【作者】 刘宝琼；

【导师】 杨期东；

【作者基本信息】 中南大学 ， 神经病学， 2009， 博士

【摘要】 背景与目的:脑血管病是当今世界公认的三大主要疾病死因之一,脑出血(Cerebral hemorrhage,CH)占其中10～55%,其病死率、致残率高,严重危害人类健康。脑出血的病理机制复杂。目前认为,脑出血主要是由于多个易感基因的单核苷酸多态性(single nucleotidespolymorphisms,SNPs)微效作用的累加,并与环境因素共同作用而发病。对脑出血候选基因SNPs的研究将有助于阐明脑出血的遗传本质,为脑出血易感患者的早期检出和预防提供坚实的理论依据和实践指导。动脉粥样硬化是脑出血的危险因素。而极低密度脂蛋白(Very lowdensity lipoprotein,VLDL)及低密度脂蛋白(Low density lipoprotein,LDL)是目前公认的重要致动脉粥样硬化因子,内源性VLDL/LDL代谢途径失调节,可能导致VLDL/LDL水平升高及动脉粥样硬化,增加脑出血的风险。迄今,有关VLDL/LDL代谢途径相关基因与脑出血的关系国内外少见报道。本研究旨在探讨该途径载脂蛋白AⅠ-CⅢ-AⅣ(Apolipoprotein AⅠ-CⅢ-AⅣ,ApoAⅠ-CⅢ-AⅣ),载脂蛋白B(Apolipoprotein B,ApoB)、脂蛋白脂酶(Lipoprotein lipase,LPL)、低密度脂蛋白受体(Low density lipoprotein receptor,LDLR)、人类枯草溶菌素转化酶9(Proprotein convertase subtilisin/kexin type 9,PCSK9)基因与长沙地区汉族人群脑出血的关系。方法:研究对象均来自长沙地区汉族人群,包括:1.有家族聚集现象的脑出血家系组(cerebral hemorrhage with family history,CHFH):共38个家系,包括家系成员200例,分为以下亚组:①家系组脑出血患者组:61例,男34例女27例,平均年龄53.26±10.60岁。②Ⅰ级亲属组:95例,男50例,女45例,平均年龄51.15±14.45岁。③Ⅱ级亲属组:23例,男9例女14例,平均年龄36.82±18.68岁。④健康无血缘关系亲属:共21例,男12例女9例,平均年龄52.32±12.74岁,来自家系中与先证者无血缘关系的家庭成员,为先证者及其Ⅰ、Ⅱ级亲属的配偶。2.散发性脑出血(Sporadic cerebralhemorrhage,SCH)组:273例,男138例,女135例,平均年龄55.38±12.08岁。3.正常对照(control)组:140例,其中男74例,女66例,平均年龄53.62±13.28岁。血脂的检测:采用氧化酶法测定甘油三脂(Triglyceride,TG)、总胆固醇(Total cholesterol,TC);测定高密度脂蛋白(High densitylipoprotein,HDL)先用沉淀剂沉淀,再用氧化酶法测定上清液中的胆固醇;低密度脂蛋白(Low density lipoprotein,LDL)由Friedwald公式:LDL=(CHO-HDL)-TG/5计算。基因分型:利用多重单碱基延伸技术与DNA测序结合相互印证的方法检测所有研究对象VLDL/LDL代谢途径5个基因12个SNPs位点多态性,包括:ApoAⅠ-CⅢ-AⅣ基因rs12721026、rs2854116、rs45487004、rs670,ApoB基因rs1367117、rs11279109,LPL基因rs320,LDLR基因rs2569542、rs688、rs5925,PCSK9基因rs11206510、rs505151。统计:采用SPSS软件对散发性脑出血与正常对照组进行统计分析,两组间基因型与等位基因频率的差异用x~2检验,不同基因型亚组间血脂比较采用ANOVA方差分析。采用FBAT软件对有家族聚集现象脑出血家系的多态性位点及其构建的单体型进行传递不平衡检验(Transmission disequilibrium test,TDT)。结果:1.①长沙地区汉族人群存在ApoAⅠ-CⅢ-AⅣ基因rs12721026、rs2854116、rs45487004、rs670位点多态性。②散发性脑出血组ApoAⅠ-CⅢ-AⅣ基因rs12721026、rs2854116、rs45487004、rs670多态各基因型及等位基因频率分布与对照组比较差异无统计学意义(P>0.05)。③TDT分析未发现ApoAⅠ-CⅢ-AⅣ基因rs2854116、rs45487004、rs670多态性位点的等位基因存在传递不平衡现象(P>0.05)。④对rs12721026、rs2854116、rs45487004、rs670构成的单体型的TDT分析未发现单个特异单体型存在过度传递现象(P>0.05)。⑤散发性脑出血组及对照组rs12721026多态TT基因型亚组的HDL水平显著高于GT/GG基因型亚组(P<0.05),rs670多态AA基因型亚组的HDL水平显著高于GG、GA基因型亚组(P<0.05)。2.①长沙地区汉族人群apoB基因存在rs1367117、rs11279109位点多态性。②散发性脑出血组apoB基因rs1367117、rs11279109多态各基因型及等位基因频率分布与对照组比较差异无统计学意义(P>0.05)。⑧TDT分析未发现apoB基因rs1367117、rs11279109多态性位点的等位基因存在传递不平衡现象(P>0.05)。④对rs1367117、rs11279109构成的单体型的TDT分析未发现单个特异单体型存在过度传递现象(P>0.05)。⑤散发性脑出血组及对照组rs11279109多态DD/DI基因型亚组的TC水平显著高于Ⅱ基因型亚组(P<0.05),对照组rs1367117多态GA/AA基因型亚组的TC水平显著高于GG基因型亚组(P<0.05)。3.①长沙地区汉族人群存在LPL基因rs320位点多态性。②散发性脑出血组与对照组的LPL基因rs320位点基因型分布有统计学差异(x~2=6.95,p=0.031),散发性脑出血组T等位基因频率显著高于对照组(x~2=5.05,p=0.025)。③TDT结果显示LPL基因rs320T等位基因传递显著增多(Z＝2.144,P=0.034)。④散发性脑出血组rs320位点TT/GT基因型亚组的TG水平显著高于GG基因型亚组(P<0.05)。4.