Yan Xu, Deqin Geng

 [H1] 
Abstract: Ischemic stroke is a polygenetic disease with complex pathogenesis, as a result of interaction and mutual influence of genetic and enviromental factors. As the genome project completed and the development of molecular genetics is growing, hot discussion on pathogenesis from genetic point of view has become an intriguing issue. A lot of candidate genes related to ischemic stroke have been widely studied, in which, the genetic polymorphism studies on ischemic stroke helps to find out the pathogenesis and discover high risk groups and individuals in advance. Therefore, such studies can prevent the disease in advance and ease the medical burden of both the society and family.
Keywords:  Ischemic stroke；polymorphism；genetic；candidate gene；mononucleotide
Citation: Xu Y, et al. The progress of related study on gene polymorphism of ischemic stroke. J Gen Neuro 2016; 1(1): ; http://dx.doi.org/
^*Correspondence to: Yan Xu, address[H2] , email address.[H3] 
Received:                          Accepted:                     Online Published:
 
 
 
Introduction
Ischemic stroke is a global public health problem that threatens human life. With high incidence of morbidity and mortality’s rate, ischemic stroke has brought a heavy burden to patients, families and societies. It is generally believed that the pathogenesis of ischemic stroke is very complex due to genetic and environmental factors, which is also as the result of interaction and mutual influence of those factors. Genetic factors play an important role in the onset of stroke^[1]. Studies have found that stroke has familial aggregation phenomenon and identical twins stroke incidence is several times higher compare to fraternal twins. Traditional lifestyle and environmental factors are easy to control and intervene, but genetic factors have relative stability and are not easy to change. So, in comparison to other related metabolic index, the genetic index is more reliable to provide important basis on early detection of ischemic stroke associated with pathogenesis and high-risk groups.
Genetic polymorphism or gene polymorphism is define as a biological group that have two or more discrete variant genotypes or alleles at the same time, which mainly displays in the forms of DNA fragment length polymorphism, repeated DNA sequence polymorphism, and single nucleotide polymorphisms. Genome-wide association study (GWAS) has identified a series of candidate genes related to both genetic and risk factors of ischemic stroke, which mainly includes: the related factors of inflammatory reaction gene, platelet activating factor acetyl hydrolase (PAF-AH) gene, renin angiotensin system (RAS) related genes, lipid metabolism related genes, NOTCH3 gene, and etc. Candidate genes of ischemic stroke will be mainly discussed below in order to have comprehensive understanding on disease-causing genes of ischemic stroke and genetic susceptibility of ischemic cerebrovascular disease.
 
