| BackgroundWith the development of the society and changes in people's diet structure,the incidence of cerebrovascular disease(CVD)has continuously increased with high morbidity and mortality,causing heavy burden to the family and society.This disease has become a very serious social health problem.In recent years,statistics in China has revealed that CVD is the second largest cause of death,and ischemic CVD(ICVD)can reach as high as 80%.Hence,there is an imminent need to study the etiology and pathogenesis of ICVD,and search for good treatment.Since the cause of ICVD is more complicated,the risk factors of cognition for people remain imperfect.At present,the known causes are high blood pressure,diabetes,hyperlipidemia,smoking,obesity,insulin resistance,etc.High homocysteine levels or hyperhomocysteinemia(HHcy)has gained increasing attention in recent years,in relation to the onset of ICVD.As is known the CVD is a combination of genetic and environmental factors caused by complex genetic diseases,and its pathogenesis is very complex.Although environmental factors such as smoking and drinking alcohol can increase its incidence,genetic factors are fundamentally important.Hence,genetic factors have become an important direction in studying the pathogenesis of ICVD.The study of the association of ICVD and homocysteine(Hcy)with metabolic enzyme gene polymorphisms has gained much attention.Hcy metabolism involves the participation of a variety of metabolic enzymes and cofactors,including cystathionine ?-synthase(CBS),methylenetetrahydrofolate reductase(MTHFR),methionine synthase(MS),methionine synthase reductase(MTRR),etc.In the process of folate metabolism,Hcy is catalyzed by MTRR into CBS and MS,and Hcy levels in plasma with CBS and MS adjust MTRR enzyme activity.Thus,Hcy-related metabolic enzymes have become important direction for determining CVD risk factors.Serine hydroxymethyltransferase(SHMT)in folate metabolism,through the four-hydrogen reversible catalytic serine and folic acid,synthesize glycine and folinic acid(methyl,participates in methylation reaction in many organism,DNA synthesis and methylation and Hcy metabolism are closely correlated).Vitamin B6,as well as CBS and SHMT,are important factors that affect the metabolic process.These also play an important role in the development of CVD.However,the role of these factors in CVD remains unclear.Hence,clinical trials with a large number of the samples are needed to discuss and clarify this role.Therefore,on the basis of previous studies,the present study further explored the Hcy metabolic pathways related to enzyme gene polymorphism and cofactor vitamin B6 in their role in CVD.ObjectiveThis study aims to explore vitamin B6 and Hcy CBS C1080 T,and the influence of MS-related enzyme gene A2756 G,TRR A66 G,SHMT C1420 T gene polymorphisms on the metabolism of Hcy,and investigate the factors and relationship between ICVD,in order to search for ICVD susceptibility genes,and provide theoretical basis for its prevention and control.MethodsA total of 200 patients with ICVD,who were admitted in the Neurology Department of our hospital from October 2012 to December 2014,were included into the study.These patents were assigned as the case group.In addition,120 patients with ICVD,who underwent a physical examination,were included into the study.These patients were assigned as the control group.All subjects did not have severe liver and kidney disease,epilepsy,malignant tumors,or immune system disease.All patients were instructed to arrive in the morning on an empty stomach.Three ml of elbow venous blood was collected and placed in a test tube with 2% EDTA,mixed with an anticoagulant,and stored in a refrigerator at 40°C for whole blood genomic DNA extraction.Inclusion criteria:(1)case group,ICVD,including lacunar cerebral infarction,atherosclerosis,thrombosis cerebral infarction and transient ischemic attack,and all patients diagnosed in accordance with the revised ICVD diagnosis standards of the Fourth National Conference on Cerebrovascular Diseases of the Chinese Medical Association,and confirmed by skull CT and/or head MRI scans;(2)control group,random individuals and patients with ICVD who received a health examination.Exclusion criteria:(1)case group,patients without cardiac cerebral embolism,patients with low blood volume of cerebral infarction,patients with asymptomatic cerebral infarction,hemorrhagic cerebral infarction,cerebral hemorrhage,or peripheral vascular thrombosis disease,and patients with artery inflammation,blood disease,and vascular malformations caused by cerebral infarction;(2)subjects with severe liver and kidney disease,epilepsy,malignant tumors and