固醇调节元件结合蛋白-2激活相关通路基因与冠心病的关联研究

【作者】 刘晓丽；

【导师】 顾东风；

【作者基本信息】 中国协和医科大学 ， 流行病与卫生统计学， 2008， 博士

【摘要】 研究背景和目的冠状动脉粥样硬化性心脏病(冠心病,Coronary heart disease,CHD)是一种重大慢性非传染性疾病,受多种环境因素和遗传因素共同影响。已证实血脂代谢异常,尤其是高胆固醇血症,是心血管疾病最重要和可控制的危险因素之一,在CHD的一级及二级预防试验中均显示降脂治疗能明显降低心血管疾病的危险性。因此,血脂代谢相关的基因成为CHD遗传易感性研究重要的候选基因。固醇调节元件结合蛋白(Sterol regulatory element binding protein,SREBP)属于转录因子超家族,可激活一系列涉及胆固醇、不饱和脂肪酸、甘油三酯等生物合成及脂质摄取所需要的酶的转录。已经被确定的成员包括三个:SREBP1a,SREBP1c和SREBP2,其中SREBP2基因主要调节参与胆固醇代谢酶的转录。SREBP生成后以无活性的蛋白质前体形式固定于内质网膜上,需从内质网转移到高尔基体被水解激活释放出活性片段,进入细胞核调节转录。这个转运激活过程需要两种(三个)蛋白的参与调节:固醇调节元件结合蛋白裂解激活蛋白(SREBPcleavage-activating protein,SCAP)和胰岛素诱导基因1(Insulin induced gene 1,INSIG1)、胰岛素诱导基因2(Insulin induced gene 2,INSIG2)。本研究拟以SREBP2基因及其激活相关通路的上述三个基因为候选基因,在CHD病例-对照样本中,探讨这几个基因是否独立与CHD相关及是否存在交互作用。材料和方法随机入选1997年10月至2001年12月期间阜外心血管病医院收治的居住在北京地区的853例CHD患者,包括存活的急性心肌梗死患者和经冠状动脉造影证实的CHD患者。所有患者均发病3个月或3个月以上,且病情稳定,并经各项检查排除心脏瓣膜疾病、先天性心脏病、心力衰竭、严重的肾脏及肝脏疾病、继发性高血压、心肌病、家族性高胆固醇血症和严重肝肾及甲状腺疾病患者。对照样本来自亚洲国际心血管疾病协作研究(International Collaborative Study ofCardiovascular Disease in Asia,InterASIA)的北京样本。选择居住在北京,年龄(±2岁)、性别与病例匹配的社区人群948例作为对照组。对照组入选标准:心电图无缺血性改变;无胸痛、胸闷等心脏病症状;经Rose问卷、临床检查及CHD病史来排除CHD。所有参加者均为中国汉族人,无血缘关系。对所有入选者采用统一的调查表详细进行调查,包括人口统计学和一般情况、心血管疾病的个人史(包括现病史和既往史)及家族史、吸烟与饮酒史、服药情况。测量血压、身高和体重;计算体重指数(Body mass index,BMI),腰臀比值。检测血糖、血脂等各项生化指标。从HapMap网站下载这四个候选基因序列范围内(上下游各扩展2kb)的45个中国人的单核苷酸多态(Single nucleotide polymorphism,SNP)信息,选择常见SNP计算连锁不平衡状态并构建单体型,使用Haploview4.0软件包选择标签SNP。使用聚合酶链式反应(Polymerase chain reaction,PCR)和限制性片段长度多态(Restriction fragment length polymorphism,RFLP)方法对1801个研究对象进行基因型鉴定。t检验、方差分析及x~2检验用于单变量分析,非条件Logistic回归分析用于检验多态位点与表型的独立关联,以上分析过程由SPSS13.0来完成。