脂蛋白相关磷脂酶A_2(Lp-PLA_2)基因多态性与冠心病关联研究
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摘要
背景
冠状动脉粥样硬化性心脏病是一种多基因疾病,遗传因素在冠状动脉粥样斑块病变的发展及其并发症发生中也发挥着至关重要的作用,研究CAD的遗传危险因素对于开展有针对性的人群预防,延缓或防止冠心病的发生发展具有重要的理论意义。
在动脉粥样硬化的形成和发展过程中,氧化和炎症扮演着重要角色。LDL中的磷脂被氧化脂质(如脂肪酸)氧化,产生的过氧化物导致轻微修饰的LDL的产生,并启动一系列步骤形成早期的脂纹。脂质过氧化物的不断形成和聚集,最终将加速动脉粥样硬化的发展。高密度脂蛋白作为脂蛋白中颗粒最小,密度最高的一种,被认为具有抗动脉粥样硬化的功能,除了是因为它具有促进胆固醇逆转运的作用外,还因为它具有抗氧化、抗炎的功能。HDL所含的几种酶类参与了它的此项功能,脂蛋白相关磷脂酶A_2(Lipoprotein-associated phospholipase A_2,Lp-PLA_2)即是其中一种。
Lp-PLA_2是磷脂酶A_2超家族中的一种亚型,也被称为血小板活化因子乙酰水解酶(PAF-AH)。流行病学研究揭示,Lp-PLA_2与冠心病发病风险存在关联,但并不清楚该现象是对动脉粥样硬化炎症刺激的一种应答反应还是导致动脉粥样硬化的直接原因,或者是一种发病风险标识。一方面,Lp-PLA_2可以水解血小板活化因子(PAF)和LDL氧化或氧化应激导致产生的氧化脂质产物,因此被认为具有抗动脉粥样硬化作用;但是另一方面,研究发现,Lp-PLA_2还能水解氧化磷脂产生溶血磷脂酰胆碱(lyso-PC)和自由氧化脂肪酸,而这些物质均具有促动脉粥样硬化的作用。
对Lp-PLA2基因多态性的研究发现,第9外显子V279F位点和第11外显子A379V位点的变异具有功能意义,这两个位点的变异与冠心病存在关联。关于Lp-PLA2基因多态性位点与冠心病的关系,在中国人群中尚未进行过大样本的研究。因此本研究主要关注Lp-PLA_2基因V279F和A379V这两个位点在中国汉族冠心病患者和健康对照人群中的基因多态性,以及它们与冠心病的发病及严重程度之间的关系。
目的
1.研究中国汉族人群冠心病患者和健康对照人群中Lp-PLA_2 V279F和A379V多态性位点的分布特点,比较冠心病患者和健康人群之间上述两个位点基因型和基因频率的差异;
2.分析在中国汉族人群中Lp-PLA_2 V279F和A379V两个多态性位点与冠心病患者的传统危险因素、脂类代谢情况、冠状动脉病变特点以及冠心病严重程度之间的相关性,进而揭示Lp-PLA_2在冠心病发病过程中发挥的作用。
方法
1.收集经冠脉造影确诊或有心梗病史的冠心病患者及同期体检的健康对照。收集各研究对象的冠心病传统危险因素相关信息,包括年龄、性别、吸烟史、家族史以及既往病史(高血压、糖尿病或血脂异常等病史);收集病情严重程度的评价指标,包括心肌梗死病史、冠脉病变严重程度、肌钙蛋白、脑钠肽等;
2.经静脉采取空腹血样,使用传统酚抽提法从血细胞中提取DNA;
3.Taqman探针法对Lp-PLA_2第9外显子的V279F位点和第11外显子的A379V位点进行分析,对上述两个位点的基因型和等位基因在冠心病组和正常对照组的频率进行比较分析;
4.对上述基因位点多态性与临床表型的相关性进行分析。
结果
1.研究共纳入冠心病患者806例(年龄61.51±10.74岁,男:女比例为620:186),健康对照者482例(年龄61.63±8.41岁,男:女比例为264:218),两组患者性别存在显著差异(p＜0.001)。对冠心病传统危险因素的比较分析发现,冠心病组BMI、高血压患病率、FBG和HsCRP水平显著高于正常对照组,而HDL-C、ApoA1显著低于正常对照组。
2.Lp-PLA_2 V279F位点的基因型与等位基因在两组间的分布有显著差异,冠心病组VF+FF基因型和F等位基因出现频率显著高于正常对照组(两组VF+FF基因型的频率分别为13.6%和9.3%,p=0.024;F等位基因的频率分别为6.9%和4.9%,p=0.037);而A379V位点基因型和等位基因的频率在冠心病患者和正常对照之间无显著差异。SHESIS单体型分析发现,两位点形成的单体型中TC单体型与冠心病发病相关(OR=1.449,95%CI[1.019-2.061],p=0.038)。
3.对多支病变患者与正常对照组的比较发现,多支病变患者中V279F位点的VF+FF基因型和F等位基因的出现频率均显著高于正常对照组,VF+FF基因型频率为15.2%(p=0.005);F等位基因频率为7.7%(p=0.009);同时也显著高于单支病变亚组(对应p值分别为0.027和p=0.028);
4.对单支冠脉狭窄≥70%的冠心病患者与正常对照组的比较发现,该群患者中VF+FF基因型和F等位基因的频率也显著高于正常对照组,冠脉中重度狭窄患者VF+FF基因型频率为14.1%(p=0.013),F等位基因频率为7.1%(p=0.025)。
5.对冠心病患者中有心梗病史者和无心梗病史者的比较分析发现,心梗患者中A379V位点的V379等位基因的出现频率显著高于无心梗病史的冠心病患者(分别为18.7%和14.8%,p=0.038),同时VV基因型的频率也显著高于无心梗病史患者(分别为3.3%和1.2%,p=0.043)。
6.对冠心病合并/未合并高血压患者及冠心病合并/未合并糖尿病患者的亚组间比较均未发现V279F和A379V位点变异在上述两亚组间存在显著差异。
7.冠心病发病风险分析提示V279F位点变异与冠心病发病相关(OR=1.525,95%Cl[1.056-2.202],p=0.024),经校正性别、BMI和高血压后仍显示具有相关性(OR=1.611,95%CI[1.020,2.545],P=0.041)。在冠心病患者中进行的心梗发病风险分析提示A379V位点变异与冠心病患者心梗发生相关,经校正性别、BMI、吸烟、HsCRP、HDL-C和LDL-C后仍显示具有相关性(OR=1.596,95%CI[1.104,2.306],P=0.013)。
8.比较V279F和A379V位点各基因型个体的血脂水平发现,冠心病组人群中A379V位点AV+VV基因型个体的LDL-C和ApoB水平显著高于AA基因型个体(LDL-C平均值分别为2.63mmol/l和2.48mmol/l,p=0.030,ApoB平均值分别为0.88mmol/l和0.81mmol/l,p=0.018),但正常对照人群中未发现该差异。其余血脂指标在两组人群各基因型个体之间无显著差异。
结论
1.Lp-PLA_2 V279F位点变异与冠心病相关,冠心病患者中VF+FF基因型频率显著高于正常对照,F等位基因携带者冠心病发病风险增加,冠脉病变程度更重。
2.Lp-PLA_2 A379V位点与冠心病发病无明显关联,但该位点变异与冠心病患者中心梗发生相关,有心梗病史的冠心病患者中VV基因型和V379等位基因的出现频率显著高于无心梗病史的冠心病患者,AV+VV基因型患者心梗风险增加。
3.Lp-PLA_2 A379V位点的AV+VV基因型个体的LDL-C和ApoB水平显著高于AA基因型个体。
4.Lp-PLA_2 V279F和A379V位点形成的TC单体型与冠心病发病相关。
Background
The coronary atherosclerotic disease (CAD) is a polygenic disease. The genetic factors play a key role in the development of atherosclerotic plaque and its complications. Theoretically, the investigation of genetic risk factors of CAD is very important for prevention and treatment of the CAD.
The oxidation and inflammation contribute to the formation and development of atherosclerosis. The phospholipids are oxidized by the oxidized lipids (such as fatty acids) in the LDL and the consequent peroxides result in the formation of LDL with a mild modification and initiate a series of procedures to form the early fatty streaks, which finally accelerate the development of atherosclerosis with the accumulation of lipid peroxides. The high-density lipoprotein, which is smallest in size and highest in density, is considered to exhibit capability of anti-atherosclerosis, because of its effect of promoting the reverse cholesterol transport as well as its capability of anti-oxidation and anti-inflammation. Several enzymes included in HDL participate in this function, one of which is lipoprotein-associated phospholipase A_2 (Lp-PLA_2).
