ESTABLISHMENT OF GENETIC TESTING SYSTEMS FOR THE EVALUATION OF DNA POLYMORPHISMS RELEVANT TO THE RISK OF ATHEROSCLEROSIS AND THROMBOSIS

建立评估与动脉粥样硬化和血栓形成风险相关的 DNA 多态性的基因检测系统

• 批准号: 12672250
• 负责人: MURATA Mitsuru
• 金额: $ 2.11万
• 依托单位: Keio University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12672250/
• 关键词: ATHEROSCLEROSIS; THROMBOSIS; GENETICS; POLYMORPHISM; GENETIC TESTING; RISK FEACTORS

Coronary artery disease (CAD) and ischemic cerebrovascular disease (CVD) are typical human attributes that have a complex multifactorial etiology. This study aimed to establish systems to comprehensively evaluate the contribution of genetic factors to the pathogenesis of atherosclerosis and thrombosis in the Japanese populations, where allele frequencies are known to be quite different than Caucasians, using "candidate gene approach".Polymorphisms in platelet activating factor (PAF) receptor, PAF acetylhydrolase, and platelet GPIba receptor were investigated in relation to their functional consequences and association with CVD. PAF is a lipid mediator that has potent biologic effects on a variety of cells. For platelets, binding of PAF to its specific receptor (PAF-R) results in activation of phospholipases and protein kinases, and increase in intracellular Ca^<++> concentration. A common single nucleotide polymorphism was recently identified that results in an amino-acid substitution … More in the cytoplasmic region of PAF-R, Ala/Asp at residue 224 (^<224>A/D, Fukunaga K et al, 2001). Our analysis of 280 general Japanese populations showed the genotype frequencies of 75.7%, 21.8%, and 2.5% for A/A, A/D, and D/D, respectively. We measured PAF-induced platelet aggregation in healthy volunteers with different genotypes. Two healthy volunteers with PAF-R D/D genotype showed impaired aggregating response to a wide range of PAF concentrations. Platelets from one of the two subjects did not undergo irreversible aggregation even at higher concentration of PAF (6 micro M). The frequencies in the patient group were 78.9%, 17.2%, and 3.9% for A/A, A/D, and D/D genotypes, respectively, which were not statistically different from the control group. Genetic deficiency of plasma PAF-AH is relatively common in the Japanese but is not found in white populations. PAF-AH deficiency is attributed to a single point mutation, Val279Phe. It has been reported that subjects with the homozygous mutation (Phe/Phe) have near-absent plasma PAF-AH activity, whereas heterozygotes (Val/Phe) have about half of the PAF-AH activity as compared to the wild-type (Val/Val). The clinical relevance of Val279Phe is still controversial. We examined the relationships of this mutation with in vitro platelet function, and the frequencies of Val279Phe in CVD patients and control subjects. Platelet-rich plasma from healthy volunteers with heterozygous mutation (Val/Phe) showed apparently higher platelet aggregating response to threshold concentrations (〜0.01 micro M) of PAF as compared to Val/Val, suggesting the role of PAF-AH in the regulation of platelet activation. Genotype frequencies for CVD patients were 60.3, 35.9, and 3.8% for Val/Val, Val/Phe, and Phe/Phe, respectively, which were significantly different from the control group (68.8, 27.9, and 3.4%, respectively, p<0.05 when compared between Val/Val vs Val/Phe+Phe/Phe). When analysis was confined to those with age<60, the difference was more significant (55.7, 39.3, and 4.9% for CVD and 70.6, 26.4, and 3.1% for controls, p<0.01, OR=1.9, Val/Val vs Val/Phe+Phe/Phe). The genotype-effect became stronger when subjects without smoking were compared (p<0.01, OR=2.4). The present study suggests that heterozygous deficiency of PAF-AH with ^<279>Val/Phe which is common in Japanese may induce impaired regulation of platelet activation, and is associated with increased risk of CVD at younger age. We also analyzed a Connexin 37 gene polymorphism C1016T in CVD patients, and found that the frequency of the T allele was higher in the patient group, and that hypertension interacts with this association.Combinations of multiple genetic factors are believed to be crucial for the development of thrombotic disorders. The ultimate goal of the clinical appreciation of polymorphic markers is to identify subgroups of individuals who are best prevented from developing diseases, or who respond best to dietary, behavioral, or pharmacologic interventions. For this purpose, polymorphisms need to be investigated not only in relation to disease susceptibility, but also with regard to responsiveness to treatment, and gene-environment interactions. Less

