【作者】 曾焕玉（LYNN HTET HTET AUNG）；

【导师】 尹瑞兴；

【作者基本信息】 广西医科大学 ， 心血管内科学， 2014， 博士

【摘要】 研究背景：动脉粥样硬化性心血管疾病(ASCVD)是给家庭和社会造成极大负担的一种全球性疾病。控制血脂水平对减少心血管疾病的风险具有重要作用。尽管目前对于降低血脂的手段仍然以降低低密度脂蛋白胆固醇(LDL-C)水平为主,但是近年来发现同时降低甘油三酯(TG)和低密度载脂蛋白(LDL)水平比单纯降低LDL水平更为有益。因此,人们在不断探讨影响TG代谢的因素以及调控血清TG水平的措施。血清TG浓度是受环境因素、遗传因素和两者之间相互作用影响的一个复杂多基因特征(complex polygenic trait)。一些研究表明,大约40-70%的脂质表型是由遗传因素影响的。因此,遗传因素是解释血清脂质和脂蛋白的家族聚集性的主要因素。遗传因素包括与血脂代谢有关的常见和罕见多种基因变异。因此,了解血清TG水平与基因变异的关系可以提供新的脂质调节通路,从而找到新的冠心病(CAD)防治措施。随着全基因组关联研究(GWAS)的快速发展,有许多与血脂相关的基因单核苷酸多态性(SNPs)位点不断被发现。然而,在不同人群的重复性研究中发现这些位点与血脂谱之间的关系在不同种族和民族间、高脂血症和正常血脂人群间的结果却不甚一致。最近的3个GWAS显示在西方人群中BUD13同源体基因(BUD13homolog, BUD13)和锌指蛋白259基因(Zinc finger protein259, ZNF259)几个单核苷酸基因多态性位点和血脂水平有关联,尤其是对TG水平的影响。然而,据我们所知,在我国南方人群中BUD13和ZNF259基因多态性与血脂水平关系的研究尚未见有报道。第一部分摘要广西仫佬族和汉族人群BUD13和ZNF259基因多态性与血脂水平的关系目的：本研究旨在探讨广西仫佬族和汉族人群BUD13同源体(BUD13)基因(rs10790162237+1741T>C、rs17119975c.323-575A>G、和rs11556024c.*147C>T)和锌指蛋白259(ZNF259)基因(rs2075290c.1093-336G>A rs964184c.*365+359C>G)SNPs与几种环境因素对血脂水平的影响。方法：研究对象包括仫佬族825人和汉族781人。5个BUD13和ZNF259SNPs的基因分型采用聚合酶链式反应(PCR)-限制性片段长度多态性(RFLP)方法后,用琼脂糖凝胶电泳方法检测。基因分型结果用PCR产物直接测序方法进行验证。结果：仫佬族人群ZNF259rs2075290、rs964184和BUD13rs10790162SNPs的基因型和等位基因频率(均P<0.001),以及BUD13rs17119975SNP的基因型频率(P<0.05)与汉族人群比较,差异均有统计学意义。仫佬族人群ZNF259rs2075290、rs964184和10790162SNPs基因型之间的甘油三酯(TG)水平比较差异有统计学意义(P<0.01-0.001,P值经Bonferroni校正)。汉族人群基因型间的TG(BUD13rs10790162)、载脂蛋白(Apo) Al (BUD13rs11556024)水平和ApoAl/ApoB比值(BUD13rs10790162和ZNF259rs964184)比较差异也具有统计学意义(P<0.01-0.001；P值经Bonferroni校正)。连锁分析显示ZNF259rs2075290与ZNF259rs964184SNP有连锁,ZNF259rs2075290与BUD13rs10790162SNP有连锁,ZNF259rs964184与BUD13rs10790162SNP有连锁(均r2>0.50,P<0.001)。两民族人群的A-C-G-A-C(ZNF259rs207529、rs964184、BUD13rs10790162, rs17119975和rs11556024SNPs之间单倍体几乎占50%以上的全单倍体型频率。两民族之间A-C-G-A-C和G-G-A-A-C单倍体型频率的差异具有统计学意义(P<0.01)。多因素回归分析结果还显示,一些环境因素包括年龄、性别、体重指数、饮酒及吸烟等与仫佬族、汉族及合并人群的血清血脂水平有显著相关(P<0.05-0.001)。结论：广西仫佬族人群BUDl3和ZNF259SNPs与血脂水平的关系与当地的汉族人群比较有显著差异。提示BUDl3和ZNF259SNPs与血脂水平的关系可能具有种(民)族特异性。第二部分摘要广西仫佬族和汉族人群BUD13和ZNF259基因多态性与血脂水平的性别特异性关系目的：本研究的目的是探讨广西仫佬族和汉族人群BUD13同源体(BUD13)基因(rs10790162、rs17119975、rs11556024)和锌指蛋白259(ZNF259)基因(rs2075290、rs964184) SNPs和几种环境因素与血脂水平关系是否存在性别特差异。方法：研究对象包括仫佬族788(男346/女442)名和汉族778(男307/女471)名。5个BUD13和ZNF259SNPs的基因分型采用聚合酶链式反应(PCR)-限制性片段长度多态性(RFLP)方法后,用琼脂糖凝胶电泳方法检测。基因分型结果用PCR产物直接测序方法进行验证。结果：仫佬族人群男性的血清TG和ApoB水平高于女性,高密度脂蛋白-胆固醇(HDL-C)和ApoAl/ApoB(?)于女性(P<0.05-0.01)。汉族人群男性的总胆固醇(TC)、TG、低密度脂蛋白-胆固醇(LDL-C)和ApoB水平高于女性,ApoAl/ApoB比值低于女性(P<0.05-0.001)。在汉族人群中,男女之间的ZNF259rs2075290基因型频率和ZNF259rs964184等位基因频率比较差异有统计学意义(P<0.05-0.001)。仫佬族人群男女间5个SNPs的基因型和等位基因频率比较差异无统计学意义(P>0.05)。仫佬族男性基因型间的TG (BUD13rs10790162和rs17119975)和ApoB (BUD13rs17119975)水平比较差异有统计学意义(均P<0.01)。仫佬族女性基因型间的HDL-C、 ApoA1(BUD13rs10790162. rs11556024)和ApoA1/ApoB (ZNF259rs964184和BUD13rs10790162)水平比较差异有统计学意义(P<0.01-0.001)。汉族男性基因型间的HDL-C、ApoAl (BUD13rs10790162和rs11556024)和ApoAl/ApoB (ZNF259rs964184和BUD13rs10790162)水平比较差异有统计学意义(P<0.01-0.001)。