Lp-PLA_2 is a subtype of phospholipase superfamily, also known as platelet activating factor-acetylhydrolase (PAF-AH). The epidemiology studies demonstrate that there is an association of the level of Lp-PLA_2 with the risk of CAD; however, it remains unknown that if this phenomena is a response to the stimulation of inflammation of atherosclerosis or a direct cause of atherosclerosis or the marker of risks. On one hand, the Lp-PLA_2 can hydrolyze the platelet activating factor (PAF) and the oxidized lipid products induced by the oxidation of LDL or oxidative stress, which therefore is considered to have the capability of anti-atherosclerosis. On the other hand, studies have revealed that the Lp-PLA_2 may hydrolyze oxidized phospholipids to generate lysophosphatidyl choline (lyso-PC) and free oxidized fatty acids, both of which play a critical role in atherogenesis.
The investigations of the genetic polymorphism of Lp-PLA_2 have demonstrated that the polymorphisms of V279F on the exon 9 and A379V on the exon 11 have functional effect, and are associated with CAD. No studies with large sample have been conducted to investigate the association of the polymorphism of Lp-PLA_2 gene with CAD in Chinese Han people. Therefore, our study will focus on polymorphism of V279F and A379V of Lp-PLA_2 gene in the patients with CAD and the healthy control, and the associations of polymorphism and the onset/ severity of CAD.
Objective
1. The study is designed to investigate the polymorphism of V279F on exon 9 and A379V on 11 exon of Lp-PLAV2 in Chinese CAD patients, compared with healthy control; also to compare and analyze the difference of the distribution of genotype and allele between these two populations.
2. To analyze the association of the polymorphism of V279F and A379V of Lp-PLA_2 with traditional risk factors, lipid profile and disease severity of CAD patients in Chinese Han people. Also to identify the role of Lp-PLA_2 in CAD progression.
Method
1. The patients were recruited in Peking Union Medical Hospital and FuWai Cardiovascular Disease Hospital from 03/2007 to 04/2008. Inclusion criterion was the presence of typical angina pectoris with a diameter stenosis >50% in at least one major coronary artery, or the presence of MI history. Age-matched healthy control without CAD, DM and hyperlipidemia were recruited from the health examination department of PUMC. Demographic features and traditional risk factors of CAD, including age, sex, smoking, family history, past history (with or without HTN, DM or hyperlipidemia) and lipid profile were collected. The result of CAG and the MI history were also collected to evaluate the severity of CAD.
2. The venous blood samples were drawn after an overnight fast. The genomic DNA was extracted from the whole blood cells using phenol-chloroform method.
3. Genotyping for V279F polymorphism on exon 9 and A379V polymorphism on 11 exon of Lp-PLA_2 were determined for each subject using Taqman(?) probe allele discrimination method. The frequencies of genotypes and alleles were analyzed and compared between CAD patients and healthy control.
4. The associations of SNPs with phenotypes were analyzed using relevant statistical methods.
Result
1. Total 806 patients (age of 61.51±10.74, F:M=620:186) and 482 healthy subjects (age of 61.63±8.41, F:M=264:218) were included. Those two groups were age matched, but had significant difference in sex (p<0.001). The comparison of traditional risk factors indicated that CAD patients had significantly greater level of BMI, FBG and HsCRP, lower level of HDL-C and ApoA1, and higher prevalence rate of HTN, than healthy control.
