Association Analysis Of Inflammation-related Genes Polymorphisms With The Susceptibility To Intracranial Aneurysm In168Cases

Intracranial aneurysm is local vascular abnormalities, leading to cystic swelling on the intracranial arterial wall, thus creating brain aneurysm formation like protrusions.80%of intracranial aneurysms occur in the first half of the cerebral arterial circle, which has a rupture tendency, once ruptured, it can cause cerebral subarachnoid hemorrhage, thus putting intracranial aneurysms one of the most devastating brain vascular disease. Research shows that85%of SAH due to intracranial aneurysm rupture The mortality rate after aneurysm rupture within24h reach20%, one-week40%, within a month mortality can be up to50%. However, among those survived, over1/3have severe neurological deficits left. Although the incidence of intracranial aneurysms is1%-8%, ranking No.3intracranial lesions, less than cerebral thrombosis, hypertension caused cerebral hemorrhage, its high mortality, high morbidity, all creating a heavy burden to family and societyMultiple factors contributed to intracranial aneurysm, current study suggests that physical labor, mood fluctuation, drinking, etc. can cause sudden rupture of the aneurysm.up to now factors like age, gender, smoking, alcohol consumption, hypertension has been confirmed to be intracranial aneurysm risk factors. Inflammation refers to what the defense system in the living tissue take up against damage factors, vascular reaction is a central part of the inflammatory process. Inflammatory cell mediated abnormal vascular remodeling is considered to be a key aspect of the pathology in a variety of vascular diseases. Changes in blood flow can activate endothelial cells and inflammatory cells releasing inflammatory mediators, increasing vascular wall shear stress can increase the level of IL-1β, facilitating inflammatory cell infiltration into the vessel wall, thus activated endothelial cells, smooth muscle cells jointly mediated inflammatory response. Inflammatory cells, antibody and complement etc. involved in the inflammatory response inducing a variety of proteases which can damage the vessel wall thus causing aneurysm formation. However, until now the specific signaling pathways underlying intracranial aneurysm is not clear. TNF-a released by neutrophils, is a key immunoreactive substance, can strongly activated endothelial cells, it is significantly increased in intracranial aneurysms. In addition, IL-6is also significantly increased in blood and cerebrospinal fluid after the rupture of intracranial aneurysms.Through Gene delivery future generations obtain characteristics similar to parents. DNA as a genetic material through transcription and translation process, and determines the basic characteristics of life, is also closely related to the occurrence of the disease. Genetic factors are important in the pathogenesis of intracranial aneurysms. Epidemiological survey found that some patients with intracranial aneurysms showed onset familial tendency, the risk of the occurrence among the first-degree relatives of patients of the disease was significantly higher. Studies in patients with familial intracranial aneurysm artery among eastern Finnish population found intracranial aneurysms in patients with a positive family history increases the risk rate of12%. Among the Japanese population with family history of intracranial aneurysms, the unruptured aneurysms have a significantly higher prevalence of10.5%-13.5%. Thus, genetic factors play an important role in the occurrence of intracranial aneurysms. SNP refers to genetic marker loci which is a Single nucleotide mutation in the genome. Its large quantity, rich polymorphism contributes to the third generation of genetic markers. Many human phenotypic differences like susceptibility to drugs or disease may be related with the SNP. Although most SNP located in the genome intron elements, but it may play an important role in RNA splicing process. Using whole-genome SNP scans of different races of intracranial aneurysms pedigree confirms a number of chromosomal segments1p34.3-1p36.13、7q11、19ql3.3and Xp22may be associated with the occurrence of aneurysms. In addition, traditional candidate gene study, measureing functionally related genes with intracranial aneurysms, also found a lot of susceptibility genes, such as endothelial glycoprotein gene, matrix metalloproteinase genes, IR collagen gene, otl-antitrypsin gene, elastin gene, angiotensin-converting enzyme gene, apolipoprotein genes. Some of these genes are functionally associated with the formation of the vascular wall, thus creating the molecular evidence. Some studies also suggest that certain inflammatory cytokines like IL-1β, IL-6and TNF-a are positive correlated with