CRP gene
C-reactive protein (CRP) gene in chromosome 1 long arm has two exons, and separated by introns, which encodes for 206 amino acid residues. One CRP molecule subunits can combine with 160 base pairs of DNA. This structure enables the CRP to integrate with platelet activating factor which plays a regulatory role in the control of inflammation. In the development process of the atherosclerosis and ischemic events occurrence[H4] , the inflammatory response plays an important role. The body's inflammatory state can be reflected through the acute phase of C-reactive protein (CRP). Once atherosclerosis damage appears in patients, the CRP levels will increase accordingly^[2]. However, the association of CRP to atherosclerosis mechanism is still unclear. It has been shown in several studies that CRP can be a factor to induce tissue expression of mononuclear cells, activate blood coagulation system and its complementary system, increase the risk of heart- and cerebrovascular disease^[3]. It is reported in several literatures that an independent measurement of CRP level can be  the initiator for the recurrence of cerebrovascular disease risk predictors^[4]. The study found that CRP, IL-6 and 1059 g/C have direct relationship with venous thrombosis^[5]. At the same time, existence of CRP in carotid artery and small cortical blood vessels plays an important role in disease progression, used as a predictor for ischemic stroke, and even used as risk prediction factors after lacunar cerebral stroke^[6-7]. Clinical studies have shown that concentration of plasma CRP in patients with ischemic stroke was significantly higher than the control group, in which, rs3093059 and rs3091244 loci gene polymorphisms were significantly associated with CRP elevation^[8-10]. Ben-Assayag et al. also suggested that promoter sequences of CRP gene (–717A/G single nucleotide polymorphism) associates with acute stroke and AG genotype, besides having a significantly higher GG genotype CRP level than AA genotype^[11].
MMP-3 gene
Matrix metalloproteinases (MMPs) is a type of active center protein containing zinc ions protease superfamily, with its physiological functions are mainly involved in the degradation and reconstruction of extracellular matrix in the body. The degradation of extracellular matrix runs throughout the whole process of hardening of the arteries. MMPs through the digestive ingredients undermine its fibrous cap structure accelerating the plaque rupture[H5] , is one of the important reasons of unstable plaques. MMP-3 gene, which is located on chromosome 1 lq22. 3, includes 10 exons and 9 introns with its homologous expression is mainly involved in the regulation of gene transcription. MMP-3 is one of the important members of MMPs family with a wide range of substrate recognition specificity, which can also activate other groups of MMPs through the induction of MMPs bioactive molecules^[12-13]. Atherosclerotic plaque is a process that involves MMPs which leads to chronic inflammatory^[14]. In unstable plaques, especially those that prone to rupture the plaque shoulder, the MMP-3 content and its enzyme activity also increased significantly, in which, the elevated serum levels of MMP-3 are closely related to unstable plaques. Gnasso et al. found in the Caucasian population with no significant atherosclerosis risk factors, 6A allele can increase the carotid intima-media thickness, expand the lumen and reduce local wall shear stress, which are more likely to form the atherosclerosis plaque^[15]. Ye  and co-workers also have highlighted the correlation of atherosclerosis associated with MMP-3 promoter of 5A / 6A polymorphism, in which, 6A genotype carriers are more prone in the development of atherosclerosis^[16]. Ma et al. showed that in rs3025058 site of 5A allele, the rs679620 loci alleles may increase the onset risk of large artery atherosclerosis (OR > 1), and G allele of rs522616 locus may reduce their onset risk (0R < 1)^[17]. However, the three single nucleotide polymorphisms genotypes showed no statistical significance differences in small artery occlusion type of both stroke and control group. On the other hand, this also reflects the pathogenesis of large artery atherosclerosis, whereby small artery occlusion stroke is different.
ApoE gene
Apolipoprotein E (ApoE) is composed of chyle particulate residue, in which, the low density lipoprotein receptor ligand receptor is the important component of plasma lipoproteins and determining factor of lipid metabolism and heart cerebrovascular disease. ApoE plays an important role in the metabolism of blood fat. Apolipoprotein E (ApoE) gene is located on chromosome 19, which consists of 3597 nucleotides with 4 exons and three introns, whereby the gene polymorphism in 112 and 158 loci coding for three ApoE subtypes: ApoE2, ApoE3 and ApoE4. Apolipoprotein E gene polymorphism has extremely obvious effects on blood lipid metabolism^[18]. Sing et al. reported that plasma total cholesterol and low density lipoprotein cholesterol levels of ApoE4 carriers in normal group were significantly higher than those of ApoE3 carriers^[19]. This suggests that E4 allele has an effect on cholesterol elevation, whereby high cholesterol level is one of the major risk factor for ischemic stroke. Studies have shown that ApoE genotype influences the occurrence of cerebrovascular disease^[20-21]. According to the study conducted by Qun Liu and co-workers, genotype groups carrying E2 and E4 significantly increased the onset risk of ischemic stroke, in which, the onset risk of ischemic stroke in E4 allele carriers was 1.78 times higher[H6]  than the control group^[22]. In comparison to the control group, the onset risk of ischemic stroke in E2 and E4 gene group increased nearly 40% (RR[H7]  = 1.36) and the differences were statistically significant.
 
