论文标题:MEF2A基因和ALOX5AP基因突变与冠状动脉粥样硬化性心脏病的相关性研究
论文作者
论文导师 蔡剑平,论文学位 博士,论文专业 临床检验与诊断学
论文单位 中国协和医科大学,点击次数 101,论文页数 139页File Size9493K
2006-06-01论文网 http://www.lw23.com/lunwen_494020882/
Coronary Atherosclerotic Heart Disease;; MEF2A;; ALOX5AP;; susceptibility;; single nucleotide polymorphism
背景：冠状动脉粥样硬化性心脏病是由遗传因素和环境因素相互作用而引起的多基因疾病。冠状动脉粥样硬化性心脏病的分子机制十分复杂，目前尚未完全阐明。经过全球科学家对该病分子机制的长期研究，已经历了脂代谢异常、平滑肌细胞增生、慢性炎症反应、到最新的血管内皮发育或功能障碍等研究阶段。MEF2A和ALOX5AP基因结构变异与冠状动脉粥样硬化性心脏病的易感性是近年来群体遗传学方面的研究热点，两个基因参与冠状动脉粥样硬化性心脏病的致病机制既有独立性又有相关性。本研究，我们选择了MEF2A和ALOX5AP这两个分别与血管内皮发育和慢性炎症反应机制有关的基因，进行中国汉族人群基因结构变异分析及变异蛋白功能的研究，并探讨其与冠状动脉粥样硬化性心脏病的相关性。 方法：本研究以冠状动脉造影为确诊标准，选择冠状动脉粥样硬化性心脏病阳性和阴性患者作为病例—对照研究对象，用PCR-SSCP和PCR—胶回收—测序方法进行基因型分析。用基因计数法计算各组等位基因频率和基因型频率，X~2检验是否符合Hardy-Weinberg平衡。通过重组蛋白荧光信号检测，研究△7aa缺失突变对MEF2A蛋白细胞内定位的影响；通过荧光素酶活性检测，研究△7aa缺失突变对MEF2A蛋白转录激活作用的影响。 结果：1)在899例受试者的MEF2A第7号外显子中未发现任何类型的突变。2)在1000例受试者的MEF2A基因共筛查到9个结构变异位点，其中无氨基酸改变者有3处：N297N(891C＼T)、P435P(1305G＼A)、G451G(1353G＼T)；有氨基酸改变者有6处：△7aa缺失(434—446aa)、Q(CAG)重复(420—430aa)、P431／432缺失(CCG缺失)、P433／434缺失(CCA缺失)、P435S(C1303T)、PQQ插入(1291CCGCAGCAG插入)。除N297N位于第9号外显子区，其余均位于第11号外显子区。其中P433／434缺失、P435S、PQQ431插入三个变异位点属国际上首次发现，它们的最低等位基因频率均低于1％，属于突变位点。对Q(CAG)重复(420—430aa)位点进一步统计分析显示，4-8个CAG三联碱基重复的等位基因频率在阳性组高于对照组，二者之间差异有显著性(χ~2=4.338，P=0.037)，其优势比(OR值)为1.664，95％可信区间为1.026-2.699。3)对△7aa缺失突变进一步结构分析发现，其在碱基水平有15种缺失模式，在氨基酸水平有7种缺失模式。对其功能研究显示，突变体—GFP融合蛋白绝大部分定位于Hela／293T细胞的细胞核中，仅个别细胞中突变蛋白在细胞质与细胞核中均有表达。突变体与野生型MEF2A蛋白荧光素酶活性无显著差异(p＞0.05)。exon3与exon4型MEF2A野生型、缺失突变型蛋白均定位在细胞核，它们的转录激活作用差异不显著(p＞0.05)。4)ALOX5AP基因编码区未发现遗传变异。 结论：1)MEF2A第7号外显子区突变可能不是我国汉族人群冠状动脉粥样硬化性心脏病的遗传危险因素。与国外研究结果的差异可能是地域和种族差异造成的。2)MEF2A基因第11号外显子4-8个CAG的等位基因可能是我国汉族人群冠状动脉粥样硬化性心脏病的易感因素。3)Δ7aa缺失突变有多种缺失模式，其突变区的形成可能是物种进化过程的产物。△7aa缺失区氨基酸对MEF2A蛋白的定位功能和转录激活作用可能不是重要区域。4)ALOX5AP编码区结构变异与我国汉族人群冠状动脉粥样硬化性心脏病的相关性较小。ALOX5AP基因中冠心病易感位点可能不在其蛋白编码区。
BACKGROUD Coronary artery disease has a complex aetiology, involving multiple genetic and environmental influences and interactions. Our views of the pathophysiology of CAD and MI have evolved over time. More researches focus on identification of the role of inflammation and immune system or dysfunction or abnormal development of the endothelium on the cause of CAD The mutations of myocyte enhancer factor (MEF2A) and arachidonate 5-lipoxygenase-activating protein (ALOXSAP) gene have been more implicated in the susceptibility to coronary atherosclerotic heart disease (CAD). They may contribute to CAD by the common and/or different mechanisms respectively. 5—lipoxygenase activating protein (FLAP) are essential for the cellular synthesis of leukotrienes. FLAP that appears to serve as an arachidonic acid binding and transfer protein thereby facilitating 5-LO enzyme activity. And many studies suggest links between the 5-LO pathway and atherogenesis. Mutation of ALOX5AP gene may affect the synthsis of LTs whose biological effects associated with the pathophysiology of inflammatory disorders. In this study, we sought to identify variations in the all the exons of MEF2A and ALOX5AP gene and investigate their association with CAD in