2. There was a significantly higher prevalence of the VF+FF genotype and F279 allele of Lp-PLA_2 V279F in CAD patients compared with healthy control. The frequencies of VF+FF genotype of those two groups were 13.6% and 9.3% respectively (p=0.024), and F279 allele were 6.9% and 4.9% respectively (p=0.037). The distribution of A379V variant was similar between CAD patients and healthy control. SHESIS haplotype analysis identified that, among the haplotype formed by those two variants TC haplotype was associated with CAD (OR= 1.449, 95%CI[1.019-2.061 ], p=0.038).
3. The prevalence of VF+FF genotype and F279 allele of V279F locus was significantly higher in the subpopulation with multi-vessel coronary disease, compared with healthy control. The frequencies of VF+FF genotype and F279 allele in this subpopulation were 15.2% and 7.7% with p-value 0.005 and 0.009 respectively. Those frequencies were also significantly higher than single-vessel coronary disease subpopulation.
4. The VF+FF genotype and F279 allele were also significantly more frequent in patients with a diameter stenosis >70% in at least one major coronary artery. The frequencies of VF+FF and F allele were 14.1% (p=0.013) and 7.1% (p=0.025), respectively.
5. The V379 allele of A379V was significantly more frequent in patients with MI histories (18.7%), compared with patients without MI histories (14.8%), p=0.038. Also, the prevalence of VV genotype was significantly higher in patients with MI histories (3.3% vs.1.2%, p=0.043).
6. The Comparison between patients with/without DM or HTN didn't reveal any significant difference of the distribution of V279F and A379V variants.
7. Logistic regression analysis showed the V279F polymorphism was positively associated with CAD. The VF+FF individuals had a significantly higher risk of CAD compared with VV individuals, even after adjusted for sex, BMI and HTN (OR=1.611, 95% CI[1.020, 2.545], P=0.041) . The risk analysis of MI in CAD patients revealed A379V polymorphism was positively associated with MI. The AV +VV individuals had a significantly higher risk of MI compared with AA individuals, even after adjusted for sex, BMI, smoking, HsCRP, HDL-C and LDL-C (OR=1.596, 95%CI[1.104,2.306], P=0.013) .
8. The association analysis of lipid profile and genotype showed AV+VV individuals in CAD group had significantly higher level of LDL-C and ApoB, compared with AA individuals (the level of LDL-C was 2.63mmol/l and 2.48 mmol/l respectively, p=0.030; and the level of ApoB was 0.88mmol/l and 0.81mmol/l, p=0.018). But the same difference was not identified in healthy control population.
Conclusion
1. Lp-PLA_2 V279F polymorphism is associated with CAD. The VF+FF genotype are more frequent in CAD patients, compared with healthy control. The individuals with F279 allele have increased risk of CAD and more severe coronary atherosclerosis.
2. The study didn't reveal the association of Lp-PLA_2 A379V polymorphism with CAD, but had identified this polymorphism is significantly associated with MI of CAD patients. The VV genotype and V379 allele are more frequent in patients with MI histories than patients without MI histories. The AV+VV individuals have increased risk of MI.
3. The individuals with AV+VV genotype of Lp-PLA_2 A379V in CAD patients had significantly higher level of LDL-C and ApoB, compared with AA individuals
4. The TC haplotype formed by V279F and A379V of Lp-PLA_2 is associated with CAD.
冠状动脉粥样硬化性心脏病是一种多基因疾病,遗传因素在冠状动脉粥样斑块病变的发展及其并发症发生中也发挥着至关重要的作用,研究CAD的遗传危险因素对于开展有针对性的人群预防,延缓或防止冠心病的发生发展具有重要的理论意义。
在动脉粥样硬化的形成和发展过程中,氧化和炎症扮演着重要角色。LDL中的磷脂被氧化脂质(如脂肪酸)氧化,产生的过氧化物导致轻微修饰的LDL的产生,并启动一系列步骤形成早期的脂纹。脂质过氧化物的不断形成和聚集,最终将加速动脉粥样硬化的发展。高密度脂蛋白作为脂蛋白中颗粒最小,密度最高的一种,被认为具有抗动脉粥样硬化的功能,除了是因为它具有促进胆固醇逆转运的作用外,还因为它具有抗氧化、抗炎的功能。HDL所含的几种酶类参与了它的此项功能,脂蛋白相关磷脂酶A_2(Lipoprotein-associated phospholipase A_2,Lp-PLA_2)即是其中一种。
Lp-PLA_2是磷脂酶A_2超家族中的一种亚型,也被称为血小板活化因子乙酰水解酶(PAF-AH)。流行病学研究揭示,Lp-PLA_2与冠心病发病风险存在关联,但并不清楚该现象是对动脉粥样硬化炎症刺激的一种应答反应还是导致动脉粥样硬化的直接原因,或者是一种发病风险标识。一方面,Lp-PLA_2可以水解血小板活化因子(PAF)和LDL氧化或氧化应激导致产生的氧化脂质产物,因此被认为具有抗动脉粥样硬化作用;但是另一方面,研究发现,Lp-PLA_2还能水解氧化磷脂产生溶血磷脂酰胆碱(lyso-PC)和自由氧化脂肪酸,而这些物质均具有促动脉粥样硬化的作用。
对Lp-PLA2基因多态性的研究发现,第9外显子V279F位点和第11外显子A379V位点的变异具有功能意义,这两个位点的变异与冠心病存在关联。关于Lp-PLA2基因多态性位点与冠心病的关系,在中国人群中尚未进行过大样本的研究。因此本研究主要关注Lp-PLA_2基因V279F和A379V这两个位点在中国汉族冠心病患者和健康对照人群中的基因多态性,以及它们与冠心病的发病及严重程度之间的关系。