汉族女性基因型间的TC (ZNF259rs964184)和TG (ZNF259rs2075290.rs964184和BUD13rs10790162)水平比较差异有统计学意义(P<0.01-0.001)。虽然仫佬族和汉族人群男女两性之间的血脂水平与不同的BUD13/ZNF259基因型有关,但基因型与血脂水平关系的趋势在男女显示一致。除了BUD13rs1719975SNP外,其余的4个SNPs均显示,突变基因型携带者的血脂水平高于野生基因型。结论：广西仫佬族和当地的汉族人群男女间BUD13和ZNF259SNPs与血脂水平的关系存在差异。提示BUD13和ZNF259SNPs与血脂水平的关系可能具有性别特异性。第三部分摘要BUD13和ZNF259基因多态性和基因间交互作用与血脂异常的关系目的：本研究的主要目的是：1)评价BUD13c.237+1741T> C(rs10790162)、c.323-575A> G (rs17119975)和c.*147C> T (rs11556024)及ZNF259c.1093-336G>A (rs2075290)和c.*365+359C>G (rs964184) SNPs与血脂异常的关系,2)评估这些SNPs以及它们的单倍体与患高胆固醇血症(HCH)/高甘油三酯血症(HTG)风险的关系,3)分析基因-基因相互作用对血脂异常风险的影响。方法：研究对象包括634例高脂血症患者(TC>5.17mmol/L and/or TG>1.70mmol/L)和547例非高脂血症者(TC<5.17mmol/L和TG<1.70)。5个BUD13和ZNF259SNPs的基因分型采用聚合酶链式反应(PCR)-限制性片段长度多态性(RFLP)方法后,用琼脂糖凝胶电泳方法检测。基因分型结果用PCR产物直接测序方法进行验证。采用GMDR (generalized multifactor dimensionalityreduction) software Beta0.9软件进行基因交互模型构建,评估每个模型的准确性。结果：HCH人群与非高胆固醇血症(non-HCH)人群间BUD13rs10790162SNP基因型频率和ZNF259rs964184等位基因频率比较差异有统计学意义(P<0.05-0.01)。HTG人群与非高胆固醇血症(non-HTG)人群间ZNF259rs2075290、ZNF259rs964184和BUD13rs10790162SNPs的基因型／等位基因频率和BUD13rs11556024等位基因频率比较差异有统计学意义(均P<0.001)。连锁分析显示,ZNF259rs2075290、ZNF259rs964184与BUD13rs10790162SNPs之间有连锁(r2>0.5,P<0.001)。高脂血症者人群基因型间的TC (ZNF259rs964184和BUD13rs10790162). TG (ZNF259rs2075290、ZNF259rs964184和BUD13rs10790162)和ApoB (BUD13rsl1556024)水平比较差异具有统计学意义(P<0.01-0.001)。非高脂血症者人群ZNF259rs964184和BUD13rs10790162SNPs基因型间的TG水平比较差异有统计学意义CP<0.01-0.001). BUD13rs10790162T等位基因携带者比非T等位基因携带者增加了2.23倍(OR:2.23;95%CI:1.05至4.75; P=0.015)的HCH风险。其余的4个SNPs与HCH/HTG发病的风险无明显关系(P>0.05)。单倍体G-G-A-A-C(携带ZNF259rs964184-G等位基因)分别增加患HCH(OR:1.35,95%CI:1.10至1.66, P=0.005)/HTG (OR:1.75,95%CI：1.39至2.21,P=0.000)的风险。而A-C-G-G-C (OR:0.77,95%CI:0.61至0.99,P=0.039)和A-C-A-G-T (OR:0.66,95%CI:0.47至0.94,P=0.021)单倍体(携带ZNF259rs964184-C等位基因)可减少患HCH的风险。交互作用分析显示,二个SNPs和三个SNPs交互模型为预测患HCH/HTG风险的最桂模型(P<0.01-0.001)。结论：我们发现在欧洲人群中与血脂显著相关的BUD13/ZNF259基因,几个SNPs在广西高脂血脂症和正常血脂人群中也显示与血脂水平相关。此外,我们发现BUD13和ZNF259两个基因之间的一些SNPs间存在相互作用的可能。然而,仍需要深入研究来阐明这些SNPs的生理功能及它们用如何机制影响血脂水平。更多还原

【Abstract】 Background:Atherosclerotic cardiovascular disease (ASCVD) has been an increasing disease burden worldwide. The modification of serum lipids plays an essential role in the reduction of CVD risk. Although lipid modification has been focused mainly on reducing the low-density lipoprotein cholesterol (LDL-C) level, in recent years, lowering both triglyceride (TG) and LDL-C levels has found to be more beneficial than lowering LDL-C alone. Hence, several researchattentions have been paid to regulate or control the serum TG levels. It is well established that the serum lipid levelsareco-modulated by the genetic and environmental factors. Numerous evidences have been shown that approximately40-70%of lipid trait is heritable. Thus, the familial resemblance in plasma lipids and lipoproteins is mainly determined by the genetic factors. Serum TG concentration is a complex polygenic trait that is determined