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，全面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贡献，日本人群的等位基因频率已知与高加索人有很大差异。研究了血小板活化因子（PAF）受体、PAF乙酰水解酶和血小板GPIba受体的多态性与它们的功能后果和与CVD的关系。PAF是一种脂质介质，对多种细胞具有强大的生物学作用。对于血小板，PAF与其特异性受体（PAF- r）结合导致磷脂酶和蛋白激酶的激活，并增加细胞内Ca^<++>浓度。最近发现了一种常见的单核苷酸多态性，导致PAF-R细胞质区域的氨基酸取代，Ala/Asp在残基224处（^<224>A/D, Fukunaga K et al ., 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%。我们在不同基因型的健康志愿者中测量了paf诱导的血小板聚集。两名PAF- r D/D基因型的健康志愿者对各种PAF浓度的聚集反应都受到损害。两名受试者之一的血小板即使在较高浓度的PAF（6微米）下也未发生不可逆聚集。A/A、A/D、D/D基因型在患者组的出现频率分别为78.9%、17.2%、3.9%，与对照组比较差异无统计学意义。血浆PAF-AH遗传缺陷在日本人中相对常见，但在白人人群中没有发现。PAF-AH缺陷归因于单点突变Val279Phe。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的临床相关性仍存在争议。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Val279Phe的频率。健康杂合突变志愿者富血小板血浆（Val/Phe）对PAF阈值浓度（~ 0.01微M）的血小板聚集反应明显高于Val/Val，提示PAF- ah对血小板活化的调节作用。CVD患者Val/Val、Val/Phe、Phe/Phe基因型频率分别为60.3、35.9、3.8%，与对照组差异有统计学意义（Val/Val、27.9、3.4%，Val/Val与Val/Phe+Phe/Phe比较，p<0.05）。当分析局限于年龄<60岁的患者时，差异更为显著（CVD组为55.7、39.3和4.9%，对照组为70.6、26.4和3.1%,p<0.01， OR=1.9, Val/Val vs Val/Phe+Phe/Phe）。与不吸烟的受试者相比，基因型效应更强（p<0.01， OR=2.4）。目前的研究表明，PAF-AH杂合缺乏（^<279>Val/Phe）在日本很常见，可能会导致血小板活化调节受损，并与年轻时心血管疾病的风险增加有关。我们还分析了CVD患者中Connexin 37基因多态性C1016T，发现患者组中T等位基因的频率更高，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对血栓性疾病的发展至关重要。临床评价多态性标记的最终目标是确定最能预防疾病发展的个体亚群，或对饮食、行为或药物干预反应最好的个体亚群。为此，多态性不仅需要研究与疾病易感性的关系，还需要研究对治疗的反应性和基因与环境的相互作用。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