目的
1.研究中国汉族人群冠心病患者和健康对照人群中Lp-PLA_2 V279F和A379V多态性位点的分布特点,比较冠心病患者和健康人群之间上述两个位点基因型和基因频率的差异;
2.分析在中国汉族人群中Lp-PLA_2 V279F和A379V两个多态性位点与冠心病患者的传统危险因素、脂类代谢情况、冠状动脉病变特点以及冠心病严重程度之间的相关性,进而揭示Lp-PLA_2在冠心病发病过程中发挥的作用。
方法
1.收集经冠脉造影确诊或有心梗病史的冠心病患者及同期体检的健康对照。收集各研究对象的冠心病传统危险因素相关信息,包括年龄、性别、吸烟史、家族史以及既往病史(高血压、糖尿病或血脂异常等病史);收集病情严重程度的评价指标,包括心肌梗死病史、冠脉病变严重程度、肌钙蛋白、脑钠肽等;
2.经静脉采取空腹血样,使用传统酚抽提法从血细胞中提取DNA;
3.Taqman探针法对Lp-PLA_2第9外显子的V279F位点和第11外显子的A379V位点进行分析,对上述两个位点的基因型和等位基因在冠心病组和正常对照组的频率进行比较分析;
4.对上述基因位点多态性与临床表型的相关性进行分析。
结果
1.研究共纳入冠心病患者806例(年龄61.51±10.74岁,男:女比例为620:186),健康对照者482例(年龄61.63±8.41岁,男:女比例为264:218),两组患者性别存在显著差异(p＜0.001)。对冠心病传统危险因素的比较分析发现,冠心病组BMI、高血压患病率、FBG和HsCRP水平显著高于正常对照组,而HDL-C、ApoA1显著低于正常对照组。
2.Lp-PLA_2 V279F位点的基因型与等位基因在两组间的分布有显著差异,冠心病组VF+FF基因型和F等位基因出现频率显著高于正常对照组(两组VF+FF基因型的频率分别为13.6%和9.3%,p=0.024;F等位基因的频率分别为6.9%和4.9%,p=0.037);而A379V位点基因型和等位基因的频率在冠心病患者和正常对照之间无显著差异。SHESIS单体型分析发现,两位点形成的单体型中TC单体型与冠心病发病相关(OR=1.449,95%CI[1.019-2.061],p=0.038)。
3.对多支病变患者与正常对照组的比较发现,多支病变患者中V279F位点的VF+FF基因型和F等位基因的出现频率均显著高于正常对照组,VF+FF基因型频率为15.2%(p=0.005);F等位基因频率为7.7%(p=0.009);同时也显著高于单支病变亚组(对应p值分别为0.027和p=0.028);
4.对单支冠脉狭窄≥70%的冠心病患者与正常对照组的比较发现,该群患者中VF+FF基因型和F等位基因的频率也显著高于正常对照组,冠脉中重度狭窄患者VF+FF基因型频率为14.1%(p=0.013),F等位基因频率为7.1%(p=0.025)。
5.对冠心病患者中有心梗病史者和无心梗病史者的比较分析发现,心梗患者中A379V位点的V379等位基因的出现频率显著高于无心梗病史的冠心病患者(分别为18.7%和14.8%,p=0.038),同时VV基因型的频率也显著高于无心梗病史患者(分别为3.3%和1.2%,p=0.043)。
6.对冠心病合并/未合并高血压患者及冠心病合并/未合并糖尿病患者的亚组间比较均未发现V279F和A379V位点变异在上述两亚组间存在显著差异。
7.冠心病发病风险分析提示V279F位点变异与冠心病发病相关(OR=1.525,95%Cl[1.056-2.202],p=0.024),经校正性别、BMI和高血压后仍显示具有相关性(OR=1.611,95%CI[1.020,2.545],P=0.041)。在冠心病患者中进行的心梗发病风险分析提示A379V位点变异与冠心病患者心梗发生相关,经校正性别、BMI、吸烟、HsCRP、HDL-C和LDL-C后仍显示具有相关性(OR=1.596,95%CI[1.104,2.306],P=0.013)。
8.比较V279F和A379V位点各基因型个体的血脂水平发现,冠心病组人群中A379V位点AV+VV基因型个体的LDL-C和ApoB水平显著高于AA基因型个体(LDL-C平均值分别为2.63mmol/l和2.48mmol/l,p=0.030,ApoB平均值分别为0.88mmol/l和0.81mmol/l,p=0.018),但正常对照人群中未发现该差异。其余血脂指标在两组人群各基因型个体之间无显著差异。
结论
1.Lp-PLA_2 V279F位点变异与冠心病相关,冠心病患者中VF+FF基因型频率显著高于正常对照,F等位基因携带者冠心病发病风险增加,冠脉病变程度更重。
2.Lp-PLA_2 A379V位点与冠心病发病无明显关联,但该位点变异与冠心病患者中心梗发生相关,有心梗病史的冠心病患者中VV基因型和V379等位基因的出现频率显著高于无心梗病史的冠心病患者,AV+VV基因型患者心梗风险增加。
3.Lp-PLA_2 A379V位点的AV+VV基因型个体的LDL-C和ApoB水平显著高于AA基因型个体。
4.Lp-PLA_2 V279F和A379V位点形成的TC单体型与冠心病发病相关。
Background
The coronary atherosclerotic disease (CAD) is a polygenic disease. The genetic factors play a key role in the development of atherosclerotic plaque and its complications. Theoretically, the investigation of genetic risk factors of CAD is very important for prevention and treatment of the CAD.
The oxidation and inflammation contribute to the formation and development of atherosclerosis. The phospholipids are oxidized by the oxidized lipids (such as fatty acids) in the LDL and the consequent peroxides result in the formation of LDL with a mild modification and initiate a series of procedures to form the early fatty streaks, which finally accelerate the development of atherosclerosis with the accumulation of lipid peroxides. The high-density lipoprotein, which is smallest in size and highest in density, is considered to exhibit capability of anti-atherosclerosis, because of its effect of promoting the reverse cholesterol transport as well as its capability of anti-oxidation and anti-inflammation. Several enzymes included in HDL participate in this function, one of which is lipoprotein-associated phospholipase A_2 (Lp-PLA_2).
Lp-PLA_2 is a subtype of phospholipase superfamily, also known as platelet activating factor-acetylhydrolase (PAF-AH). The epidemiology studies demonstrate that there is an association of the level of Lp-PLA_2 with the risk of CAD; however, it remains unknown that if this phenomena is a response to the stimulation of inflammation of atherosclerosis or a direct cause of atherosclerosis or the marker of risks. On one hand, the Lp-PLA_2 can hydrolyze the platelet activating factor (PAF) and the oxidized lipid products induced by the oxidation of LDL or oxidative stress, which therefore is considered to have the capability of anti-atherosclerosis. On the other hand, studies have revealed that the Lp-PLA_2 may hydrolyze oxidized phospholipids to generate lysophosphatidyl choline (lyso-PC) and free oxidized fatty acids, both of which play a critical role in atherogenesis.
