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    The Progress of Related Study on Gene Polymorphism of Ischemic Stroke
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    The Progress of Related Study on Gene Polymorphism  of Ischemic Stroke

                       Yan Xu   Deqin Geng

           
    Abstract  Ischemic stroke is a polygenetic disease with complex pathogenesis and resulted by interaction and mutual infulunce of genetic and enviromental factors. As the completation of the genome project and the development of molecular genetics,discussing the pathogenesis from genetic level has become a hot question.A lot of canidate gene related to ischemic stroke has been widely studied and the genetic polymorphism study on ischemic stroke helps to find out the pathogenesis and discover high risk groups and individuals in advance.Therefore, preventing in advance and easing the medical burden of both the society and the family.
    Key words  Ischemic stroke；polymorphism；genetics；candidate gene；mononucleotide
     
    Ischemic stroke is global pulic health problem threatening human life ,With high incidence and high morbidity and mortality, and has brought a heavy burden to patients, families and society . Now it is generally believed that the pathogenesis of ischemic stroke is very complex, genetic factors and environmental factors is the result of the interaction and mutual influence. 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 of fraternal twins. Traditional lifestyle and environmental factors are easy to control and intervention, but genetic factors have relative stability and not easy to change. So comparing to other related metabolic index, the genetic index is more stable to  provide important basis for ischemic stroke’s early detection of pathogenesis and high-risk groups.
    Genetic polymorphism or gene polymorphism means in a biological group, there are two or more discrete variant genotypes or alleles at the same time, mainly displaying in the DNA fragment length polymorphism, repeated DNA sequence polymorphism, single nucleotide polymorphisms. Genome-wide association study (GWAS) has identified a series of candidate genes related to genetic factors and risk factors of ischemic stroke , mainly includes: the related factors of inflammatory reaction gene, platelet activating factor (PAF - AH) acetyl hydrolase gene, renin angiotensin system (RAS) related genes, lipid metabolism related genes, NOTCH3 gene, etc. Candidate genes of ischemic stroke will be mainly discussed below in order to have a more comprehensive understanding of disease-causing genes  of ischemic stroke and get more comprehensive understanding to the genetic susceptibility of ischemic cerebrovascular disease.
    1 CRP gene
    C-reactive protein (CRP) gene in chromosome 1 long arm has two exons, separated by a introns, encoding 206 amino acid residues. 1 CRP molecule subunits can combine with 160 base pairs. This structure enables the CRP to integrate with platelet activating factor which plays a regulatory role in the control of inflammation. In atherosclerosis and ischemic events occurred in the process of the development , the inflammatory response plays an important role.The body's inflammatory state can reflect through the acute phase of c-reactive protein (CRP).Once atherosclerosis damage appears in patients ,the CRP levels will rise accordingly [2]. But the CRP to atherosclerosis mechanism is unclear.It has been shown throgh studies that CRP can induce expression of tissue factor of mononuclear cells, activatie blood coagulation system and the complement system, increase the risk of heart, cerebrovascular disease [3]. It is reported by literatures that an independent CRP level measurement can be used as a starting or recurrence of cerebrovascular disease risk predictors [4]. The study found that CRP, IL - 6 and 1059 g/C hace a relation with venous thrombosis [5]. At the same time, CRP in carotid artery and small cortical blood vessels plays an important role in disease progression and can be used as a predictor of ischemic stroke, and even can be used as risk prediction factors after lacunar cerebral stroke [6-7]. And clinical studies have shown that plasma CRP concentration in patients with ischemic stroke group is significantly higher than control group in the crowd, rs3093059 and rs3091244 loci gene polymorphisms are significantly associated with elevated CRP [8-10]. Ben – Assayag’s studies also suggest that CRP gene promoter sequences - 717 - a/G single gene mutation associates with acute stroke and AG, GG genotype CRP level is significantly higher than AA type [11].
    2 MMP-3 gene
    Matrix metalloproteinases (MMPs) is a kind of active center containing zinc ions protease superfamily whose main physiological functions are involved in the degradation and reconstruction of extracellular matrix in the body. The degradation of extracellular matrix runs through the whole process of hardening of the arteries. MMPs through the digestive ingredients undermine its fibrous cap structure accelerating the plaque rupture, is one of the important reasons of unstable plaques. MMP - 3 gene located on chromosome 1 lq22. 3, includs 10 exons and 9 introns of homology with its expression mainly in the regulation of transcription. MMP - 3 is one of the important members of MMPs with a wide range of specificity of substrate, can also activate other kinds of MMPs, initiating in the MMPs bioactive action [12-13]. Atherosclerotic plaque is a process involving the MMP inflammatory [14]. In unstable plaques, especially prone to rupture of the plaque shoulder, MMP - 3 content is higher and the activity of enzyme was also significantly increased, which follows that elevated serum levels of MMP - 3 are closely related with unstable plaques. Gnasso et al. have found that in the health of the Caucasian population with no significant atherosclerosis risk factors, 6 A 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 [16] and others also put forward the correlation of atherosclerosis of the MMP - 3 promoter 5 A / 6 A polymorphism and 6 A6A genotype carriers are more prone to the progress of atherosclerosis.  Ma Aijun[17] and others from China’s study shows that rs3025058 site 5 A allele, A rs679620 loci alleles may increase the onset risk of large artery atherosclerosis (OR > 1), and G allele rs522616 locus may reduce their onset risk (0 r < 1). But the three single nucleotide polymorphisms genotypes have no difference in small artery occlusion type stroke group and control group has no statistical significance. From another side, this also reflects the pathogenesis of large artery atherosclerosis and small artery occlusion stroke is different.
    3 ApoE gene
    Apolipoprotein E (ApoE) as chyle particulate residue and low density lipoprotein receptor ligand receptor is the important component of plasma lipoproteins and determining factor of lipid metabolism and heart cerebrovascular disease, playing an important role in the metabolism of blood fat. Apolipoprotein E (ApoE) gene located on chromosome 19 ,consists of 3597 nucleotides with four exons and three introns, whose gene polymorphism in 112 and 158 loci coding three ApoE subtypes: ApoE2, ApoE3 and ApoE4. Apolipoprotein E gene polymorphism has very obvious effects on blood lipid metabolism [18]. Sing and others reported that plasma total cholesterol and low density lipoprotein cholesterol levels of ApoE4 carriers in normal crowd  were significantly higher than those of ApoE3/3 carriers [19]. This suggests that the E4 allele has the effect of elevated cholesterol, and 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 of Qun Liu [22] and others, genotype groups carrying E2 and E4 significantly increased the onset risk of ischemic stroke and the onset risk of ischemic stroke in E4 allele carriers was 1.78 times of the control group. Comparing to the control group, the onset risk of ischemic stroke in E2 and E4 gene group increases neary 40% (RR = 1.36) and the differences were statistically significant.
     