PAF-AH gene
Platelet activating factor acetyl hydrolase (PAF-AH) is a kind of calcium which is not dependent on the existence of phospholipase A, with specific effect on the bottom of the phospholipid content of sn-two short acetyl groups. The de-acetylation reactions are catalyzed by the hydrolase, which can cause the generation of platelet activating factor (PAF) and oxidized phospholipids losing activity^[23]. By having PAF as a variety of intracellular phospholipid metabolites, strong platelet activation and inflammatory mediated effect, PAF is considered as the strongest platelet aggregation revulsant detected so far and the most active in vivo inflammatory factors involved in atherosclerosis early inflammatory reaction, as well as the occurrence and development of cerebral infarction and secondary brain injury^[24]. Through hydrolysis of PAF and oxidization of phospholipids, PAF-AH shows the antioxidant and anti-inflammatory effect, and its resistance to atherosclerosis. Mutations of PAF gene can lead to the reduction or loss of catalytic activity of PAF–AH, and promote the development of atherosclerosis and the formation of blood clots^[23]. Platelet activating factor acetyl hydrolase (PAF- AH) gene is located on 6p12-22.1 with 12 exons in total, which encodes 441 amino acids.
There are multiple genes polymorphism loci in PAF-AH. White Europeans have -275-G/T/C, A, -593-1136-T/C polymorphism at the 4th, 7th, and 11th exons whereas Asians only have -1001-A/G and -994-G/T polymorphism of the 9th exon, which help to lower the activity of PAF-AH, reduce the RAF hydrolysis, boost the PAF activity to a higher level and cause blood vessel damage^[25]. Hiramoto et al. found that 52 cases out of 120 patients with cerebral thrombosis in Japan (43.4%) had PAF-AH gene mutation (CT or TT), in which, it was 25.4% significantly higher than the control group^[26]. The differences between the group showed statistically significant and PAF-AH gene mutation was thought to be a risk factor for ischemic stroke. Zhang et al. analyzed the PAF-AH-994-g/T gene polymorphism of Chinese Han population found that T allele frequency was significantly higher than that of control group in LAA[H8]  of ischemic stroke group with TT genotype, however, the differences between corresponding SAA[H9]  type of ischemic stroke group and CE[H10]  type of ischemic stroke group, to the control group is not statistically significant^[27].
 
NOTCH3 gene
NOTCH3 gene is located on chromosome 19p13 spanning of 41.35 kb, and composed of 33 exons encoding a combined receptors and signal transduction function of transmembrane proteins. The protein has 2321 amino acid residues. A number of studies have pointed out that almost all patients who were not classified as NOTCH3 single nucleotide polymorphisms’ carrier did not have similar symptoms as CADASIL[H11]  patients, which shows that the mutations of cysteine residues play a core role in this disease^[28]. In the earlier discussion of this topic, we have found for the first time in international scope for G446T pathogenic mutations of CADASIL. NOTCH3 gene exon 4 mutation is the cause of the disease of CADASIL patients in the * *[H12]  area, and can be used as diagnostic for molecular biology indicator of CADASIL patients.
The study on NOTCH3 transgenic mice found a mature NOTCH3-/-mice’s smooth muscle cells were still lacking of contractile of protein and desmin expression, but the expression of vimentin is relatively high, which suggests that the smooth muscle cells was in its immature state and may turn the conversion process of synthetic to deflating appearance into disorder^[29]. These changes are mainly seen in the small diameter resistance arteries and the large diameter of the elastic arteries, in which, the veins are almost unaffected, thereby suggesting that the NOTCH3 gene mutation affects only in the differentiation of small artery of smooth muscle cell^[30] and then subsequently shows cerebral hemodynamic abnormalities and diffuse brain hypoperfusion, causing extensive damage to the brain’s white matter or tiny cerebral infarction^[31]. Using magnetic resonance imaging scanner, the changes in artery of lacunar infarction was shown. Schmidt et al. reported that conventional NOTCH3 gene mutations increased the risk of hypertension in patients with age-related white matter changes (ARWMC), while strong relationship was found between these 4 variants (rs1043994 rs10404382, rs10423702 and rs1043997) and cerebral white matter lesions^[28]. NOTCH3 gene-defected mice model shows that lack of NOTCH3 gene can cause dilation of blood vessels, reduction of elastin protein, smooth muscle cells dysfunction, and vascular maturation disorders after the birth as well as declination in regulation of cerebrovascular function^[32]. Therefore, NOTCH3 gene mutations can lead to cerebrovascular disease and vascular system degeneration on the whole. Ross et al. revealed that NOTCH3 p.R1560P (rs78501403; Exon 25) is the protective factors of ischemic stroke in the Caucasian and Swiss population (OR: 0.50, p = 0.50), with p.P380P (rs61749020; Exon 7) reduced the risk of large vascular stroke (OR: 0.35, p = 0.35) in the Caucasian population^[33].
 
