The Correlation Of Cholesteryl Ester Transfer Protein Gene Polymorphism And Susceptibility To Cerebral Haemorrhage And Study On The Mechanism

Backgrounds:In recent years,the incidence of cardiovascular and cerebrovascular diseases in Chinese increased gradually,serious diseases such as intracerebral hemorrhage(ICH)is threatening the health and lives of people.The main clinical treatments of ICH was conservative treatments and surgeries but the mortality rate and disability rate of ICH was still really high so prevention was still the key on reducing the risk of ICH.ICH was defined as cerebral injury caused by nontraumatic cerebral vessels rupture,which main caused by hypertension,hyperlipidemia,atherosclerosis and genetic factors.Gene polymorphism was the main form of influence on diseases of genetic factors,so the study on the relationship of key gene polymorphism and diseases was significant for the early prevention of diseases.The current researches indicated that cholesteryl ester transfer protein(CETP)was involved in the cardiovascular diseases and lipid metabolism associated diseases so its polymorphism may be correlated with the attack of ICH.There was few current researches about the relationship of CETP polymorphism and ICH and there was no clear conclusions about how different genetype influence the diseases.Hence,exploring the correlation of CETP polymorphism and ICH and the possible mechanisms has positive clinical significance for the early prevention and treatments for ICH.Part One Detection of CETP genetype and its correlation of ICHObjectives:To preliminarily discuss the correlation of CETP single nucleotide polymorphism sites TaqIB(rs708272),I405V(rs5882),D442G(rs2303790),R451Q(rs1800777),A373P(rs5880)and ICH susceptibility,and to analyze the interaction among these SNPs.Methods:1.236 ICH cases were collected as case group and 284 healthy control were collected as the control group.The generally information was collected and compared which included sex,age,family history,education,smoking,drinking and complications.The ICH patients were divided into mild,moderate and severe stages according to the performance at admission and oICH score;2.The blood pressure,blood glucose,total cholesterol,triglyceride,low density lipoprotein cholesterin(LDL-C),high density lipoprotein cholesterin(HDL-C)of the two groups were detected to diagnose weather the participants have hypertension,hyperlipidemia and diabetes or not;3.The genetype of CETP TaqIB,I405V,D442G,R451Q,A373P in the two groups were detected and if the genetype distribution was in agreement with Hardy-Weinberg genetic equilibrium law was tested then.The distribution of each SNP in the two groups were compared respectively,and the distribution among different clinical ICH stages were compared as well.P<0.05 was considered as significant different.4.Logistic regression analysis involving generally information and genetype was conducted to pick out the independent risk factors of ICH attack and illness stage;5.Haploview was used to analyze the linkage disequilibrium of there SNPs and conduct the haplotype analysis.Results:1.The family history,smoking,hypertension,hyperlipidemia rate in the case group and control group were significant different(P<0.05);The hyperlipidemia rate among different ICH stages was different(P<0.05);2.The distribution in the two groups were agreement with Hardy-Weinberg balance.The distribution frequency of TaqIB B1B1,B1B2,B2B2 genetypes in the case group and control group were 84(35.6%),113(47.9%),39(16.5%)and 88(31.0%),144(50.7%),52(18.7%)respectively,and there was no significant statistical difference(?~2=1.270,P=0.530);The distribution frequency of I405V II,IV,VV genetypes in the two groups were 56(23.7%),138(58.5%),42(17.8)?94(33.1%),152(53.5%),38(13.4%)respectively and the difference was statistical significant(?~2=6.124,P=0.048);The distribution frequency of D442G DD,DG genetypes in the two groups were 218(92.4%),18(7.6%),0(0%)and272(95.8%),12(4.2%),0(0%),therewasnostatistical different(?~2=4.744,P=0.098)?The R451Q and A373P genetype in the two groups were both RR and AA and there was no mutant genotype;3.The genetype and allele distribution of I405V among different ICH stages were different(?