Chinese Han population. In addition, we also carried out the functional analysis of 7aa-deletion mutation to identify their probable mechanism which is involved in CAD. METHODS Case-control design was applied in this study. We recruited independent patients with unequivocal diagnosis of CAD based on cardiac angiography (CAG):Single-strand conformation polymorphism (SSCP) and DNA sequence analyses were used to identidy mutations in MEF2A and ALOX5AP. The nuclear localization signal of MEF2A mutation was identified by expression of green fluorescent protein (GFP)/MEF2 fusion proteins in the transfected 293T/Hela cells. And, the effect of mutation of MEF2A on transcriptional activation activity was analysed using the dual luciferase reporter gene assay. RESULTS 1) No mutation was detected in exon 7 of MEF2A gene in all the 899 samples. 2) We found 9 variations in all exons that one variation in exon 9 and the others in exon 11 of MEF2A gene in the 1000 samples. There are 3 SNPs (N297N,P435P, G451G ) which didn"t alter the amino acid sequence of the MEF2A protein, and 6 variations which altered the amino acid sequence of the MEF2A protein in exon 11, which included polyglutamine tandem repeats varied from 4 and 15, the proline tandem repeats (P431/432 deletion or P433/434 deletion),△7aa-del (21-bp deletion),△3aa(PQQ 430) insertion and one missense mutation (P435S). Statistical analysis showed that the shorter CAG repeat lengths (4～8 repeats), carried by approximately 7.64% of alleles in CAD patients in Chinese, has a significant association with CAD [p=0.037, OR=1.664 (95%CI, 1.026-2.699)]. 3) Further genetic analysis of△7aa-deletion mutation showed that there may be several possible models for the 7-aa deletion. The wild MEF2A/GFP proteins were strictly expressed in nuclear, and, the considerable proportion of△7aa-deletion/GFP fluorescence were located in the nucleus. Western blot analysis showed that both wild type and△7aa-deletion mutant proteins were successfully expressed in transfected cells. And both types of MEF2A proteins activated transcription of the HRC promoter, as well as, the ANF promoter. Moreover,△7aa-deletion didn"t reduce the transcriptional activation activity of MEF2A protein. And two alternatively MEF2A proteins express same transcriptional activation ability. 4) We didn"t identify any mutations in ALOX5AP coding regions. CONCLUSION 1) Mution in exon 7 of MEF2A gene may not be genetic risk factor leading to CAD in the Chinese Han population. The discrepancy between the foreign and our research may be due to area- and race-specific. 2) The alleles with CAG repeat numbers from 4 to 8 may be regarded as a susceptible molecular marker of risk for CAD. 3) A C-terminal region encompassing aa 434-446 is not necessary for fully localizing GFP/MEF2A in the nucleus of 293T or Hela cell. This area formation maybe due to evolution. 4) The genetic variations in ALOX5AP coding region may not contribute to CAD in the Chinese Han population.
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