| Part I Study of genetic susceptibility to ischemic strokeStroke is a leading cause of death and disability worldwide. It is a major social and financial burden. The majority stroke is ischemic, accounting for 80% of all strokes. Increasing evidence shows that IS is a complex multifactorial and polygenic disease arising from a wide number of gene-gene and gene-environment interactions. Besides the prevention of traditional risk factors, much more attention should be paid on gene risk factors. Identifying susceptible genes for ischemic stroke is expected to illuminate the etiology of stroke and to guide clinical treatment. Some Genome-Wide Association study (GWAS) study reported risk genes associated with ischemic stroke in populations with European and Japanese. Meanwhile, lots of researchers analyze the candidate gene for traditional risk factors of ischemic stroke. However, differences in genetic susceptibility between ethnicities have yet to be fully elaborated. In this study, we made an analysis for the association of candidate gene and ischemic stroke in a northwest Chinese Han population.In this hospital-based retrospective study,515 ischemic stroke cases and 515 control subjects were recruited from the second hospital of Lanzhou University. The subjects in two groups were matched well with age and sex.13 candidate gene SNPs were selected in this study according to the reference literatures. And MassARRAY iPLEx (sequenom, San Diego, CA) platform was used to accomplish allele discrimination and subsequent genotyping.The results showed that there were 5 susceptible genes for ischemic stroke identified in this study, which are MTHFR (rs1801133), Clorf156 (rs10489177), HDAC9 (rs2107595, rs2240419, rs2389995), RNLS (rs10887800) and XYLB (rs17118). Through the analysis of susceptibility genes in northwest China’s population of ischemic stroke, this study provides the basic data for screening populations and molecular genetics mechanism at risk of ischemic strokePart Ⅱ Meta-analysis of methylenetetrahydrofolate reductase (MTHFR) gene polymorphisms and susceptibility to ischemic strokeModerately elevated plasma homocysteine levels have been established as independent risk factors in vascular disease, including ischemic stroke(IS).Recently, a common mutation (C677T) in the 5,10-methylenetetrahydrofolate reductase (MTHFR) gene reducing the activity of MTHFR and increasing homocysteine levels in plasma was reported. The C677T MTHFR mutation may be a risk factor for ischemic stroke, but the results of previous studies have been conflicting. We conducted a meta-analysis to derive a more precise estimation of the relationship between MTHFR gene C677T polymorphism with ischemic stroke.We collected all of the relevant studies from Pubmed, Embase, Chinese Wan Fang database and CNKI databases to October 2014. The available data was analyzed by Stata software 12.0. We used odds ratios (ORs) and corresponding 95% confidence intervals (CIs) to present the strength of the association. Heterogeneity was evaluated by the Q-test and I2 statistic. Different genetic models, subgroup analysis, publication bias and sensitivity analysis were used to improve the comprehensive understanding.A total of 34 case-control studies including 6535 cases and 7704 controls were included in this meta-analysis. Our meta-analysis showed a significant association between the MTHFR gene C677T polymorphism and IS among total population(TC vs. CC:OR=1.29,95% CI 1.16-1.43,I2=35%, Pheterogeneity=0.025; TT vs. CC:OR= 1.66,95% CI 1.38-2.01,I2= 58.8%,Pheterogeneity<0.001; T vs C:OR= 1.29,95% CI 1.18-1.41, I2= 62.9%,Pheterogeneity<0.001; TT/CT vs CC:OR= 1.38,95% CI 1.23-1.54,I2=51.4%,Pheterogeneity< 0.001; TT vs CT/CC:OR=1.41,95% CI 1.21-1.64,I2=50.7%,Pheterogeneity<0.001).To better clarify the heterogeneity among our studies, we used subgroup analysis in the ethnicity and HWE.In subgroup analysis based on ethnicity, a significantly increased risk was found among Asians(TC vs. CC: OR=1.23,95% CI 1.11-1.37,I2=26.4%, Pheterogeneity= 0.117; TT vs. CC:OR=1.61, 95% CI 1.30-2.00,I2=63.6%, Pheterogeneity< 0.001;T vs C:OR=1.27,95% CI 1.15-1.41,I2=66.7%, Pheterogeneity<0.001; TT/CT vs CC:OR= 1.33,95% CI 1.17-1.51,I2=51.2%, Pheterogeneity= 0.002; TT vs CT/CC:OR=1.41,95% CI 1.18-1.67,I2= 55.5%, Pheterogeneity=0.001) and among Caucasians(TC vs. CC:OR=1.55, 95% CI 1.23-1.96,I2=0%, Pheterogeneity= 