~2=16.109,P=0.003),and there was no difference in other SNPs(P>0.05);4.The results of Logistic regression showed that hypertension,hyperlipidemia,smoking and 405V carrier were the independent risk factors of ICH;hyperlipidemia,405V genetype,442G genetype were the possible influence factors of ICH illness stage.5.Haplotype analysis showed that there was mild linkage disequilibrium between TaqIB and I405V and there was moderate linkage disequilibrium between I405V and D442G(D'>0.5).The frequency of haplotype ranked as B1-I-D(34.8%),B2-V-D(22.1%)?BI-V-D(21.5%),B2-I-D(19.6%),B1-I-G(2%)and there was statistical difference on the B1-V,I-D,V-D,B1-V-D haplotype between the case group and control group(P<0.05)?Conclusion:1.Hypertension,hyperlipidemia,smoking and 405V carrier may be the independent risk factors of ICH,and hyperlipidemia,and I405V were the influence factors of severity of ICH attack.D442G may affect the occurrence and development of ICH.2.There was different levels of linkage disequilibrium among TaqIB,I405V and D442G sites.Part Two Study on the influence biology mechanism of CETP polymorphism on ICHObjectives:To analyze the relationship of CETP SNPs and glucolipid metabolism indexes,and to discuss the possible mechanism that how CETP SNPs influence the ICH.Methods:1.The plasma CETP level of the cases group and control group were detected by ELISA and the corresponding CETP level of each genetype were compared.The TC,TG,LDL-C and HDL-C level of each genetype were compared as well.2.The correlation of genetype,CETP concentration and all blood lipid indexes were analyzed by correlation coefficient.3.Bioinformatics analysis of CETP SNPs were conducted,which concluded NCBI database,ProtParam basic physical and chemical properties,PSORT subcellular localization,KEGG Pathway,3D Ligandsite for general feature and Phyre,RCSB PDB database,Smart structure domains analysis for basic structure.The models of partial structure were built by Quark and the change before and after the mutation were analyzed.Results:1.There was no difference of the plasma CETP level between the case group and the control group(2.23±1.58ug/ml vs 2.30±1.30ug/ml,P>0.05).There was either no difference of CETP among different genetypes(P>0.05).2.The results of blood liquid indexes comparison showed that there was statistical difference among different gentype of I405V in the case group(P<0.05),and there was no difference of all blood liquid indexes among different genetypes of other SNPs(P>0.05).3.The result of correlation analysis showed that the TaqIB sites correlated to plasma CETP level(r=0.105,P=0.016<0.05);There was a positive correlation between the plasma CETP level and LDL-C(r=0.091,P=0.039<0.05);There was a correlation between the I405V genetype and TC and LDL-C,both the TC and LDL-C of V carrier were higher than that of I carrier;There was no correlation between the D442G and any lipid metabolism indexes.4.The result of Bioinformatics showed that:CETP was lipophilic and hydrophobic,and its structure was stable,and it located outside the cell and participated in cholesterol metabolism signaling pathway.CETP's major function was promoted the conversion of different density lipoprotein.CETP was mainly composed of two functional domains.There were two sites of the I405V and D442G located in the CETP's N-terminal domain and nearby the binding site of the CETP and the glycolipid metabolites.It had been changed of the local structure of I405V by the before and after the mutation,but the structure of D442G by the before and after the mutation did not have significantly changed.Conclusions:1.The polymorphism of TaqIB site of CETP gene might affect the transcription and expression of CETP,but the site of I405V and D442G were not effected the expression of CETP gene.2.The reasons of its association with intracerebral hemorrhage might be that the influence of the I405V polymorphism site to the level of the blood lipid--TC and LDL-C.3.The site of I405V may change the binding activity of CETP to the receptor through influencing the local structure of CETP,and it may through affecting the function of CETP in glycolipid metabolism to change the blood lipid level,therefore affecting the development of related diseases.Overall ConclusionsThe overall conclusions of this study are as follows:the I405V site of CETP genotype had a certain correlation with the occurrence and development of intracerebral hemorrhage,that it may the reason of leading the change of the binding activity of CETP and glycolipid metabolites is by the different genotypes corresponded to local structure difference,and it affected the level of TC and LDL-C of blood lipid,therefore affecting the occurrence and development of cerebral hemorrhage which is vascular related diseases.There is also a correlation between D442G and cerebral hemorrhage,and its mechanism may be that related to the linkage disequilibrium reaction between the sites of D422G and I405V.