①长沙地区汉族人群LDLR基因存在rs2569542、rs688、rs5925位点多态性。②LDLR基因rs2569542、rs688、rs5925多态各基因型及等位基因频率分布与对照组比较差异无统计学意义(P>0.05)。③TDT分析未发现LDLR基因rs2569542、rs688、rs5925多态性位点的等位基因存在传递不平衡现象(P>0.05)。④对rs2569542、rs688、rs5925构成的单体型的TDT分析未发现单个特异单体型存在过度传递现象(P>0.05)。⑤散发性脑出血组及对照组LDLR基因rs2569542、rs688、rs5925位点不同基因型TG、TC、HDL及LDL水平无显著差异(P>0.05)。5.①长沙地区汉族人群存在PCSK9基因rs11206510、rs505151位点多态性。②散发性脑出血组PCSK9基因rs11206510位点CC/CT基因型及C等位基因频率显著高于对照组(x~2=4.19,p=0.041;x~2=4.28,p=0.039)。散发性脑出血组rs505151位点基因型及等位基因频率分布与对照组比较差异无统计学意义(P>0.05)。③散发性脑出血组及对照组rs505151多态GA/GG基因型亚组的LDL水平显著高于AA基因型亚组(P<0.05)。结论:1.LPL基因rs320多态T等位基因、PCSK9基因rs11206510多态C等位基因可能与长沙汉族人群脑出血发病有关。2.ApoAⅠ-CⅢ-AⅣ基因rs12721026、rs2854116、rs45487004、rs670,ApoB基因rs1367117、rs11279109,LDLR基因rs2569542、rs688、rs5925,PCSK9基因rs505151多态性可能与长沙汉族人群脑出血无关。3.ApoAⅠ-CⅢ-AⅣ基因rs12721026、rs2854116、rs45487004、rs670,ApoB基因rs1367117、rs11279109,LDLR基因rs2569542、rs688、rs5925位点构建的单体型可能与长沙汉族人群脑出血无关。4.ApoAⅠ-CⅢ-AⅣ基因rs670、rs12721026,ApoB基因rs11279109、rs1367117,LPL基因rs320,PCSK9基因rs505151多态性可能与长沙汉族人群的血脂代谢有关。更多还原

【Abstract】 Background and objective:Cerebrovascular disease is among the three leading causes of death worldwide,of which cerebral hemorrhage(CH) represents 10%to 55% with high morbidity and disability.CH is a heterogeneous disease caused by different pathogenic mechanisms.There are probably many alleles with small effect sizes interacting with environmental factors. Identification of gene polymorphisms that confer susceptibility to CH and the development of genetic risk diagnosis systems may contribute to the personalized prevention of these conditions.Atherosclerosis is an important risk factor of CH.It is acknowledged that very low density lipoprotein(VLDL) and Low density lipoprotein(LDL) are important risk factors for atherosclerosis. Imbalance disturbance of endogenous VLDL/LDL metabolic pathway may influence VLDL/LDL levels and the processe of atherosclerosis, which in turn may increase the risk of CH.The relationship between genes associated with endogenous VLDL/LDL metabolic pathway and CH has been rarely reported.The aim is to study the relationship between genes associated with endogenous VLDL/LDL metabolic pathway and CH in Han people of Changsha area,which include apolipoprotein AⅠ-CⅢ-AⅣ(ApoAⅠ-CⅢ-AⅣ),apolipoprotein B(ApoB),lipoprotein lipase(LPL),low density lipoprotein receptor(LDLR),and proprotein convertase subtilisin/kexin type 9(PCSK9) genes.Methods:All subjects came from Changsha Han population which were composed of 3 groups:(1) 200 members of 38 family with CH aggregation,including:①61 CH patients with family history:34 men and 27 women,averagely aged 53.26±10.60 years;②95 first-degree relatives:50 men and 45 women,averagely aged 51.15±14.45 years;③23 second-degree relatives:9 men and 14 women,averagely aged 36.82±18.68 years;④21 relatives with no kinship with proband:12 men and 9 women,averagely aged 52.32±12.74 years,who are spouses of the proband or the first-degree and second-degree relatives.