目的:本研究改进基于脱氧核糖核酸(DNA)连接酶的中低通量基因分型方法,并用此方法进行核苷酸结合寡聚结构域(NOD)样受体基因NOD1和NOD2与冠心病的关联分析。方法:通过多重聚合酶链式反应(PCR)预扩增增加连接模板分子数量,提高特异性连接效率,优化DNA探针设计,摸索连接反应温度、时间、循环圈数及连接酶种类实现等位基因特异性连接,通过荧光掺入PCR和毛细管电泳实现多种等位基因特异性产物一次性检测。Sanger测序验证此方法准确性后,利用此方法对NOD1和NOD2基因上的单核苷酸多态性(SNP)位点在1 555例冠心病患者和1887例对照受试者中进行分型和关联分析。结果:通过优化反应条件和等位基因特异性探针设计原则,实现10 ng DNA样本一次性分型30个等位基因多态性位点。基于改进的DNA连接酶中低通量基因分型方法,NOD1、NOD2基因与冠心病的关联分析发现,NOD2基因上rs1861759和rs751271位点在非高血压条件下与冠心病相关(P均<0.05),经Bonferroni多重检验校正后,相关性仍然显著(P均<0.05)。结论:本研究优化了DNA连接酶链式反应技术在设计上的关键点,联合使用多重PCR和毛细管电泳,建立了一种高准确性、低成本、满足基于中低通量位点分型的临床分子诊断和科研需求的基因分型新方法,并用该方法发现NOD2基因上的位点rs1861759和rs751271在非高血压条件下与冠心病相关。
Advanced age is an independent risk factor for ageing-related complex diseases, such as coronary artery disease, stroke, and hypertension, which are common but life threatening and related to the ageing-associated vascular dysfunction. On the other hand, patients with progeria syndromes suffer from serious atherosclerosis, suggesting that the impaired vascular functions may be critical to organismal ageing, or vice versa. However, it remains largely unknown how vascular cells, particularly endothelial cell, become senescent and how the senescence impairs the vascular functions and contributes to the age-related vascular diseases over time. Here, we review the recent progress on the characteristics of vascular ageing and endothelial cell senescence in vitro and in vivo, evaluate how genetic and envi- ronmental factors as well as autophagy and stem cell influence endothelial cell senescence and how the senescence contributes to the age- related vascular phenotypes, such as atherosclerosis and increased vascular stiffness, and explore the possibility whether we can delay the age-related vascular diseases through the control of vascular ageing.
目的本研究将结合转录组学和全基因组关联分析,鉴定新的冠状动脉心脏病(冠心病)易感基因。方法利用冠心病全基因组关联研究(genome-wide association study,GWAS)数据,结合动脉粥样硬化相关转录组学分析,初步筛选得到候选基因和位点,然后进行多中心病例-对照遗传关联分析。通过等位基因特异性DNA连接酶链式反应,在两个独立人群中进行候选位点的分型,验证其与冠心病的相关性。结果初步筛选得到21个候选基因上的21个候选位点;经过包括2650例汉族冠心病病例和对照的两个独立人群验证后,ROR1基因的rs11208367位点显示与冠心病显著的相关性。Meta整合分析发现rs11208367位点的P值为2.00×10^-4,达到Bonferroni多重检验P值阈值(0.05/21=2.38×10^-3)。结论ROR1基因上的rs11208367位点与冠心病相关,ROR1基因可能是新的冠心病易感基因。
Alternative polyadenylation （APA）, a phenomenon that RNA molecules with different 3＇ ends originate from distinct polyadenylation sites of a single gene, is emerging as a mechanism widely used to regulate gene expression. In the present review, we first summarized various methods prevalently adopted in APA study, mainly focused on the next-generation sequencing （NGS）-based techniques specially designed for APA identification, the related bioinformatics methods, and the strategies for APA study in single ceils. Then we summarized the main findings and advances so far based on these methods, including the preferences of alternative polyA （pA） site, the biological processes involved, and the corresponding consequences. We especially categorized the APA changes discovered so far and discussed their potential functions under given conditions, along with the possible underlying molecular mechanisms. With more in-depth studies on extensive samples, more signatures and functions of APA will be revealed, and its diverse roles will gradually heave in sight.