The investigations of the genetic polymorphism of Lp-PLA_2 have demonstrated that the polymorphisms of V279F on the exon 9 and A379V on the exon 11 have functional effect, and are associated with CAD. No studies with large sample have been conducted to investigate the association of the polymorphism of Lp-PLA_2 gene with CAD in Chinese Han people. Therefore, our study will focus on polymorphism of V279F and A379V of Lp-PLA_2 gene in the patients with CAD and the healthy control, and the associations of polymorphism and the onset/ severity of CAD.
Objective
1. The study is designed to investigate the polymorphism of V279F on exon 9 and A379V on 11 exon of Lp-PLAV2 in Chinese CAD patients, compared with healthy control; also to compare and analyze the difference of the distribution of genotype and allele between these two populations.
2. To analyze the association of the polymorphism of V279F and A379V of Lp-PLA_2 with traditional risk factors, lipid profile and disease severity of CAD patients in Chinese Han people. Also to identify the role of Lp-PLA_2 in CAD progression.
Method
1. The patients were recruited in Peking Union Medical Hospital and FuWai Cardiovascular Disease Hospital from 03/2007 to 04/2008. Inclusion criterion was the presence of typical angina pectoris with a diameter stenosis >50% in at least one major coronary artery, or the presence of MI history. Age-matched healthy control without CAD, DM and hyperlipidemia were recruited from the health examination department of PUMC. Demographic features and traditional risk factors of CAD, including age, sex, smoking, family history, past history (with or without HTN, DM or hyperlipidemia) and lipid profile were collected. The result of CAG and the MI history were also collected to evaluate the severity of CAD.
2. The venous blood samples were drawn after an overnight fast. The genomic DNA was extracted from the whole blood cells using phenol-chloroform method.
3. Genotyping for V279F polymorphism on exon 9 and A379V polymorphism on 11 exon of Lp-PLA_2 were determined for each subject using Taqman(?) probe allele discrimination method. The frequencies of genotypes and alleles were analyzed and compared between CAD patients and healthy control.
4. The associations of SNPs with phenotypes were analyzed using relevant statistical methods.
Result
1. Total 806 patients (age of 61.51±10.74, F:M=620:186) and 482 healthy subjects (age of 61.63±8.41, F:M=264:218) were included. Those two groups were age matched, but had significant difference in sex (p<0.001). The comparison of traditional risk factors indicated that CAD patients had significantly greater level of BMI, FBG and HsCRP, lower level of HDL-C and ApoA1, and higher prevalence rate of HTN, than healthy control.
2. There was a significantly higher prevalence of the VF+FF genotype and F279 allele of Lp-PLA_2 V279F in CAD patients compared with healthy control. The frequencies of VF+FF genotype of those two groups were 13.6% and 9.3% respectively (p=0.024), and F279 allele were 6.9% and 4.9% respectively (p=0.037). The distribution of A379V variant was similar between CAD patients and healthy control. SHESIS haplotype analysis identified that, among the haplotype formed by those two variants TC haplotype was associated with CAD (OR= 1.449, 95%CI[1.019-2.061 ], p=0.038).
3. The prevalence of VF+FF genotype and F279 allele of V279F locus was significantly higher in the subpopulation with multi-vessel coronary disease, compared with healthy control. The frequencies of VF+FF genotype and F279 allele in this subpopulation were 15.2% and 7.7% with p-value 0.005 and 0.009 respectively. Those frequencies were also significantly higher than single-vessel coronary disease subpopulation.
4. The VF+FF genotype and F279 allele were also significantly more frequent in patients with a diameter stenosis >70% in at least one major coronary artery. The frequencies of VF+FF and F allele were 14.1% (p=0.013) and 7.1% (p=0.025), respectively.
5. The V379 allele of A379V was significantly more frequent in patients with MI histories (18.7%), compared with patients without MI histories (14.8%), p=0.038. Also, the prevalence of VV genotype was significantly higher in patients with MI histories (3.3% vs.1.2%, p=0.043).
6. The Comparison between patients with/without DM or HTN didn't reveal any significant difference of the distribution of V279F and A379V variants.
7. Logistic regression analysis showed the V279F polymorphism was positively associated with CAD. The VF+FF individuals had a significantly higher risk of CAD compared with VV individuals, even after adjusted for sex, BMI and HTN (OR=1.611, 95% CI[1.020, 2.545], P=0.041) . The risk analysis of MI in CAD patients revealed A379V polymorphism was positively associated with MI. The AV +VV individuals had a significantly higher risk of MI compared with AA individuals, even after adjusted for sex, BMI, smoking, HsCRP, HDL-C and LDL-C (OR=1.596, 95%CI[1.104,2.306], P=0.013) .
8. The association analysis of lipid profile and genotype showed AV+VV individuals in CAD group had significantly higher level of LDL-C and ApoB, compared with AA individuals (the level of LDL-C was 2.63mmol/l and 2.48 mmol/l respectively, p=0.030; and the level of ApoB was 0.88mmol/l and 0.81mmol/l, p=0.018). But the same difference was not identified in healthy control population.
Conclusion
1. Lp-PLA_2 V279F polymorphism is associated with CAD. The VF+FF genotype are more frequent in CAD patients, compared with healthy control. The individuals with F279 allele have increased risk of CAD and more severe coronary atherosclerosis.
2. The study didn't reveal the association of Lp-PLA_2 A379V polymorphism with CAD, but had identified this polymorphism is significantly associated with MI of CAD patients. The VV genotype and V379 allele are more frequent in patients with MI histories than patients without MI histories. The AV+VV individuals have increased risk of MI.
3. The individuals with AV+VV genotype of Lp-PLA_2 A379V in CAD patients had significantly higher level of LDL-C and ApoB, compared with AA individuals
4. The TC haplotype formed by V279F and A379V of Lp-PLA_2 is associated with CAD.
引文
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45.Bell,R.,Collier,D.A.,Rice,S.Q.,Roberts,G.W.,MacPhee,C.H.,Kerwin,R.W.,Price,J.and Gloger,I.S.(1997) Systematic screening of the LDL-PLA2 gene for polymorphic variants and case-control analysis in schizophrenia.Biochem.Biophys.Res.Commun.,241,630-635.