intracranial aneurysms susceptibility.Although it has been reported TNF-a, IL-1β, and IL-6are associated with the susceptibility of intracranial aneurysms, they have not been verified in large GWAS samples. In the Italian population, IL-6is not a intracranial aneurysm susceptibility gene. Thus, TNF-a, IL-1β, and IL-6is a susceptibility gene intracranial aneurysms remains uncertain, it may be genetic differences between ethnic groups, or the small number of the sample genome research, lax quality control and not tight statistical analysis strategy leading to an error information. To further confirm that TNF-a, IL-1β, and IL-6gene polymorphism and intracranial aneurysms correlation in Chinese Han population, we collected200cases of intracranial aneurysm patients and200matched peers with strict normal physical examination. The tag SNP loci nearby TNF-a, IL-1(3, and IL-6gene were genotyped, and after strict quality control and rigorous statistical analysis, we try to explore the association of TNF-α, IL-1β, IL6gene polymorphisms with the onset ofntracranial aneurysms among chinese Han population. The implementation of this study will not only made finding the pathogenesis of intracranial aneurysms possible, but also provided important basic molecular biology underlying intracranial aneurysm, facilitate screening among susceptible population and early diagnosis and treatment. Above all, it is of great significance as to raising the detection rate and improving survival and quality of life of patients.Chapter one:Association study of inflammation-related gene polymorphisms and susceptibility of intracranial aneurysms1. ObjectiveTo explore the allele frequencies and genotype frequency distributions of the SNPs in the inflammation-related genes among intracranial aneurysms patients. To investigate the relationship between the inflammation related gene polymorphisms with the severity of intracranial aneurysms after SAH, the clinical relevance of outcomes and the incidence number (solitary/multiple) of intracranial aneurysms.2. Materials and methods2.1Object of study:The168aneurysmal SAH patients with intracerebral hematoma companied or not were choosed as Study group were collected from neurosurgery ward in our NanFang hospital from2010to2012. All Patients choosed were with a onset lower than72hours and older than18years old.they were confirmed by clinical symptoms and then subject to whole brain digital subtraction angiography or CT angiography for the diagnosis of intracranial aneurysms. The control group was184healthy Han people from our hospital’s medical department, all of them had no history of intracranial aneurysms. The three representative SNPs (rs1800795, rs1143627and rs1799964) of tNF-α, IL-1β, and the IL-6gene region were picked out from the NCBI database2.2Research Methods:The clinical data of outpatient intracranial aneurysm patients, hospitalized patients with intracranial aneurysms and healthy Volunteers were all recorded into Excel. Whole blood genomic DNA was extracted using Tigen wholly genomic DNA extraction kit. Then it was tested with agarose gel electrophoresis identified whether it was degraded and measured concentration in the Nanodrop ND-2000spectrophotometer. PCR primers of the corresponding SNP loci were designed according to the nucleotide sequences in NCBI database, then they were used to amplify the sequences which will later used for sequencing in Biotech companies.2.3Statistical analysis:Chi-square test of the SPSS statistical software were used to analysis the allele and genotype frequencies. The allele and genotype frequencies of the SNP loci in TNF-a, IL-1β, and IL-6gene region between cases and controls were compared with the X2test. Mean plus or deviation standard deviation were show for Measurement data, after the homogeneity of variance test (P>0.05), they were compared using ANOVA. P<0.05was considered statistically significant. The progressive model of "Logist regression model" were used to identify the SNP loci of primary effect and assess whether there is a cumulative effect between susceptibility SNP loci and risk factors for intracranial aneurysms. Haploview4.2software were Used for Kazakhstan-Weinberg equilibrium tests and linkage disequilibrium analysis on specific SNP loci segments among the Chinese and Japanese population, LD diagram of the section was drawn.3. ResultsOur study collects168study group intracranial aneurysm patients with male74 cases (44.05%), female94cases (55.95%), mean age (51.67±18.71) years and BMI (23.21±2.16). There were no significant difference between the two groups in general, including history of hypertension, high cholesterol, diabetes, smoking and drinking history (all P>0.05). After treatment the outcomes of the Intracranial aneurysm patients were