0.465; TT vs. CC:OR=2.02,95% CI 1.17-3.50,I2=58.1%, Pheterogeneity=0.036;T vs C:OR=1.42,95% CI 1.12-1.82,I2= 55.8%, Pheterogeneity= 0.045; TT/CT vs CC:OR= 1.63,95% CI 1.24-2.14,I2=31.4%, Pheterogeneity=0.200), however, no significant association was observed in African population. Furthermore, when stratifying by HWE, significantly elevated risks were observed for IS in two population (Yes and No HWE). Sensitivity analysis was conducted by estimating the influence of each study on the overall OR, and the result showed that no individual study affected the overall OR dominantly. There was no publication bias in our meta-analysis.This meta-analysis realed a significant association between the MTHFR gene C677T polymorphism and IS,and the MTHFR allele T polymorphism was significantlyassociated with increased risk of ischemic stroke.PART III The plasma proteomics research of Ischemic stroke with MTHFR C677T genotypeIn the first part of the research results confirmed polymorphism of MTHFR, a common polymorphism exists in the gene encoding the catalytic domain of this enzyme, in which a C'T substitution at the 677 position results in the replacement of alanine into valine at position 222 of the protein. The 677 polymorphism in the MTHFR gene has been often referred as a risk factor for cardiovascular diseases.This is principally due to its association with moderately elevated plasma level of homocysteine, an independent risk factor for occlusive arterial disease in the coronary, cerebral, and peripheral vessels and also a risk factor for venous thrombosis. So far, the direct involvement of MTHFR in cardiovascular diseases’development has not been demonstrated, although molecule epidemiological studies associated an elevated risk of Ischemic stroke with the MTHFR 677TT genotype. Proteomics, known as an important systems biology tool in the post-genomics era, provided a novel approach for dynamic analysis of different genetic background. In the present study, it was investigated whether the C677T polymorphism is associated with some plasma protein changes, different from the MTHFR itself, useful as predictive or diagnostic markers of cardiovascular diseases. For this purpose, a proteomic approach for the analysis of the plasma protein differential expression was used, based on 2-DE and MS identification.The blood samples of different genotypes Ischemic stroke patients has been collected. High abundance ratios of protein to remove by kit. The protein concentration were measured by Bradford method. The proteins were separated by means of two dimensional gel electrophoresis (2-DE) based on 17cm immobilized pH gradient. After silver staining and image scanning,2-DE image comparison was performed to analyze the differentially expressed proteins including theoretical Mr, theoretical pI value and 3D analysis using PDQuest8.0 software. Furthermore, mass spectrometry was applied to identify the differentially expressed proteins and the data were searched against NCBInr database by an in-house Mascot search engine. Functional clustering were filtered using Database for Annotation, Visualization and Integrated Discovery (DAVID), and through the STRING software to analyze protein interactions.A total of 28 protein spots in 2-DE images of plasma were found to be differentially expressed between 2 groups, of which 25 spots were up-regulated,3 spots down-regulated. Finally,5 spots were successfully identified to be HPT、TRFE、 ALBU by MALDI-TOF/TOF and Mascot software. This results was consistent with SWISS-PROT database. DAVID tool set is used to analysis the difference of protein cluster analysis showed that most of the differences in protein belongs to inflammatory immune response. KEGG pathway analysis found that differences in protein has three involved in the complement cascade reaction of the system. STRING interaction analysis confirmed by molecular genetic analysis highlight the novel association between the C677T MTHFR genotype with the DBP. Moreover, we found a quantitative reduction of Apolipoprotein A-4 in mutant individualsThe preliminary results revealed, the phenotype of MTHFR genotype effect may be related to multiple protein has a synergistic effect.This research can provide some ideas to the research of gene phenotypes, provides some references for the mechanism of Ischemic stroke. |
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