study of Badheka et al. [37] which conducted that overweight patients were associated with a higher risk of ischemic stroke, and obese patients were associated with a higher risk of thromboembolism compared with normal weight patients.
Kurth and his colleagues [38] mentioned that obesity and overweight are well-known cardiovascular risk factors and associated with an increased risk of IS. They have proved that each unit increase of BMI is independently associated with 6% increase in the relative risk of IS.
Chen et al. [39] study stated that obesity is strongly and positively correlated with ischemic stroke, mainly through its effect on blood pressure.
The genotype distribution results of the 1306 C/T SNP of MMP2 gene have shown significant differences (p<0.05) between controls and ischemic stroke patients. A large number of studies have verified that the genetic polymorphisms within the MMP genes that alter expression levels of the enzymes, implying a possible role in IS development. In particular, the1306 polymorphic
site of MMP2 is located upstream of the MMP2 gene and may affect the protein expression by modulating its transcription, hence leading to the occurrence of many human diseases, such as bladder cancer and sclerosing cholangitis [40, 41]; a variety of transcription factors, such as activator protein-1 (AP-1), specificity protein-1 (SP-1) and activator protein-2 (AP-2), have binding sites at the MMP2 promoter region to regulate transcription of the MMP2 gene [42, 43].
MMP2 gene promoter -1306C/T polymorphism, may affect gene transcription and lead to changes in gene function. The transition C-T at locus 1306 in MMP2 gene could disrupt a Sp-1 binding site (and other transcription factors) [44, 45].
Rs243865 (1306C/T) as a mutation in promoter region of MMP2, it is found to be possibly associated with the high transcription level and affect enzyme activity [46].
These results were consistent with the study of Zhang et al. [47] which showed that single polymorphism in MMP2 was explored the association with stroke. MMP2 1306C/T polymorphisms were revealed to be associated with the onset risk of stroke, not only genotype but allele.
These findings are comparable to the findings of Price et al. [48] study which showed that promoter region contains 1306C/T polymorphism may be closely related to the occurrence and development of stroke.
Study in Chinese population [49] has evaluated the1306 C/T promoter of MMP2 in IS patients and found the polymorphism resulted in modulating transcriptional activity.
On other hand, these results were in disagreement with the results of Rosenberg et al. [50] which identified no significant change in CC genotype and C allele frequencies for MMP2 1306C/T in IS patients compared to healthy controls. Therefore, IS incidence was not significantly correlated with 306C/T polymorphism. This may be due to increased MMP2 transcription levels from the C allele, resulting in increased MMP2 expression within infarcted brain tissues. While the results of Gonzalez-Hernandez et al. [51] study showed that the MMP2 1306C/C genotype increased IS risk principally in the hypertensive individuals of the population studied.
Conclusion
Based on the results and findings, the study has drawn the following conclusions: the prevalence rate of ischemic stroke is significantly higher among the older age groups, which means that the incidence of stroke increase with age among Iraqi population. Generalized overweight is associated with ischemic stroke patients. Finally the study revealed that 1306 C/T MMP2 gene polymorphism and T allele frequency in Iraqi patients may play a big role in the pathogenesis of ischemic stroke.
Acknowledgement
The authors wish to express their appreciation to the authorities of University of Kufa, Faculty of science for according permission to carry out this research work in the laboratory of molecular biology in the Department of Biology.
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