Study On The Correlation Of Methylenetetrahydrofolate Reductase Polymorphism And Cerebral Infarction And Its Mechanism

Background: In recent years,with the aging of the population,the incidence and mortality of cerebrovascular diseases in China are also increasing,among which cerebral infarction(ischemic stroke)accounts for the highest proportion.Cerebral infarction refers to the tissue ischemia and hypoxia caused by cerebral blood circulation disorder,which leads to necrosis and softening of localized brain tissue.At present,it is believed that cerebral infarction is the result of the interaction and influence of environmental and genetic factors,and gene polymorphism is the main manifestation of genetic factors.Methylene tetrahydrofolate reductase(MTHFR)gene polymorphism has been reported to be associated with cancer,pregnancy-related hypertension,diabetes and vascular diseases,and it has been suggested that MTHFR gene polymorphism can affect the occurrence and development of cerebral infarction by affecting the level of homocysteine.Therefore,the study on the correlation between MTHFR gene polymorphism and cerebral infarction in cerebrovascular patients and its biological mechanism has positive clinical significance for the prevention and treatment of cerebral infarction.Part I: MTHFR genotype detection and correlation analysis with cerebral infarction Objectives:To discuss the correlation of polymorphism of MTHFR C677 T and A1289 C and susceptibility of cerebral infarction.Methods:1.204 cerebral infarction patients who visited the department of neurology,Xianyang hospital of Yan’an Univer sity from October 2017 to October 2018 were collected as observation group and210 health examination controls without cerebrovascular history were collected as control group.General data and clinical indicators were collected and compared b etween two groups,including age,height,weight,sex,hypertension,diabetes,total cholesterol(TC),triglycerides(TG),high-density lipoprotein(HDL),low density l ipoprotein(LDL),fasting glucose,homocysteine(Hcy).2.Genotypes of MTHFR C677 T and A1298 C sites in the two groups were detected and analyzed to test wheth er the genotype distribution was in according with the Hardy-Weinberg equilibrium.Genotypes and alleles frequencies were compared,and P<0.05 was considered as statistically significant.3.Logistic regression was performed to analyze the indepen dent influencing factors of cerebral infarction for the indicators with statistically si gnificant differences between the two groups.4.Haploview was used to analyze th e linkage disequilibrium relationship between C677 T and A1298 C sites.Results:1.T here were significant differences in Hcy and hypertension between the observation group and the control group(all P<0.05).2.The genotype distribution of C677 T and A1298 C sites in the two groups was consistent with Hardy-Weinberg equilibri um,and the genotype distribution differences of C677 T and A1298 C sites were st atistically significant(P<0.05).3.Logistic regression analysis showed that high Hcy level,CC and CT genotypes at C677 T,CC genotypes at A1298 C,and hypertensi on were risk factors for cerebral infarction.4.Linkage disequilibrium analysis show ed that there was linkage disequilibrium between C677 T and A1298 C sites,and th e haplotype distribution frequencies were AT=54.3%,AC=29.3%,CC=13.7%,CT=2.7%,respectively.Conclusion:1.Hcy level,C677 T locus polymorphism,A1298 C locus polymorphism and hypertension were independent influencing factors of cerebral infarction.2.The C677 T and A1298 C sites of MTHFR gene had a linkage disequilibrium relation ship.Part Ⅱ: Study on the biological mechanism of MTHFR gene polymorphism and cerebral infarction Objectives:Combined with bioinformatics,the biological mechanism of C677 T and A1298 C may influence cerebral infarction was analyzed.Methods:1.The nucleic acid,protein sequences and gene polymorphism sites of MTHFR were searched in NCBI database,and ProtParam was used to analyze the basic physicochemical properties of MTHFR protein.2.PSORT was used to analyze the subcellular localization of MTHFR.3.KEGG signaling pathway and STRING protein interaction network were used to analyze the signaling pathways and possible biological functions which MTHFR involved in.4.NCBI conservative domain analysis was used to analyze the conservative domain of MTHFR,and Phyre2 was used to analyze its secondary structure.5.3D Ligandsite was used to analyze the ligand binding sites of MTHFR protein to understand its basic structural characteristics.6.Quark modeling was conducted to model the protein structure with different C677 T and A1298 C genetype and compare the differences.Results:1.The analysis of basic physical and chemical properties showed that MTHFR had a total of656 amino acid residues,was lipophilic and hydrophilic,and had unstable structure.The physical and chemical properties with different C677 T and A1298 C sites did not change.2.Subcellular localization showed that MTHFR was mainly located in the cytoplasm.3.Analysis of KEGG signaling pathway showed that MTHFR was mainly involved in the folic acid metabolism pathway.4.Conservative domain analysis showed that MTHFR had only one MTHFR superfamily domain,and C677 T site was located in the corner,while A1298 C site was located in the site helix.5.3D Ligandsite analysis showed that MTHFR ligands were FAD,SAH and CIT,and C677 T site was close to FAD ligand binding region.6.Local modeling of gene polymorphism sites showed that theprotein structure before and after mutation at C677 T site changed from a gene helix and a fold to a gene helix and random curl.There was no significant difference in the structure of different A1298 C site,both of which were two helix structures.Conclusions:1.MTHFR may influence the occurrence and development of cerebral infarction by participating in folic acid metabolism pathway.2.The polymorphism of MTHFR C677 T locus may affect the binding of MTHFR and its ligands by influencing the protein structure,thus affecting the occurrence and development of cerebral infarction.Full text conclusion:The overall conclusion of this study is as follows: the polymorphism of MTHFR C677 T and A1298 C sites were independent influencing factors for the occurrence of cerebral infarction of cerebrovascular disease patients,and the influencing mechanism may be that the C677 T site polymophism may cause changes in the local structure of the protein,which affects the binding of MTHFR and its ligands,thus affecting the occurrence of cerebral infarction.The correlation between A1298 C site and cerebral infarction may also be related to the linkage imbalance between C677 T site.