应用Haplo.stats软件分析校正环境因素后单体型与表型的关联。采用多因子降维法(Multifactor dimensionality reduction,MDR)方法分析基因之间的交互作用。结果病例组平均年龄及男性比例高于对照组,病例组高血压患者、糖尿病患者及吸烟者比例也均高于对照组。此外,病例组收缩压(Systolicblood pressure,SBP)、低密度脂蛋白胆固醇(Low density lipoprotein cholesterol,LDL-C)、总胆固醇(Total cholesterol,TC)、甘油三酯(Triglyceride,TG)、体重指数(Body massindex,BMI)和血糖(Glucose,Glu)水平均高于对照组,高密度脂蛋白胆固醇(High density lipoprotein cholesterol,HDL-C)水平低于对照组。基于HapMap数据,分别选择了3个、4个、2个SNP作为INSIG1、INSIG2、SCAP基因的单体型标签SNP(Haplotype taging SNP,htSNP);同时选择SREBP2基因编码区的一个常见功能位点rs4822063为本研究关注的位点。结果发现:(1)与CHD的单点分析未发现与CHD相关的位点;(2)与血糖水平的单个位点分析显示,INSIG1基因SNP rs9769826位点与血糖水平显著相关,少见等位基因G携带者(GG+AG基因型)的血糖水平高于AA基因型携带者,分别为5.74±2.03mmol/L和5.45±1.37 mmol/L(P=0.015);(3)与血脂水平的单位点分析显示,SREBP2基因rs4822063位点与血LDL-C、TC水平相关。在对照组中CC基因型携带者的LDL-C、TC水平均较常见等位基因携带者(GG+GC基因型)水平高。LDL-C水平分别为3.44±0.90mmol/L和3.17±0.84mmol/L(P=0.031);TC水平分别为5.43±1.06mmol/L和5.13±0.97mmol/L(P=0.040)。(4)单体型与CHD的分析显示,INSIG1基因的单体型Hap3(TGA,位点按照rs10271719-rs9719268-rs9769826顺序,以下顺序同此)在病例组频率显著低于对照组(0.162 VS 0.203,校正的P=0.0102,simulation P=0.0107),而Hap4(TAA)在病例组频率高于对照组(0.129 VS 0.085,校正的P=0.00013,simulation P=0.00001)。与单体型Hapl(GGA)相比,INSIG1基因的单体型Hap4(TTA)与CHD危险增加相关,比值比(Odds ratio)为1.59(P=0.0006),双体型分析也发现了与CHD危险增加相关的双体型;Hap3(TGA)与降低的CHD患病危险相关(OR=0.74,P=0.006),同时携带Hap3的双体型Dip3和Dip5也显示与降低的CHD危险相关。(5)MDR和Logistic回归分析显示,2-SNP模型,包括INSIG1基因的rs10271719和rs9719268,显示最高的预测准确度(56.09%,P=0.002);另一个4-SNP模型,包括INSIG1-rs10271719、INSIG1-rs9719268、INSIG2-rs9308762、SCAP-rs4858868,也显示了交互作用。结论本研究显示SREBP2基因激活相关通路的INSIG1基因的单体型变异与CHD相关;INSIG1、INSIG2及SCAP基因间存在交互作用,与CHD发病危险相关;INSIG1基因的常见变异与血糖水平相关,而SREBP2基因的常见变异与血LDL-C及TC水平相关。希望我们的结果能得到其他独立样本的重复和进一步的功能验证。更多还原