    4 PAF-AH gene
     
    Platelet activating factor (PAF - AH) acetyl hydrolase 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 actyl actions catalyzed by the hydrolase can cause platelet activating factor (PAF) and oxidized phospholipids losing activity [23]. PAF as a variety of intracellular phospholipid metabolites, having strong platelet activation and inflammatory mediated effect, is 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]. By hydrolysis of PAF and oxidized phospholipids, PAF - AH shows the antioxidant and anti-inflammatory effect and the resistance to atherosclerosis. Mutations of PAF gene function can lead to the catalytic activity of PAF - AH reduction or loss and promote the development of atherosclerosis and the formation of blood clots [23]. Platelet activating factor (PAF - AH) acetyl hydrolase gene located in 6 p12-22.1, with a total of 12 exons, encoding 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 of the 4th, 7th, 11th exon and asians only have  - 1001 - A/G and - 994 - G/T polymorphism of the 9th exons,which can lower the activity of PAF - AH, reduce the RAF hydrolysis,make the PAF activity at a higher level and cause blood vessel damage [25]. Hiramoto [26] and the ohers found that 52 cases out of 120 patients with cerebral thrombosis in Japan (43.4%) had PAF - AH gene mutation (CT or TT), significantly 25.4% higher than the control group.The difference was statistically significant and PAF - AH gene mutation was thought to be a risk factor for ischemic stroke. Xiong Zhang [27] and others through analying the PAF - AH - 994 - g/T gene polymorphism of Han Chinese, found that LAA ischemic stroke group TT genotype, T allele frequency was significantly higher than that of control group, but the difference between corresponding SAA type ischemic stroke group and CE type ischemic stroke group  and control group is not statistically significant.
     
