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

冠状动脉疾病（CAD）和缺血性脑血管疾病（CVD）是具有复杂多因素病因的典型人类属性。本研究的目的是建立一个系统来综合评估遗传因素对日本人群动脉粥样硬化和血栓形成发病机制的作用，其中等位基因频率已知与高加索人有很大差异，使用“候选基因方法”。血小板活化因子（PAF）受体、PAF乙酰水解酶、和血小板GPIba受体的功能后果和与CVD的关联进行了研究。PAF是一种脂质介质，对多种细胞具有有效的生物学效应。对于血小板，PAF与其特异性受体（PAF-R）的结合导致磷脂酶和蛋白激酶的活化，以及细胞内Ca^++浓度的增加。最近发现一种常见的单核苷酸多态性导致氨基酸替换 关于我们 在PAF-R的胞质区域中，在残基224处为Ala/Asp（^<224>A/D，Fukunaga K等，2001）。我们对280个日本普通人群的分析显示，A/A、A/D和D/D的基因型频率分别为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。两名健康志愿者与PAF-R D/D基因型显示受损的聚集反应的PAF浓度范围广泛。即使在较高浓度的PAF（6 μ M）下，两名受试者之一的血小板也没有发生不可逆的聚集。A/A、A/D和D/D基因型在患者组中的频率分别为78.9%、17.2%和3.9%，与对照组相比无统计学差异。血浆PAF-AH的遗传缺陷在日本人中相对常见，但在白色人群中未发现。PAF-AH缺陷是由于一个单一的点突变，Val 279 Phe。据报道，具有纯合突变（Phe/Phe）的受试者具有几乎不存在的血浆PAF-AH活性，而杂合子（瓦尔/Phe）具有与野生型（瓦尔/瓦尔）相比约一半的PAF-AH活性。Val 279 Phe的临床意义仍有争议。我们研究了这种突变与体外血小板功能的关系，以及CVD患者和对照组中Val 279 Phe的频率。来自具有杂合突变（瓦尔/Phe）的健康志愿者的富血小板血浆显示出对PAF阈值浓度（约0.01 μ M）的血小板聚集反应明显高于瓦尔/瓦尔，这表明PAF-AH在血小板活化的调节中的作用。CVD患者的瓦尔/瓦尔、瓦尔/Phe和Phe/Phe的基因型频率分别为60.3%、35.9%和3.8%，与对照组相比有显著差异（分别为68.8%、27.9%和3.4%，当在瓦尔/瓦尔与瓦尔/Phe+Phe/Phe之间比较时，p&lt;0.05）。当分析仅限于年龄&lt;60岁的患者时，差异更显著（CVD组为55.7、39.3和4.9%，对照组为70.6、26.4和3.1%，p&lt;0.01，OR=1.9，瓦尔/瓦尔vs瓦尔/Phe+Phe/Phe）。与不吸烟者相比，基因型效应更强（p&lt;0.01，OR=2.4）。目前的研究表明，日本人常见的PAF-AH与^瓦尔/Phe的杂合缺陷<279>可能导致血小板活化调节受损，并与年轻时CVD风险增加相关。我们还分析了C1016 T基因多态性在心血管疾病患者中的分布，发现T等位基因在患者组中的频率较高，并且高血压与这种关联相互作用，多种遗传因素的组合被认为是血栓性疾病发生的关键。多态性标志物临床评价的最终目标是确定最能预防疾病发生或对饮食、行为或药物干预反应最好的个体亚组。为此，多态性不仅需要研究疾病易感性，而且还需要研究对治疗的反应性以及基因-环境相互作用。少

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