冠状动脉疾病(CAD)和缺血性脑血管疾病(CVD)是典型的人类属性，具有复杂的多因素病因。本研究旨在利用“候选基因方法”建立系统，综合评估遗传因素在日本人群动脉粥样硬化和血栓形成发病机制中的作用。日本人群的等位基因频率与高加索人群截然不同。研究了血小板活化因子(PAF)受体、PAF乙酰水解酶和血小板GPIba受体基因多态性与功能后果的关系及其与心血管疾病的关系。PAF是一种脂质介质，对多种细胞具有强大的生物效应。对于血小板而言，PAF与其特异性受体(PAF-R)结合可激活磷脂酶和蛋白激酶，并增加细胞内钙离子浓度。最近发现了一种常见的单核苷酸多态，导致氨基酸替换…更多在PAF-R的细胞质区域，Ala/Asp在残基224(^&lt；224；A/D，Fukunaga K等人，2001年)。我们对280名日本普通人群的分析显示，A/A、A/D和D/D的基因频率分别为75.7%、21.8%和2.5%。我们检测了不同基因型的健康志愿者PAF诱导的血小板聚集。两名携带PAF-RD/D基因的健康志愿者对较大范围的PAF浓度表现出聚集反应受损。两个受试者之一的血小板即使在较高浓度的PAF(6微米)下也不发生不可逆聚集。患者组A/A、A/D、D/D基因频率分别为78.9%、17.2%、3.9%，与对照组比较差异无统计学意义。血浆PAF-AH基因缺陷在日本人中相对常见，但在白人人群中未发现。PAF-AH缺乏症是由Val279Phe单点突变引起的。据报道，纯合突变(Phe/Phe)的受试者几乎没有血浆PAF-AH活性，而杂合子(Val/Phe)的PAF-AH活性约为野生型(Val/Val)的一半。Val279Phe的临床意义仍存在争议。我们研究了该突变与体外血小板功能的关系，以及在CVD患者和对照组中Val279Phe的频率。与Val/Val相比，具有杂合突变的健康志愿者富含血小板血浆(Val/Phe)对PAF阈值浓度(~0.01微米)的血小板聚集反应明显增强，提示PAF-AH在调节血小板活化中起作用。CVD患者Val/Val、Val/Phe、Phe/Phe的基因频率分别为60.3%、35.9%和3.8%，与对照组(Val/Val与Val/Pal+Phe+Phe/Phe相比分别为68.8%、27.9%和3.4%)相比，差异有统计学意义(P<0.05)。当分析限于年龄和60岁的人时，差异更显著(CVD组55.7、39.3和4.9%，对照组70.6、26.4和3.1%，P&lt；0.01，OR=1.9，Val/Val vs Val/Phe+Phe/Phe)。当不吸烟的受试者被比较时，基因效应变得更强(P&lt；0.01，OR=2.4)。本研究提示，日本人中常见的PAF-AH基因杂合性缺失可导致血小板活化调节功能受损，并与年轻时心血管疾病的风险增加有关。我们还分析了CVD患者连接蛋白37基因C1016T的多态性，发现T等位基因在患者组中的频率较高，高血压与这种关联存在交互作用，多种遗传因素的组合被认为是血栓性疾病发生的关键因素。临床评价多态标记物的最终目标是确定哪些个体亚群的疾病预防效果最好，或者哪些个体对饮食、行为或药物干预的反应最好。为此，不仅需要研究多态与疾病易感性的关系，而且需要研究对治疗的反应以及基因与环境的相互作用。较少

项目成果

Rosenberg N, Murata M, Ikeda Y, Opare-Sem O et al.: "The Frequent 5,10-Methylenetetrahydrofolate Reductase C677T Polymorphism Is Associated with a Common Haplotype in Whites, Japanese, and Africans"Am J Hum Genet.. 70. 758-762 (2002)

Rosenberg N、Murata M、Ikeda Y、Opare-Sem O 等人：“常见的 5,10-亚甲基四氢叶酸还原酶 C677T 多态性与白人、日本人和非洲人的常见单倍型相关”Am J Hum Genet.. 70。

Kawano K, Yoshino H, Aoki N, Udagawa H, et al.: "Shear-induced platelet aggregatioon increase in patients with proximal and severe coronary artery disease"Clin Cardiol. 25. 154-160 (2002)

Kawano K、Yoshino H、Aoki N、Udakawa H 等人：“近端和严重冠状动脉疾病患者中剪切诱导的血小板聚集增加”Clin Cardiol。

Ishii K, Ogushi S, Murata M, Mitsuyoshi Y, et al.: "Activated factor XII levels are dependent on factor XII 46C/T genotypes and factor XII zymogen levels, and are associated with vascular risk factors in patients and healthy subjects"Blood Coagulation and

Ishii K、Ogushi S、Murata M、Mitsuyoshi Y 等人：“活化的 XII 因子水平取决于 XII 因子 46C/T 基因型和 XII 因子酶原水平，并且与患者和健康受试者的血管危险因素相关”血液

Kawano K, Yoshino H, Aoki N, Udagawa H, Watanuki A, Hioki Y, Hasumura Y, Yasumura T, Homori M, Murata M, Ikeda Y, Ishikawa K: "Shear-induced platelet aggregatioon increase in patients with proximal and severe coronary artery disease"Clin Cardiol. 25. 154-

Kawano K、Yoshino H、Aoki N、Udakawa H、Watanuki A、Hioki Y、Hasumura Y、Yasumura T、Homori M、Murata M、Ikeda Y、Ishikawa K：“近端和严重冠心病患者中剪切诱导的血小板聚集增加

Meguro S, Takei I, Murata M, Hirose H, Takei N, Mitsuyoshi Y, Ishii K, Oguchi S, Shinohara J, Takeshita E, Watanabe K, Saruta T: "Cholesteryl ester tranffer protein polymorphism is associated with macroangiopathy in Japanese type 2 diabetes mellitus"Ather

Meguro S、Takei I、Murata M、Hirose H、Takei N、Mitsuyoshi Y、Ishii K、Oguchi S、Shinohara J、Takeshita E、Watanabe K、Saruta T：“胆固醇酯转运蛋白多态性与日本 2 型大血管病相关