【Abstract】 BackgroundCoronary heart disease (CHD) is a complex disease where both genetic and environmental factors interact to produce the phenotype. Dyslipidemia, especially hypercholesterolemia, is one of established major modifiable risk factors. It has been reported that lipid- lowering treatment could significantly decrease the risk of CHD in both primary and secondary prevention. Therefore, many genes involving in lipids metabolism have been considered to be important candidate genes to elucidate the genetic basis of CHD. Sterol regulatory element binding proteins (SREBPs), as a family of membrane-bound transcription factors, play important roles in feed back regulation of cellular cholesterol and fatty acid metabolism by activating related genes. The SREBPs include SREBP-1a, SREBP-1c, and SREBP-2, of which the SREBP2 preferentially activates cholesterol metabolism by binding sterol regulatory element (SRE) of the target genes involved in cholesterol synthesis such as 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, HMG CoA synthase and uptake such as low density-lipoprotein (LDL) receptor (LDLR). The SREBPs are synthesized as inactive precursors residing in the endoplasmic reticulum (ER) membrane and activation of SREBPs require cleavage by two proteases in the Golgi apparatus. The transportation of SREBPs from ER to Golgi needs another two kinds of proteins, namely SREBP cleavage-activating protein (Scap) and insulin induced gene (Insig) proteins including Insig1 and Insig2. In present study, we conducted a case-control study in Chinese Han population to investigate the relationship between the genetic variants of the SREBPs activating pathway, involving SREBP2, SCAP, INSIG1 and INSIG2 genes, and risk of CHD.MethodsA total of 1801 unrelated subjects were included in this study. We recruited 853 patients with CHD from patients hospitalized at the Fuwai Hospital and Cardiovascular Institute (Beijing, China) between October 1997 and December 2001. Eligible patients were those who survived an acute myocardial infarction or were documented by coronary angiography to have evidence of left main coronary artery≥50% and/or at least a 70% stenosis in any major coronary artery. Patients with congenital heart disease, cardiomyopathy, valvular disease, familial hypercholesterolemia and renal or hepatic disease were excluded. A total of 948 age-matched (±2 years) and gender-matched control subjects were randomly selected from individuals residing in Beijing and participating in the InterASIA (International Collaborative Study of Cardiovascular Disease in Asia). The controls were judged to be free of ischemic changes by ECG, without symptoms of chest pain and to be free of CHD by medical history, the Rose questionnaire and clinical examination. All subjects were Chinese Han nationality. Details of medical history were obtained from all participants by standardized questionnaire, together with information of drug intake, cigarette smoking, and alcohol consumption. Blood pressure, height, weight, waistline and were measured. Concentrations of serum lipids and glucose were determined by standard protocols.Haplotype-tagging SNPs (htSNPs) were chosen for SCAP, INSIG1 and INSIG2 with Haploview 4.0 software based on the genotyped SNPs in the Han Chinese of Beijing (CHB) of the HapMap project (the PhaseⅡdatabase, Apr 2007) within each gene and located in 2kb from 5’ flanking upstream and 3’ flanking downstream, respectively. One nonsynonymous-coding SNP rs4822063 in SREBP2 gene was selected. All SNPs were further genotyped in all subjects by using PCR-RFLP protocols. Association analyses were done separately for each of the SNPs and followed up by haplotype analyses. The Haplo.stats approach was used to test the association of statistically inferred haplotypes with CHD. The gene-gene interaction was examined using both the Multifactor Dimensionality Reduction (MDR) program (non-parametric) and Logistic regression model (parametric).ResultsCompared with the control group, the CHD group had more male and older patients and more individuals with hypertension, diabetes, alcohol consumption. Moreover, the patients had higher mean BMI and SBP, higher levels of serum triglycerides (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) and fasting glucose, and lower high density lipoprotein cholesterol (HDL-C) levels than the controls. Based on the genotyped SNPs in the Han Chinese of Beijing (CHB) of the HapMap project, we selected 3, 4, 2 htSNPs for the INSIG1, INSIG2 and SCAP genes. Our results: (1) Single polymorphism analyses on risk of CHD didn’t show any association with CHD. (2) Single polymorphism analyses on plasma glucose showed that the SNP rs9769826 of the INSIG1 gene was significantly associated with plasma glucose among controls. The minor allele G carriers had higher glucose level than individuals with major AA homozygous genotype (5.74±2.03mmol/L versus 5.45±1.37mmol/L, p=0.015). (3) Single polymorphism analyses on plasma lipid levels showed that the rs4822063 of SREBP2 was associated with LDL-C in the controls. The genotype CC carriers had higher LDL-C and TC than the major allele G carriers (GG+GC) (3.44±0.90mmol/L versus 3.17±0.84mmol/L, p=0.031 for LDL-C; 5.43±1.06mmol/L versus 5.13±0.97mmol/L,p=0.040 for TC). (4) Haplotype analysis showed that haplotype Hap3 (TGA) of the INSIG1 gene had lower frequency in CHD patients compared with controls (0.162 vs 0.203, simulation p=0.0107). However, the haplotype Hap4 (TTA) had higher prevalence in the CHD patients (0.129 vs 0.085, simulation p=0.0001). The haplotype Hap4 significantly associated with an increased risk of CHD (adjusted OR=1.59, 95%CI: 1.22-2.06, p=0.0006), while the haplotype Hap3 significantly associated with a decreased risk of CHD (adjusted OR=0.74, 95%CI: 0.60-0.92,p=0.006), compared with the reference haplotype Hap1 (GGA). (5) Multi-loci interaction analyses indicated that the 2-locus model involving the rs10271719 and rs9719268 of the INSIG1 gene showed the highest level of testing accuracy (56.09%, p=0.002 on 1000 permutations) as well as the 4-locus model showed significant interaction underlying the pathgenesis of CHD by MDR and Logistic regression model.ConclusionThe present study was the first time to illustrate the relationship between genetic variants from the SREBP2 activating-related pathway and CHD. Our results demonstrated a significant association between the INSIG1 gene variation and CHD. Our study also demonstrated a significant association between the SREBP2 gene variant and plasma lipid levels; and the INSIG1 gene variant was associated with plasma glucose. Moreover, our results provided evidence of interaction between the genes from SREBP2 activating-related pathway on risk of CHD for first time. Our findings warranted further study to replicate our results in other population and to elucidate the biological mechanism.更多还原