by environmental and genetic factors including common and rare variants in various genes. Therefore, identification of the genes related to TG metabolism could provide a clue to search for novel pathway in lipid regulation, and thereby discoveringnoval therapeutic or preventive methods for CAD. With the rapid progress in genome-wide association studies (GWAS), a great number of TG-related loci have been reported. As the frequency of the susceptible等位基因s and their effect size on serum lipid levels may vary across world populations or between hyperlipidemic and normolipidemic populations, replication of GWAS signals across diverse ethnic groups and assessment of their association with the risk of hyperlipidemia have become fundamental in validation of these signals. Recently, three studies have found a genome-wide significant association between single nucleotide polymorphisms (SNPs) in the BUD13homolog (BUD13)/zinc finger protein259(ZNF259) genes and one or more serum lipid traits in the European populations. However, such association remains elusive in the Chinese populations.Part Ⅰ:Associations of Polymorphisms in the BUD13and ZNF259Genes and Serum Lipid Levels in the Mulao and Han PopulationsObjective:This study’s objective was to detect the association between5SNPs (BUD13rs10790162c.237+1741T> C, rs17119975c.323-575A> G, and rs11556024c.*147C> T and ZNF259rs2075290c.1093-336G> A,and rs964184c.*365+359C> G) in the BUD13and ZNF259genes and environmental factors with serum lipid levels in the Chinese Mulao and Han populations.Methods:This study comprised of825of Mulao and781of Han participants. Genotyping of5SNPs in the BUD13and ZNF259genes was performed with the polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) combined with gel electrophoresis, andthen confirmed by direct sequencing. Results:The genotype and allele frequencies of ZNF259rs2015290and rs964184and BUD13rs10790162were different between the Mulao and Han populations (P<0.001for all). After the Bonferroni P values correction, in the Mulao population,the levels of TG(ZNF259rs2075290and rs964184and BUD13rs10790162) among the genotypes were significantly different (P<0.01-0.001for all); whereas in the Han population, the levels of TG(BUD13rs10790162), ApoA1(BUD13rs11556024) and the ApoA1/ApoB ratio(BUD13rs10790162and ZNF259rs964184) among the genotypes were different (P<0.01-0.001for all). Significant linkage disequilibrium (LD) was noted betwee ZNF259rs2075290and rs964184, ZNF259rs2075290and BUD13rs10790162, and ZNF259rs964184and BUD13rs10790162(r2>0.50, P<0.001). The A-C-G-A-C (among the ZNF259rs2075290and rs964184, and BUD13rs10790162, rs17119975and rs11556024SNPs) haplotype accounted for over half of the%haplotype of each ethnic group. Significant difference in the frequencies of the A-C-G-A-C and G-G-A-A-C haplotypes between the two ethnic groups was detected (P<0.01). On multiple linear regression analyses, a significant correlation between the serum lipid parameters and numerous environmental factors including age, gender, height, weight, waist circumference, body mass index, smoking, alcohol consumption, blood pressure, and fasting blood glucose levels was noted in both ethnic groups (P<0.05-0.001).Conclusions:The BUD13and ZNF259SNPs were associated with different serum lipid parameters in the two ethnic groups, suggesting that the association of these variants with serum lipid levels might have ethnic specificity. Part II:Sex Specific Associations of Polymorphisms in the BUD13and ZNF259Genes and Serum Lipid Levels between the Mulao and Han populationsObjective:This aim of the current study was to detect the association of the BUD13and ZNF259SNPs and environmental factors with serum lipid levels between males and females in the Mulao and Han populations.Methods:The study population composed of788(346males/442females) of Mulao and778(307males/471females) of Han participants.Genotypes of5SNPs (BUD13rs10790162, rs17119975, and rs11556024and ZNF259rs2075290, and rs964184) were determined by polymerase chain reaction(PCR) and restriction fragment length polymorphism(RFLP) combined with gel electrophoresis, and then confirmed by direct sequencing.Results:In the Mulaoethnic group, male population had higher levels of serum TG and ApoB, but lower HDL-C and ApoAl/ApoB ratio than the female population (P<0.05-0.01). Inthe Hangroup, males had higher levels of TC, TG, LDL-C and ApoB, but lower ApoAl/ApoB ratio than the females (P<0.05-0.001). The genotype frequency of ZNF259rs2075290and allele frequency of ZNF259rs964184differed significantly between the gendersin the Han population (P<0.05-0.001for all) but not in the Mulao population (P>0.05). Serum levels of TG(BUD13rs10790162and rs17119975) and ApoB (BUD13rs17119975) in Mulao males, the levels of TC and TG (ZNF259rs2075290and rs964184, and BUD13rs10790162) in Mulao females were different between the genotypes (P<0.01-0.001). On the other hand, the levels of HDL-C and ApoA1(BUD13rs10790162and rs11556024), and ApoA1/B (ZNF259rs964184and BUD13rs10790162) in Han males, and the levels of TC (ZNF259rs964184) and TG (ZNF259rs2075290and rs964184, and BUD13rs10790162) in Han females were different between the genotypes (P<0.01-0.001). The trend of association is that compared to the major homozygous genotype, the minor homozygous genotype and heterozygous genotype was associated with the higher lipid levels. This trend was consistent for all the SNPs except BUD13rs17119975, which showed a reverse-trend in males but not in females.Conclusions:The BUD13/ZNF259SNPs are associated with the different serum lipid parameters between males and females in the Mulao and Han ethnic groups. These findings imply that the correlation between the BUD13/ZNF259SNPs