Renin angiotensin system (RAS) related genes
Renin angiotensin system (RAS) is crucial in the body fluid control system and plays an important role in the water and salt metabolism balance, and the vascular tension adjustment. In addition, heart, brain, blood vessels, kidneys and other organs also has its local RAS system. Substrate angiotensin (AGT), key enzyme ACE[H13]  and AT1 receptor of RAS system also have been reported for gene polymorphism.
 
AGT gene
AGT is an important determinant of the RAS activity which also acts as the limiting factor formed by the only substrate AngII of RAS. Human AGT gene is a single-copy gene with the length of 13 kb, and located in chromosome 1q42-43, which composed of 5 exons and 4 introns. Several studies have reported that Met 235 genetic mutation is associated with the onset of thrombus diseases^[34], and the M235T genes and its alleles have close relationship with the onset of ischemic stroke^[35]. The study on the relationship between the brain cavity terrier and RAS gene polymorphism conducted by Guo et al. shows that AGT gene-704-t/C is closely related to the number of SAA subtypes of infarction lesions and AGT gene-704-t/C is the independent risk factor of the SAA type ischemic stroke^[36].
 
ACE Gene
Human angiotensin converting enzyme (ACE) gene is located on chromosome 17q23 (gbd119840). Among the insertion / deletion of a 287 bp sequence in the 16 introns which makes ACE gene to be polymorphism are namely DD type, II type and ID type. ACE is an important rate limiting enzyme in the renin angiotensin aldosterone (RAS) system, which directly affects the process of atherosclerosis. Several studies have indicated that ACE gene polymorphism is associated with hypertension, diabetes mellitus, coronary heart disease and atherosclerosis, which possesses high risk factors of cerebrovascular disease^[37-39]. According to the study done by Rao et al., the plasma levels of ACE in the stroke group showed that DD type X-ray > II type > ID type; with the DD genotype cases and D allele frequency of the stroke group were significantly higher than that of the control group; and DD genotype was an independent risk factor for stroke of SAA^[40]. Yao et al. proved that the frequencies of DD genotype and D allele in the ischemic stroke group were significantly different from that in the control group through the control study involving ischemic stroke, hypertensive hemorrhagic stroke and healthy people^[41]. They also indicated that the polymorphism of the ACE gene is a risk factor for ischemic stroke and hemorrhagic stroke in patients with hypertension.
 
AT1 Receptor Gene
In the RAS system, the main receptor for the biological activity of angiotensin II is AT1, which is a typical 7 transmembrane segment of G protein with 359 amino acids. AT1R gene is a 1 kb gene located on chromosome 3. AT1R gene has only one exon without any intron structure in it. There was a study showing that AT1R gene polymorphism is an independent risk factor for ischemic stroke^[42]. Other studies have also indicated that there is a synergistic effect between the substrate and gene polymorphisms of the key enzymes and receptors of RAS system, which subsequently has an effect on the disease.
In addition, plasma fiber protein causes sub (FG[H14] ), serine protease inhibitory protein 3 (phylogenetic 3[H15] ) gene, cyclooxygenase-2 (COX-2) gene, lipid oxygen synthetic enzyme activity protein (ALOX5AP) gene, phosphodiesterase 4D (PDE4D) gene, interleukin-6 (IL-6) gene polymorphism and etc may also associated with ischemic stroke.
The pathogenesis of ischemic stroke is associated to several factors, and as the result of multiple factors. In recent years, with the improvement of genetics and molecular biology methods, as well as microarray and bioinformatics technology that continue to emerge, new opportunities have been brought into the research associated with stroke and genetic polymorphism, in which, a number of great achievements have been accomplished. However, it is still a long way to go in order to reveal the pathogenesis of ischemic stroke, and there are still a lot of works to be done in order to identify the causative gene. The study on ischemic stroke susceptibility genes has two benefits: firstly, the discussion of risk factors and pathogenesis of ischemic stroke from the perspective of gene level makes it possible to screen high-risk groups to perform early prevention; secondly, the category of ischemic stroke could be drawn from the point of genetics based on the different susceptible gene of different subtypes to perform individual therapy.
Conflict of interest
The authors declared no potential conflict of interest with respect to the research, authorship, and/or publication of this article.
 
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