【作者】 毕昊；

【导师】 屈伸；

【作者基本信息】 华中科技大学 ， 生物化学与分子生物学， 2010， 博士

【摘要】 心房颤动(atrial fibrillation, AF)简称房颤,是一种最为常见的心律失常。中国目前约有一千万房颤患者,房颤已成为严重影响我国公众健康的疾病。对其发病机制的深入研究,一直是心律失常研究领域的热点。关于该病的发生机制,先后提出了“局灶驱动”、“多环折返”和“自旋波理论”等学说。近来还发现一些非离子通道基因突变与房颤的发生紧密关联,这不仅丰富了对房颤发生机制的认识,也为将来实现个体化治疗提供了理论基础。过去的研究主要依赖于房颤家系的基因连锁分析。但这些病例只占到所有房颤患者的很少一部分,更多的是普通房颤,为多基因疾病,其发病机制可能是由多个基因或者基因-环境相互作用引起。以往,传统的多基因疾病的研究模式,是以假说为导向,即首先假设某基因为致病候选基因,再对其进行深入研究,无异于大海捞针,即使成功也存在很多偶然性。随着人类基因组计划的完成和后续工作的展开,生命科学研究已经进入了快速、准确、低耗地分析遗传和表达的信息时代,新一代测序技术和生物信息分析平台的诞生使多基因疾病的研究手段逐渐加强。目前方兴未艾的全基因组关联分析(Genome-Wide Association Studies,GWAS)正是基于人类基因组计划(Human Genome Project, HGP)和人类基因组单体型图计划(The International HapMap Project, HapMap)的研究成果发展起来的一种研究手段。该技术能快速、简便地在覆盖全基因组的范围内提供与疾病表现型(Phenotype)相关联的遗传差异(Genotype),为易感基因的寻找提供了准确的定位信息。2007年,冰岛人大规模的房颤“病例-对照”全基因组关联分析发现位于4号染色体长臂2区5带(4q25)的单核苷酸多态性(single nucleotide polymorphism, SNP)rs2200733与心房颤动高度关联,随后在多个种族人群包括我们所报道的中国汉族人群中,证实与房颤紧密关联。然而在rs2200733所处的连锁不平衡区域内并无已知功能基因的存在。因此,正如大部分报道所认为,位于rs2200733上游约150 kb处PITX2基因可能是潜在的易感基因,因为已知PITX2是指导机体多个器官包括心脏发育的一种重要信号分子。但到目前为止,无论是从基因水平或是基因表达水平,仍然缺乏直接证据可证实PITX2参与影响房颤发生。"PITX2基因是否参与房颤的发生?”这留给我们一个亟待证实的问题。在本研究中,我们选择了PITX2基因上3个SNPsrs976568、rs994978和rs2595104,运用“病例-对照”方法,在中国汉族人群中进行了与房颤之间的关联分析,首次从遗传学角度上考证PITX2是否为中国汉族人群房颤的易感基因。目的：探讨PITX2基因上的3个SNPs:rs976568、rs994978和rs2595104与中国汉族人群心房颤动的相关性。方法：从中国汉族人群基因ID库中随机挑选371例房颤患者基因组DNA和620例非房颤患者对照基因组DNA,利用高分辨率溶解曲线(high resolution melting, HRM)基因分型方法,同时对rs976568,rs994978 and rs2595104进行基因分型,利用PLINKv1.05软件进行哈迪-温伯格(Hardy-Weinberg, HW)平衡检验。3个SNPs的基因频率及基因型频率与房颤的关联用Pearson 2x2以及2x3列联表分析,显著程度用卡方检验计算(SPSS, version 13.0)。相对危险度(odd ratio, OR)以及95%置信区间(confidence intervals, CIs)用卡方检验计算(SPSS, version 13.0)。回归性p值用Logistic多因素回归分析(SPSS, version 13.0),以性别、年龄、2型糖尿病、高血压等指标作为协变量。当“病例-对照”群体进行分层后,p值将会使用Bonferroni’s correction来进行矫正。利用PLINK v 1.05软件进行100,000次Monte Carlo模拟以衡量经验性p值。利用Haploview v4.2构建单倍体型和连锁不平衡(linkage disequilibrium, LD)模型。单倍体型频率与房颤的关联用Pearson 2×2列联表分析,显著程度用卡方检验计算(SPSS, version 13.0)。OR以及95%置信区间用卡方检验计算(SPSS, version 13.0).统计效力的分析使用nQuery Advisor 6.0软件。结果：统计效力分析显示该研究的样本数能提供足够的统计效率。3个SNPs的对照组基因型均符合HW平衡。其中,rs2595104与房颤的关联具有极显著统计学意义,观察P值为4.88×10-4,风险等位基因为T等位基因,OR值是1.39,OR的95%置信区间为1.15到1.66。rs976568与房颤的关联接近统计学显著意义,观察P值为0.054。rs994978与房颤不具有统计学显著意义,观察P值分别为0.078。3个SNPs的基因型均显示与房颤的关联。在隐性遗传模式下,rs976568、rs994978及rs2595104的基因型均获得极显著关联,观察P值分别为0.003、0.004及0.002。通过构建rs976568-rs994978-rs2595104单倍体,发现可观察到5种单倍体形式,G-T-T为50.0%,T-C-G为41.1%,G-C-G为3.5%,G-T-G为2.8%,T-C-T为1.2%。其中只有G-T-T和G-T-G单倍型与房颤的关联显著,具有统计学意义。观察P值分别为0.005和0.003。结论：PITX2:基因为房颤的易感基因目的建立蛋白质错误折叠循环扩增(protein misfolding.cyclic amplification, PMCA)技术,体外筛选能抑制细胞型朊蛋白(cellular prion protein, PrPc)向感染性朊蛋白(scrapie prion protein, PrPsc)转化的药物。方法将感染羊瘙痒因子H22#发病仓鼠脑组织匀浆与正常仓鼠脑组织匀浆混合,经反复超声-孵育后,Western Blot检测扩增产物中PrPsc的含量,优化最佳扩增条件,用于体外抑制药物筛选；利用针对人PrPc不同结构域的抗体研究抑制药物对PrPc空间构象的影响。结果氮芥(Mechlorethamine, MCT)联合二硫苏糖醇(Dithiothreitol, DTT)能体外抑制PrPc向PrPsc的转化,抑制作用具有明显量效关系；MCT联用DTT可使抗体6H4的抗原表位位点隐蔽。结论PMCA技术可成功用于TSE治疗药物的体外筛选；MCT联用DTT能体外抑制PrPc向PrPsc的转化,对其机制的深入探讨,有助于新一类TSE治疗药物的研发。更多还原