assessed using the Glasgow Outcome Scale (Glasgow Outcome Scale, GOS), with GOS4～5into a good prognosis and GOS1～3into poor prognosis.122patients with good prognosis (representing72.62%),46patients with poor prognosis (representing27.38%) are found. We first find the the region of the tested genes TNF-a, IL-1β, and IL-6in UCSC library. Then Looking for SNP loci located within the area of10kb range (upstream and downstream5kb) of the three genes in Chinese Han population. At last, we chose rs1799964located upstream of TNF-a gene, rs1143627upstream of the IL-1βgene, rs1800795upstream of the IL-6gene with the most representatives of the region to be examined.The concentration of DNA of whole blood samples among intracranial aneurysms and healthy subjects was between100ng-1μg with good quality. Agarose gel electrophoresis show size about15000kb above with no smearing confirming no degradation. PCR amplification fragmen were shown by agarose gel electrophoresis, all are unity bands with no nonspecific amplification products. The PCR product was genotyped of the SNP loci for each sample. The allele frequency of rs1799964, rs1143627and rs1800795corresponding to IL-6, IL-1β, TNF-a show significant difference between intracranial aneurysm group and normal control group (X2=5.256, P=0.022)(X2=186, P=0.023)(X2=4.368, P=0.037). The allele frequency of rs1143627and rs1800795locus was associated with the severity of illness of intracranial aneurysms after SAH (x2=4.257, P=0.039)(X2=083, P=0.043). In addition rs1143627locus was associated with the clinical outcomes after intracranial aneurysm related SAH.4. Conclusion The polymorphism of inflammatory factor IL-6, IL-1β, TNF-a gene were associated with intracranial aneurysms susceptibility in Chinese Han population. It thus lay foundation for further study of the role IL-6, IL-1β, TNF-αin played in intracranial aneurysm.Chapter two:A preliminary study of the molecular mechanisms of the inflammation related SNP loci which is associated with the of intracranial aneurysms susceptibility in Han population1. ObjectiveExplore molecular mechanisms of the inflammation related SNP loci which is associated with the of intracranial aneurysms susceptibility in Han population and for provide new ideas for the prevention and treatment of intracranial aneurysms.2. Materials and methods2.1Object of study:24healthy volunteers were collected from hospital medical center older than18years old. All are Han population with no history of intracranial aneurysm, intracranial aneurysm family hypertension, cerebrovascular disease, cancer, brain trauma. All did not take anti-inflammatory drugs recently. All with no histroy of cardiovascular, brain, liver, kidney related disease, no diabetes, thyroid disease history, no recent infection and inflammation. Participants were taken50ml whole blood through the cubital vein, sodium citrate anticoagulation, used for analysis gene expression levels and serum markers. SNP loci rs1799964/rs1143627and rs1800795are those associated with intracranial aneurysms which have been verified in the previous section2.2Research Methods:Blood samples from healthy subjects were collected and separated PBMCs, the total RNA were extracted with Trizol. Their content were Measured on a Nanodrop ND-2000spectrophotometer and purity was identified on1%agarose gel electrophoresis to confirm whether the RNA degradated. The expression amount of a reverse transcription to cDNA library was constructed by reverse transcription kit and the target gene mRNA level was mesured by fluorescent quantitative PCR.3. ResultsAfter lymphocyte separation medium by PBMC, we extracted the white blood cells RNA using Trizol, there are28S,18S clear,5S faint bands, indicating no degradation of total RNA, thus ensuring the efficient and reliable reverse transcription. By real-time PCR technology we detect the expression lever of TNF-a, IL-1β, and IL-6gene of the healthy control with different genotypes. We found that the TNF-a gene rs1799964genotype sites, IL-1β gene rs1143627locus rs1800795, and IL-6gene loci have no effect on the expression of each gene. Using real-time enzyme-linked immunosorbent assay we detect the serum protein level of TNF-a, IL-1β and IL-6of the healthy volunteers with different genotypes. TNF-a gene locus rsl799964, IL-1β gene locus rs1143627and IL-6gene locus rs1800795had no effect on the expression of inflammatory cytokines.4. ConclusionNo correlation between the rs1799964locus and TNF-a mRNA and serum TNF-a protein levels. No correlation between the rs1143627locus and IL-1β mRNA and serum IL-1βprotein levels. No correlation between the rs1800795locus and IL-6mRNA and serum IL-6protein levels. What role of these SNP loci to play through in intracranial aneurysms needs further study to confirm it.