46.张慧平,孙福成等.血小板活化因子乙酰水解酶基因V279F变异与冠心病的关系.中华老年医学杂志.2006;25(12):895-899
47.Jang Y,K.im OY,Koh SJ,et al:The Va1279Phe variant of the lipoprotein-associated phospholipase A2 gene is associated with catalytic activities and cardiovascular disease in Korean men.J Clin Endocrin Metab 2006;91:3521-3527.
48.Kruse S,Mao XQ,Heinzmann A,et al.The Ile198Thr and Ala379Val variants of plasmatic PAF-acetylhydrolase impair catalytical activities and are associated with atopy and asthma. Am J Hum Genet. 2000;66:1522-1530
49. Ewa Ninio, David Tregouet, Jean-Luc Carrier, et al. Platelet-activating factor-acetylhydrolase and PAF-receptor gene haplotypes in relation to future cardiovascular event in patients with coronary artery disease. Human Molecular Genetics. 2004;13(13):1341-1351
50. Abuzeid, A.M., Hawe, E., Humphries, S.E. and Talmud, P.J. (2003) Association between the Ala379Val variant of the lipoprotein associated phospholipase A2 and risk of myocardial infarction in the north and south of Europe. Atherosclerosis, 168, 283-288.
51.P.-Y. LIU, Y.-H. LI, H.-L. WU, et al. Platelet-activating factor-acetylhydrolase A379V (exon 11) gene polymorphism is an independent and functional risk factor for premature myocardial infarction. Journal of Thrombosis and Haemostasis; 2006;4:1023-1028.
52. Shao-Yan ZHANG, Hiroshi SHIBATA, Kenji KARINO, et al. Comprehensive Evaluation of Genetic and Environmental Factors Influencing the Plasma Lipoprotein-Associated Phospholipase A2 Activity in a Japanese Population. Hypertens Res. 2007;30(5):403-409
53. Subramanian VS, Goyal J, Miwa M, et al: Role of lecitincholesterol acyltransferase in the metabolism of oxidized phospholipids in plasma: studies with platelet-activating factor acetylhydrolase-deficient plasma. Biochim Biophys Acta 1999;1439: 95-109.
54. Yamada Y, Izawa H, Ichihara S, et al: Prediction of the risk of myocardial infarction from polymorphisms in candidate genes. N Engl J Med 2002; 347: 1916-1923.
55. Johnson A, Zalewski A, Janmohammed S, et al. Lipoprotein associated phospholipase A2(Lp-PLA2) activity, an emerging CV risk marker, can be inhibitied in atherosclerotic lesions and plasma by novel pharmacologic intervention: the results of a multicenter clinical study. Circulation 2004; 110:111-590.
56. Holvoet P, De Geest B, Van Linthout S, et al. The Arg123-Tyr166 central domain of human ApoAI is critical for lecithin:cholesterol acyltransferase-induced hyperalphalipoproteinemia and HDL remodeling in transgenic mice. Arterioscler Thromb Vasc Biol. 2000;20:459-66.
57. Peelman F, Vanloo B, Perez-Mendez O, et al. Characterization of functional residues in the interfacial recognition domain of lecithin cholesterol acyltransferase(LCAT). Protein Eng. 1999;12:71-8.
58. Francone OL, Fielding CJ. Structure-function relationships in human lecithin:cholesterol acyltransferase.Site-directed mutagenesis at serine residues 181and 216.Biochemistry.1991;30:10074-7.
59.Hoeq Jm,Santamarina-Fojo S,Berard AM.Overexpression of lecithin:cholesterol acyltransferase in transgenic rabbits prevents diet-induced atherosclerosis.Proc Nat1Acad Sci USA.1996;93:11448-53.
60.Santamarina-Fojo S,Lambert G.Heq JM,et al.Lecithin-cholesterol acyltmnsferase:role in lipoprotein metabolism,reverse cholesterol transport and atherosclerosis.Curr Opin Lipidol.2000;11:267-75
61.Cargill M,Altshuler D,Ireland J,et al.Characterization of single-nucleotide polymorphisms in coding regions of human genes.Nat Genet.1999;22:231-8.
62.张克兰,张思仲,郑克勤等人.卵磷脂胆固醇酰基转移酶基因单核苷酸多态性与冠心病脂代谢易感性的关联研究.中华医学遗传学杂志.2003;20:135-7.
63.Zhang K,Zheng K,He Y,et al.Novel P143L polymorphism of the LCAT gene is associated with dyslipidemia in Chinese patients who have coronary atherosclerotic heart disease.Biochem Biophys Res Commun,2004,318:4-10.
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5. Watson A, Berliner J, Hama S, et al. Protective effect of high density lipoprotein associated paraoxonase: inhibition of the biological activity of minimally oxidized low density lipoprotein. J Clin Invest. 1995;96:2882-2891.
6. Nilsson J, Dahlgren B, Ares M, et al. Lipoprotein-like phospholipid particles inhibit the smooth muscle cell cytotoxicity of lysophosphatidylcholme and platelet-activating factor. Arterioscler Thromb Vasc Biol. 1998;18:13-19.
7. Yanoshita, R., I. Kudo, K. Ikizawa, et al. Hydrolysis of platelet-activating factor and its methylated analogs by acetylhydrolases. J. Biochem. 1988;103:815-819.
8. Stafforini, D.M., S.M. Prescott, et al. Platelet-activating factor acetylhydrolase activity in human tissues and blood cells. Lipids. 1991;26:979-985.
9. Stafforini DM, McIntyre TM, Zimmerman GA, et al. Platelet-activating factor, a pleiotrophic mediator of physiological and pathological processes. Crit Rev Clin Lab Sci. 2003;40:643-672.
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12. Kruse S, Mao XQ, Heinzmann A, et al. The Ile198Thr and Ala379Val variants of plasmatic PAF-acetylhydrolase impair catalytical activities and are associated with atopy and asthma. Am J Hum Genet. 2000;66:1522-1530
13. Yamada, Y, S. Ichihara, T. Fujimura, and M. Yokota. Identification of the G994/T missense mutation in exon 9 of the plasma platelet-activating factor acetylhydrolase gene as an independent risk factor for coronary artery disease in Japanese men. Metabolism.1998;47:177-181.