【Abstract】 Atrial fibrillation (AF) is the most common arrhythmia. In China’s mainland, approximately 10 million people are affected with AF, threatening the public health. The researches on the pathogenesis of AF benifit the correct and effective prevention and treatment of it, being the focus in the rearch fields of arrhythmia. Up to date, three crucial arrhythmia mechanisms have been held to be involved in AF:1) focal ectopic activity,2) multiple-circuit reentry and 3) single-circuit reentry with fibrillatory conduction. But the researches on AF are still gonging on, especially the finding of some un-ionic channels gene with gene mutation related with AF indicated that much unknown factors beside ionic channels participate in the development of AF. These researches will not only rich the knowledge of the pathogenesis of AF, but also afford the theoretic instruction for the individal treatment in the future. It has been certain that heredity is the important correlated factor of AF. In the past years, great achievement has been obtained in the fundamental Genetics researches in AF, most of which denpened on the researches in the family AF, but account for only a small proportion of all AF cases. Most of AF are common AF which are multigenic disease, caused by gene-gene interaction or gene-enviroment interaction. Thus, identification of common AF susceptive genes becomes the most favoured direct methods to understand the mechanisms.It is interested that although the association of single nucleotide polymorphism (SNP) rs2200733 on 4q25 with AF has been replicated in many study cohorts, including our previously reported in Chinese GeneID population, there’s no gene in the linkage disequilibrium block containing this SNP. In the AF GWA reports, PITX2 was taken as a candidate gene in this 4q25 locus because it is the nearest gene to rs2200733 and lies 150 kb upstream. But so far, there’s no evidence to support the direct association between PITX and atrial fibrillation, whatever through genomic association or expression level association. This leaves us a question on the role of PITX2 gene into the development of AF.In this study, we selected three high minor allele frequency SNPs in PITX2 gene locus and test the association between AF and PITX2 in the GeneID Chinese Han population by the"case-controll" association study, anticipating in providing direct evidence in the relation of PITX2 with AF in heredity level.Aim:To study the association between single nucleotide polymorphism PITX2 gene polymorphism including rs976568, rs994978 and rs2595104 with Atrial Fibrillation in the Chinese Han Population.Method:Genomic DNA of 371 AF patients and 620 non-AF controls were collected randomly from the GeneID Chinese mainland Han population.3 SNPs in PITX2 including rs976568, rs994978 and rs2595104 were genotyped by High Resolution Melt (HRM) methods. The 3 SNPs were tested for Hardy Weinberg equilibrium among controls using PLINK v1.05. Haplotypes construction and frequencies were estimated by softwawre Haploview v4.2. Allelic and genotypic associations of SNPs with AF were assessed using Pearson’s 2×2 and 2×3 contingency tableχ2 test (PLINK v1.05). Odds ratios (ORs) and 95% confidence intervals (CIs) were estimated by theχ2 test (PLINK vl.05). Multivariate analysis was performed