(2)Sporadic cerebral hemorrhage(SCH) group:273 patients with sporadic cerebral hemorrhage,138 men and 135 women,averagely aged 55.38±12.08 years.(3) Control group:140 healthy controls,74 men and 66 women, averagely aged 53.62±13.28 years.Serum lipid levels were tested:Total cholesterol(TC),High density lipoprotein(HDL) and triglycerides(TG) were mesured by standard enzymatic methods.LDL cholesterol levels were calculated with the Friedewald formula(LDL=TC-HDL-TG/5).Multiplex SNaPshot genotyping analysis and DNA sequencing were used to detect 12 SNPs in 5 genes associated with endogenous VLDL/LDL metabolism of all subjects,including:rs12721026、rs2854116、rs45487004、rs670 of Apo AⅠ-CⅢ-AⅣgene,rs1367117、rs11279109 of ApoB gene,rs320 of LPL gene,rs2569542、rs688、rs5925 of LDLR gene and rs11206510、rs505151 of PCSK9 gene.The frequencies of the alleles and genotypes between the SCH group and control group were analyzed by x~2 analysis,and lipid levels were compared among genotypes by ANOVA using SPSS.The SNP and haplotype transmissions of the CH pedigrees were analyzed by transmission disequilibrium test(TDT) using FBAT computer program.Results:1.①There are apoAⅠ-CⅢ-AⅣgene rs12721026,rs2854116, rs45487004 and rs670 polymorphisms in Changsha Han population.②There is no significant difference of genotype and alleles frequencies of rs12721026,rs2854116,rs45487004 and rs670 between SCH group and control group(P＞0.05).③Application of TDT to genotype data from rs12721026,rs2854116,rs45487004 and rs670 polymorphisms revealed no evidence for transmission disequilibrium with CH(P＞0.05).④Haplotype analysis of rs12721026,rs2854116,rs45487004 and rs670 revealed no evidence for transmission disequilibrium with CH(P＞0.05).⑤Both in SCH group and in control group,the levels of HDL of the TT gene type of rs12721026 were significantly higer than that of the GT/GG gene type(p＜0.05),while the levels of HDL of the AA gene type of rs670 were significantly higer than those of the GG、GA gene type.2.①There are ApoB gene rs1367117 and rs11279109 polymorphisms in Changsha Han population.②There is no significant difference of genotype and alleles frequencies of rs1367117 and rs11279109 between SCH group and control group(P＞0.05).③Application of TDT to genotype data from rs1367117 and rs11279109 polymorphisms revealed no evidence for transmission disequilibrium with CH(P＞0.05).④Haplotype analysis of rs1367117 and rs11279109 revealed no evidence for transmission disequilibrium with CH(P＞0.05).