and serum lipid levels might have sex-specificity. Part III:Association of the Variants in the BUD13and ZNF259Genes and their haplotypes with the Risk of DyslipidemiaObjectives:Our objectives were1) to detect the association between the BUD13/ZNF259SNPs and serum lipid levels in the hypercholesterolemia (HCH)/hypertriglyceridemia (HTG) populations,2) to evaluate the association betweenthe BUD13/ZNF259haplotypes and the risk of HCH and HTG, and3) to explore the possible interactions among the BUD13/ZNF259SNPs.Methods:Genotypes of5SNPs (BUDI3rs120790162, rs17119975, and rs11556024and ZNF259rs2075290, and rs964184) in634hyperlipidemic and547normolipidemic subjects were determined by polymerase chain reaction (PCR)and restriction fragment length polymorphism (RFLP) combined with gel electrophoresis, and then confirmed by direct sequencing. GMDR (generalized multifactor dimensionalityreduction) software Beta0.9was used for construction of gene-gene interaction model and the accuracy of possible association model was tested.Results:The genotype frequency of the BUD13rs10790162SNP and the allele frequencies of the ZNF259rs964184, BUD13rs10790162and BUD13rs17119975SNPs significantly differed between the HCH and non-HCH populations (P<0.05-0.01). On the other hand, the genotypic and allelic frequencies of the ZNF259rs2075290, ZNF259rs964184and BUD13rs10790162SNPs and the allelic frequency of the BUD13rs11556024SNP significantly differed between the HTG and non-HTG groups (P<0.001for each). Significant linkage disequilibrium (LD) was noted among the ZNF259rs2075290, ZNF259rs964184and BUD13rs10790162SNPs (r2>0.5, P<0.001).The ZNF259rs2075290, ZNF259rs964184and BUD13rs10790162SNPs were significantly associated with serum lipid levels in both HCH and non-HCH populations(P<0.008-0.001). On single locus analysis, only BUD13rs10790162was associated with HCH (odd ration, OR:2.23,95%CI:1.05to4.75,P=0.015). The remaining4SNPs did not reachstatistically significant association levelwith either HCH or HTG risk (P>0.05). The G-G-A-A-C haplotype, carrying rs964184-G-allele, was associated with increased in HCH risk (OR:1.35,95%CI:1.10,1.66, P=0.005) and HTG (OR:1.75,95%CI:1.39to2.21, P=0.000). The A-C-G-G-C and A-C-A-G-T haplotypes, carrying rs964184-C-allele, were associated with decreased in HCH risk (OR:0.77,95%CI:0.61,0.99, P=0.039and OR:0.66,95%CI:0.47to0.94, P=0.021, respectively). On multi-locus analyses, the two-to-three locus models showed a significant association with HCH and HTG (P<0.01-0.001).Conclusions:The BUD13/ZNF259SNPs, which were significant in the European populations, are also replicable in the Guangxihyperlipidemic and normolipidemic populations. Moreover, inter-locus interactions may exist among these SNPs. However, further functional analysis is required to clarify how these SNPs and genes actually affect the serum lipid levels.更多还原