by incorporating age, sex, hypertension (HT), and CAD as covariates by using multivariate logistic regression (PLINK v1.05). Empirical P values were determined using the PLINK v1.05 program with 100,000 Monte-Carlo simulations. linkage disequilibrium (LD) pattern constructions were done by Haploview v4.2.Result:Power analysis suggested that our sample size provides sufficient power to identify the association between these three SNPs and AF in this study.There was no deviation from Hardy-Weinberg Equilibrium of the 3 SNPs genotype distributions in the control group. rs2595104 T allele gained most significant association with AF, observed P value was 4.88×10-4 and odds ratio was 1.39 (95% confidence interval 1.15 to 1.66). rs976568 and rs994978 were in nearly significant with AF,χ2 test P values were 0.054 and 0.078 respectively. In lone AF patients, only rs2595104 reached statistical significant association with the P value of 0.011 and OR was 1.48 (95% CI 1.09-1.99). rs976568 and rs994978 were not statistical significant any more (P value were 0.158 and 0.082 respectively).For genotypic association, all 3 SNPs showed significant association with AF in Chinese GeneID population. The most significant P values were acquired under recessive model for these 3 variants at the same time,0.003 for rs976568,0.004 for rs994978 and 0.002 for rs2595104.There were in total five detectable haplotypes, accounted for 50.0%(in rs976568-rs994978-rs2595104 order G-T-T),41.1%(T-C-G),3.5%(G-C-G),2.8%(G-T-G) and 1.2%(T-C-T) of all haplotype alleles. Among these, only G-T-T and G-T-G haplotypes were significantly associated with AF. T-C-G, G-C-G and T-C-T were negative significant, with P values of 0.063,0.221 and 0.791 respectively.Conclusion:PITX2 is the susceptive gene of Atrial Fibrillation Aim To establish protein misfolding cyclic amplification (PMCA) technology and screen the drug which can inhibit the conversion of PrPc to PrPsc in vitro. Method The brain homogenate of hamster infected with sheep scrapie factor H22# was incubated with normal hamster brain homogenate. After different cycles consisted by sonication followed by incubation, amplified PK-resistant PrPsc was detected by western blotting, to determine the optimal cycle times. Optimal PMCA system was used for drug screen in vitro. By antibodies binding with different domains of PrPc, the effect of Mechlorethamine (MCT) with Dithiothreitol (DTT) on the conformation of PrPc was investigated. Result Established PMCA system has high specificity. Optimal amplification time was 42h, ensuring the best amplification efficiency. By drug screen system, MCT with DTT was found to inhibit the conversion of PrPc to PrPsc in vitro and showed significant dose-effect relationship. Epitope of antibody 6H4 was concealed after treatment by MCT with DTT, which might play a role in the mechanism of inhibition. Conclusion: PMCA technology was successfully established and can be used for the drug screening for TSE. MCT with DTT can inhibit the conversion of PrPc to PrPsc in vitro. Further research on the mechanism will benefit the development for new drug for TSE.更多还原