14.Yamada,Y.,H.Yoshida,S.Ichihara,et al.Correlations between plasma platelet-activating factor acetylhydrolase(PAF-AH) activity and PAF-AH genotype,age and atherosclerosis in a Japanese population.Atherosclerosis.2000;150:209-216.
15.Shimokata,K.,Yamada,Y.,Kondo,T.,et al.Association of gene polymorphisms with coronary artery disease in individuals with or without nonfamilial hypercholesterolemia.Atherosclerosis.2004;172:167-173.
16.张慧平,孙福成等.血小板活化因子乙酰水解酶基因V279F变异与冠心病的关系.中华老年医学杂志.2006;25(12):895-899
17.Jang Y,Kim OY,Koh SJ,et al.The Va1279Phe variant of the lipoprotein-associated phospholipase A2 gene is associated with catalytic activities and cardiovascular disease in Korean men.J Clin Endocrine Metab 2006;91:3521-3527.
18.Ewa Ninio,David Tregouet,Jean-Luc Carrier,et al.Platelet-activating factor-acetylhydrolase and PAF-receptor gene haplotypes in relation to future cardiovascular event in patients with coronary artery disease.Human Molecular Genetics.2004;13(13):1341-1351
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20.P.-Y.LIU,Y.-H.LI,H.-L.WU,et al.Platelet-activating factor-acetylhydrolase A379V (exon 11) gene polymorphism is an independent and functional risk factor for premature myocardial infarction.Journal of Thrombosis and Haemostasis;2006;4:1023-1028.
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27. Carpenter, K.L., Dennis, I.F., Challis, I.R., et al. Inhibition of lipoprotein-associated phospholipase A2 diminishes the death-inducing effects of oxidised LDL on human monocyte-macrophages. FEBS Lett. 2001;505:357-363.
28. Packard CJ, O'Reilly DS, Caslake MJ, et al. Lipoprotein-associated phospholipase A2 as an independent predictor of coronary heart disease. West of Scotland Coronary Prevention Study Group. N Engl J Med. 2000;343:1148-55.
29. Caslake, M.J., Packard, C.J., Suckling, K.E., et al. Lipoprotein-associated phospholipase A(2), platelet-activating factor acetylhydrolase: a potential new risk factor for coronary artery disease. Atherosclerosis, 2000; 150:413-419.
30. Blankenberg, S., Stengel, D., Rupprecht, H., et al. Plasma PAF-acetylhydrolase in patients with coronary artery disease. Results of a cross-sectional analysis. J. Lipid Res. 2003;44:1381-1386
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37. Subramanian VS, Goyal J, Miwa M, et al: Role of lecitincholesterol acyltransferase in the metabolism of oxidized phospholipids in plasma: studies with platelet-activating factor acetylhydrolase-deficient plasma. Biochim Biophys Acta 1999; 1439: 95-109.
38. Yamada Y, Izawa H, Ichihara S, et al: Prediction of the risk of myocardial infarction from polymorphisms in candidate genes. N Engl J Med 2002; 347: 1916-1923.
1.Shaw PX.Rethinking oxidized low-density lipoprotein,its role in atherogenesis and the immune responses associated with it.Arch Immuno Ther Exp.2004;52:225-39.
2.Jessup W.Oxidized lipoprotein and nitric oxide.Curr Opin Lipidol.1996;7:274-80.
3.Watson A,Berliner J,Hama S,et al.Protective effect of high density lipoprotein associated paraoxonase:inhibition of the biological activity of minimally oxidized low density lipoprotein.J Clin Invest.1995;96:2882-91.
4.Nilsson J,Dahlgren B,Ares M,et al.Lipoprotein-like phospholipid particles inhibit the smooth muscle cell cytotoxicity of lysophosphatidylcholine and platelet-activating factor.Arterioscler Thromb Vase Biol.1998;18:13-9.
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23. Jarvik GP, Hatsukami TS, Carlson C. et al. Paraoxonase activity, but not haplotype utilizing the linkage disequilibrium structure, predicts vascular disease. Arterioscler., Thromb Vasc Biol. 2003;23:1465-71
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25. Wheeler JG, Keavney BD, Watkins H, et al. Four paraoxonase gene polymorphisms in 11212 cases of coronary heart disease and 12786 controls: meta-analysis of 43 studies. Lancet. 2004;363:689-95.
26. SU Shao-yong, CHEN Jian-hong, HUANG Jian-feng,et al. Paroxonase gene cluster variations associated with coronary heart disease in Chinese Han women. Chinese Medical Journal. 2005; 114:1167-1174.
27. Sanghera DK, Aston CE, Saha N, et al. DNA polymorphisms in two paraoxonase genes (PON1 and PON2) are associated with the risk of coronary heart disease. Am J Hum Genet. 1998;62:36-44.
28. Chen Q, Reis SE, Kammerer CM, et al. Association between the severity of angiographic coronary artery disease and paraoxonase gene polymorphisms in the National Heart, Lung, and Blood Institute-sponsored Women's Ischemia Syndrome Evaluation (WISE) study. Am J Hum Genet. 2003 ;72:13-22.
29. Mackness M, Mackness B. Paraoxonase 1 and atherosclerosis: is the gene or the protein more important?. Free Radic Bio Med. 2004;37:1317-23.
30. Yanoshita, R., I. Kudo, K. Ikizawa, et al. Hydrolysis of platelet-activating factor and its methylated analogs by acetylhydrolases. J. Biochem. 1988; 103:815-819.
31. Stafforini, D.M., S.M. Prescott, et al. Platelet-activating factor acetylhydrolase activity in human tissues and blood cells. Lipids. 1991;26:979-985.
32. Stafforini DM, McIntyre TM, Zimmerman GA, et al. Platelet-activating factor, a pleiotrophic mediator of physiological and pathological processes. Crit Rev Clin Lab Sci. 2003;40:643-72.
33. Steinberg, D. Low density lipoprotein oxidation and its pathobiological significance. J Biol Chem., 1997;272:20963-66.