thyroid disease,patients who underwent transplantation,patients with severe malnutrition and diseases of the immune system,and patients who have eliminated the influence of plasma Hcy concentration drugs for nearly one month(such as,drug resistant epilepsy carbuncle,birth control pills,dopamine drugs,folic acid and vitamin B12).Vitamin B6 intervention trial: A total of 120 patients with ICVD were randomly divided into two groups,with 60 patients in each group.Conventional treatment with 0.5-1.0 mg per day of vitamin B6 was performed on the case group,while the control group did not receive any intervention.After one week of intervention,all patients were detected for Hcy indicators in plasma.Enzyme-linked immunosorbent assay was performed to detect the plasma Hcy levels.Polymerase chain reaction(PCR)for gene amplification,amplification segments by agarose gel electrophoresis,and reference marker standard judgment was performed.Restriction Fragment Length Polymorphism(RFLP)analysis of genotype polymorphism and gene sequencing was conducted for confirmation.The levels of vitamin B6 were analyzed by immunoassay.Results(1)The DNA samples of CBS C1080 T,MS A2756 G,MTRR A66 G,and SHMT C1420 T genotype distribution in the case group and control group were according to the Hardy-Weinberg equilibrium,with the object of study was the representative.(2)Plasma Hcy levels in the case group was significantly higher than that in the control group.Hcy levels in all CBS 1080 tt,MS 2756 gg,and MTRR 66 gg type samples were significantly higher than those in wild-type samples.Furthermore,Hcy levels in SHMT 1420 tt samples were significantly lower than those in wild–type samples.(3)The difference in DNA samples MS A2756 G,MTRR A66 G,and SHMT C1420 T in the case group and control group was significant in terms of the distribution of genotype frequency,while the difference in genotype frequency distribution with CBS C1080 T was not significant.The frequency distribution of MS A2756 G genotype AA,AG and GG was 65.5%,22.5% and 12.0%,respectively,for the case group,and 56.4%,22.6% and 21%,respectively,for the control group.The frequency distribution of MTRR A66 G genotype AA,AG and GG was 26.0%,41.5% and 32.5%,respectively,for the case group,and 14.2%,43.8% and 42.0%,respectively,in the control group.The frequency distribution of SHMT C1420 T genotype CC,CT and TT was 33.5%,45.0% and 21.5%,respectively,for the case group,and 41.8%,43.8% and 14.4%,respectively,for the control group.The frequency distribution of CBS C1080 T genotype CC,CT and TT was 41.5%,43.5% and 15.0%,respectively,for the case group,and 39.0%,40.2% and 20.8%,respectively,for the control group.(4)MS A2756 G,MTRR A66 G and SHMT C1420 T gene polymorphism was significantly associated with ICVD risk.MS A2756 G and MTRR A66 G were significantly positively correlated with ICVD,while SHMT C1420 T was significantly negatively correlated with ICVD.(5)The level of plasma vitamin B6 of patients in the case group was obviously lower than of patients in the control group.The plasma concentrations of vitamin B6 was negatively correlated with the concentration of Hcy.(6)Vitamin B6 intervention can obviously decrease plasma Hcy levels in ICVD patients.(7)For CBS C1080 T and SHMT C1420 T patients with vitamin B6 intervention,plasma Hcy levels of the wild-type object of study were significantly lower than patients without– intervention.Furthermore,the difference in plasma Hcy concentration changes in mutations in patients with vitamin B6 intervention was not significant between the two groups.For the MS A2756 G and MTRR A66 G genotype of patients,regardless of whether vitamin B6 mutations occurred after intervention,plasma Hcy levels in the object of study were significantly lower,compared to the case group.Conclusion(1)Hcy in plasma increased,and the lack of vitamin B6 was an important risk factor for ICVD.(2)MS gene A2756 G,MTRR A66 G and C1420 T SHMT gene mutations are important factors that affect the increase in plasma Hcy concentration.(3)MS gene A2756 G,MTRR A66 G and SHMT C1420 T mutations were closely associated with ICVD,and its diagnosis and treatment can be used as a molecular target.(4)Vitamin B6 intervention for MS A2756 G and MTRR A66 G genotypes significantly adjust plasma Hcy levels in patients.Furthermore,for CBS C1080 T and SHMT C1420 T wild-type genotypes,the plasma Hcy levels of patients were significantly adjusted,while for CBS C1080 T and SHMT C1420 T mutant genotypes,the adjustment of plasma Hcy levels in patients is not obvious.The clinical intervention can be adjusted according to the different genotypes of patients. |
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