    5 NOTCH3 gene
    NOTCH3 gene located on chromosome 19 p13, spanning 41.35 KB, is composed of 33 exons, encoding a combined receptors and signal transduction function of transmembrane proteins. The protein has 2321 amino acid residues. Studies have pointed out that almost all of the patients do not carry NOTCH3 single nucleotide polymorphisms didn't have similar symptoms as CADASIL patients,which shows that the cysteine residues of mutations plays a core role in this disease [28]. In the early study of this topic, we have found for the first time in international scope G446T pathogenic mutations of CADASIL.NOTCH3 gene exon 4 mutation is the cause of the disease of CADASIL patients in the * * area, and can be used as diagnostic molecular biology indicator of CADASIL patients.
    The study on Notch3 transgenic mice found a mature Notch3 - / - mice’s smooth muscle cells are still lack of contractile of protein and desmin expression, but the expression of vimentin is relative on the increase which suggests that the smooth muscle cells is in the synthesis of immature state and may run into disorder in the process of conversion from synthetic to deflating appeared [29]. These changes are mainly seen in the small diameter resistance arteries, and the large diameter of the elastic arteries and veins is almost unaffected, which suggests the Notch3 gene mutations affects small artery smooth muscle cell differentiation [30] and then shows cerebral hemodynamic abnormalities and diffuse brain hypoperfusion, causing extensive damage to the brain white matter or tiny cerebral infarction [31]. On the imaging,the change is shown through artery of lacunar infarction. Schmidt H [28] and etc. also report conventional Notch3 gene mutations increase the risk of hypertension crowds age-related white matter damage, while the relationship between these four sites rs1043994 rs10404382, rs10423702 and rs1043997 and cerebral white matter lesions is the largest. Notch3 gene defect model in mice shows that the lack of Notch3 gene can cause dilation of blood vessels and blood vessel elastin protein reduction, smooth muscle cells modoling dysfunction, vascular maturation disorders after the birth as well as the regulation of cerebrovascular function decline [32]. Therefore, Notch3 gene mutations can lead to cerebrovascular disease andin the whole vascular system degeneration. Owen, A. Ross and etc. [33] studied that NOTCH3 p.R 1560 P (rs78501403; Exon 25) is the protective factors of ischemic stroke in the Caucasus and Switzerland population(OR: 0.50, P = 0.50), p.P 380 P (rs61749020; Exon 7) reduces the risk of large vascular stroke (OR: 0.35, P = 0.35) in the Caucasus population.
    6 Renin angiotensin system (RAS) related genes
    Renin angiotensin system (RAS) is important 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 to the whole body RAS system, heart, brain, blood vessels, kidneys and other organs also has local RAS system. Substrate angiotensin (AGT), key enzyme ACE and AT1 receptor of RAS system also have gene polymorphism.
    6.1 AGT gene
    AGT as the limit factor frmed by the only substrate AngII of RAS , is an important determinant of the RAS activity. Human AGT gene is a single copy genes with length of 13 KB, located in chromosome 1 q42 -43, composed of five exons and four introns. Studies have reported that Met 235 genetic mutations is associated with the onset of thrombus diseases [34], and the studies of Yepu Su have shown that M235TT genes and its alleles have close relationship with the onset of ischemic stroke [35]. The sutdy on the relationship between the cavity terrier and RAS gene polymorphism from Guo ZS etc. [36] 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 factors of the SAA type ischemic stroke.
    6.2 ACE Gene
    Human angiotensin converting enzyme gene is located in chromosome 17q23 (gbd119840). Among the insertion / deletion of a 287bp sequence in the 16 introns makes ACE gene to be polymorphism, i.e. 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. Studies have indicated that ACE gene polymorphism is associated with hypertension, diabetes mellitus, coronary heart disease and atherosclerosis, high risk factors of cerebrovascular disease[37-39]. According to the study of Rao R et al [40], the plasma levels of ACE in the stroke group showed that DD type X-ray > type >ID type; 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. Chinese researcher Yao Lifen[41] has 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 of ischemic stroke, hypertensive hemorrhagic stroke and healthy people. She also indicated that the polymorphism of the ACE gene is a risk factor for ischemic stroke and hemorrhagic stroke in patients with hypertension.
     
    6.3 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 located on chromosome 3 and its full length is 1KB. There is only one exon and no intron structure in AT1R gene. Studies show 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 the gene polymorphisms of the key enzymes and receptors of RAS system, which has an effect on the disease.
    In addition, plasma fiber protein causes sub (FG), serine protease inhibitory protein 3 (phylogenetic 3) gene, cyclooxygenase-2 (COX-2) gene, lipid oxygen synthetic enzyme activity protein (ALOX5AP) gene, phosphodiesterase 4D (PDE4D) gene and interleukin-6 (IL-6) gene polymorphism etc. may also associated with ischemic stroke.
    The pathogenesis of ischemic stroke is related to many factors, and it is the result of multiple factors.In recent years, with the improvement of genetics and molecular biology methods, as well as microarray and bioinformatics technology continue to emerge, new opportunitieshave been brought to stroke and genetic polymorphism research, and great achievements have been made. However, it is still a long way to go to reveal thepathogenesis of ischemic stroke. And there still exists a lot to work to identify the causative gene. The study on ischemic stroke susceptibility genes has two benefits: first, the discussion of risk factors and pathogenesis of ischemic stroke from the gene levelmakes it possible to screen high-risk groups to perform early prevention; second, the category could be drawn of ischemic stroke from the point of genetics based on the different susceptible gene of different subtypes to perform individual therapy.
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