⑤Both in SCH group and in control group,the levels of TC of the DD/DI gene type of rs11279109 were significantly higer than those of theⅡgene type,while the levels of TC of the GA/AA gene type of rs1367117 were significantly higer than that of the GG gene type(p＜0.05).3.①There are LPL gene rs320 polymorphism in Changsha Han population.②There is significant difference of genotype of rs320 between SCH group and control group(x~2=6.95,p=0.031).The frequencies of T allele were significantly higher in SCH group than these in control group(x~2=5.05,p=0.025).③TDT analysis revealed that the frequency of transmission(from parent to the offspring with CH) of alleles C of rs320 polymorphism in CH increased significantly(Z=2.144, p=0.034).④The levels of TG of the TT/GT gene type of rs320 in SCH group was significantly higer than that of the GG gene type(p＜0.05).4.①There are LDLR gene rs2569542,rs688 and rs5925 polymorphisms in Changsha Han population.②There is no significant difference of genotype and alleles frequencies of rs2569542,rs688 and rs5925 polymorphisms between SCH group and control group(P＞0.05).③Application of TDT to genotype data from rs2569542,rs688 and rs5925 polymorphisms revealed no evidence for transmission disequilibrium with CH(P＞0.05).④Haplotype analysis of rs2569542, rs688 and rs5925 revealed no evidence for transmission disequilibrium with CH(P＞0.05).⑤There was no significant difference of levels of TG,TC,HDL and LDL of different gene types of rs2569542、rs688、rs5925 in SCH group and control group(p＜0.05).5.①There are PCSK9 gene rs11206510 and rs505151 polymorphisms in Changsha Han population.②There is no significant difference of genotype and alleles frequencies of rs505151 between SCH group and control group(P＞0.05).The frequencies of CC/CT genotype and C allele of rs11206510 were significantly higher in SCH group than these in control group(x~2=4.19,p=0.041;x~2=4.28,p=0.039).③The levels of LDL of the GA/GG gene type of rs505151 were significantly higer than those of the AA gene type both in SCH group and in control group(p＜0.05).Conclusions:1.A allele in LPL gene rs11206510 polymorphism and C allele in PCSK9 gene rs11206510 polymorphism are associated with risks of CH in Changsha Han population. 2.There is no association of ApoAⅠ-CⅢ-AⅣgene rs12721026、rs2854116、rs45487004、rs670,apoB gene rs1367117、rs11279109, LDLR gene rs2569542、rs688、rs5925 and PCSK9 gene rs11206510 polymorphisms with CH in Changsha Han population.3.There is no association of haplotypes of ApoAⅠ-CⅢ-AⅣgene rs12721026、rs2854116、rs45487004、rs670,ApoB gene rs1367117、rs11279109,LDLR gene rs2569542、rs688、rs5925 polymorphisms with CH in Changsha Han population.4.ApoAⅠ-CⅢ-AⅣgene rs670、rs12721026,ApoB gene rs11279109、rs1367117,LPL gene rs320,PCSK9 gene rs505151 polymorphisms may be associated with lipid metabolism in Changsha Han population.更多还原