34. Tjoelker, L.W., Wilder, C., Eberhardt, C., et al. Anti-inflammatory properties of a platelet-activating factor acetylhydrolase. Nature. 1995 ;374:549-553.
35. Noto, H., Hara, M., Karasawa, K., et al. Human plasma platelet-activating factor acetylhydrolase binds to all the murine lipoproteins, conferring protection against oxidative stress. Arterioscler Thromb Vasc Biol. 2003 ;23:829-83 5.
36. Carpenter, K.L., Dennis, I.F., Challis, I.R., et al. Inhibition of lipoprotein-associated phospholipase A2 diminishes the death-inducing effects of oxidised LDL on human monocyte-macrophages. FEBS Lett. 2001;505:357-363.
37. Packard CJ, O'Reilly DS, Caslake MJ, et al. Lipoprotein-associated phospholipase A2 as an independent predictor of coronary heart disease. West of Scotland Coronary Prevention Study Group. N Engl J Med. 2000;343:1148-55.
38.Caslake,M.J.,Packard,C.J.,Suckling,K.E.,et al.Lipoprotein-associated phospholipase A(2),platelet-activating factor acetylhydrolase:a potential new risk factor for coronary artery disease.Atherosclerosis,2000;150:413-419.
39.Blankenberg,S.,Stengel,D.,Rupprecht,H.,et al.Plasma PAF-acetylhydrolase in patients with coronary artery disease.Results of a cross-sectional analysis.J.Lipid Res.2003;44:1381-1386
40.Stafforini D M,Satoh K,Atldnson D L,et al.Platelet-activating factor acetylhydrolase deficiency.A missense mutation near the active site of an anti-inflammatory phospholipase.J Clin Invest,1996,97(12):2784-2791.
41.Yamada,Y.,S.Ichihara,T.Fujimura,and M.Yokota.1998.Identification of the G994/T missense mutation in exon 9 of the plasma platelet-activating factor acetylhydrolase gene as an independent risk factor for coronary artery disease in Japanese men.Metabolism.47:177-181.
42.Satoh,K.,H.Yoshida,T-a.Imaizumi,S.Takamatsu,and S.Mizuno.1992.Platelet-activating factor acetylhydrolase in plasma lipoproteins from patients with ischemic stroke.Stroke.23:1090-1092.
43.Yamada,Y.,Yoshida,H.,Ichihara,S.,Imaizumi,T.,Satoh,K.and Yokota,M.(2000)Correlations between plasma platelet-activating factor acetylhydrolase(PAF-AH)activity and PAF-AH genotype,age and atherosclerosis in a Japanese population.Atherosclerosis,150,209-216.
44.Shimokata,K.,Yamada,Y.,Kondo,T.,Ichihara,S.,Izawa,H.,Nagata,K.,Murohara,T.,Ohno,M.and Yokota,M.(2004) Association of gene polymorphisms with coronary artery disease in individuals with or without nonfamilial hypercholesterolemia.Atherosclerosis,172,167-173.
45.Bell,R.,Collier,D.A.,Rice,S.Q.,Roberts,G.W.,MacPhee,C.H.,Kerwin,R.W.,Price,J.and Gloger,I.S.(1997) Systematic screening of the LDL-PLA2 gene for polymorphic variants and case-control analysis in schizophrenia.Biochem.Biophys.Res.Commun.,241,630-635.
46.张慧平,孙福成等.血小板活化因子乙酰水解酶基因V279F变异与冠心病的关系.中华老年医学杂志.2006;25(12):895-899
47.Jang Y,K.im OY,Koh SJ,et al:The Va1279Phe variant of the lipoprotein-associated phospholipase A2 gene is associated with catalytic activities and cardiovascular disease in Korean men.J Clin Endocrin Metab 2006;91:3521-3527.
48.Kruse S,Mao XQ,Heinzmann A,et al.The Ile198Thr and Ala379Val variants of plasmatic PAF-acetylhydrolase impair catalytical activities and are associated with atopy and asthma. Am J Hum Genet. 2000;66:1522-1530
49. Ewa Ninio, David Tregouet, Jean-Luc Carrier, et al. Platelet-activating factor-acetylhydrolase and PAF-receptor gene haplotypes in relation to future cardiovascular event in patients with coronary artery disease. Human Molecular Genetics. 2004;13(13):1341-1351
50. Abuzeid, A.M., Hawe, E., Humphries, S.E. and Talmud, P.J. (2003) Association between the Ala379Val variant of the lipoprotein associated phospholipase A2 and risk of myocardial infarction in the north and south of Europe. Atherosclerosis, 168, 283-288.
51.P.-Y. LIU, Y.-H. LI, H.-L. WU, et al. Platelet-activating factor-acetylhydrolase A379V (exon 11) gene polymorphism is an independent and functional risk factor for premature myocardial infarction. Journal of Thrombosis and Haemostasis; 2006;4:1023-1028.
52. Shao-Yan ZHANG, Hiroshi SHIBATA, Kenji KARINO, et al. Comprehensive Evaluation of Genetic and Environmental Factors Influencing the Plasma Lipoprotein-Associated Phospholipase A2 Activity in a Japanese Population. Hypertens Res. 2007;30(5):403-409
53. Subramanian VS, Goyal J, Miwa M, et al: Role of lecitincholesterol acyltransferase in the metabolism of oxidized phospholipids in plasma: studies with platelet-activating factor acetylhydrolase-deficient plasma. Biochim Biophys Acta 1999;1439: 95-109.
54. Yamada Y, Izawa H, Ichihara S, et al: Prediction of the risk of myocardial infarction from polymorphisms in candidate genes. N Engl J Med 2002; 347: 1916-1923.
55. Johnson A, Zalewski A, Janmohammed S, et al. Lipoprotein associated phospholipase A2(Lp-PLA2) activity, an emerging CV risk marker, can be inhibitied in atherosclerotic lesions and plasma by novel pharmacologic intervention: the results of a multicenter clinical study. Circulation 2004; 110:111-590.
56. Holvoet P, De Geest B, Van Linthout S, et al. The Arg123-Tyr166 central domain of human ApoAI is critical for lecithin:cholesterol acyltransferase-induced hyperalphalipoproteinemia and HDL remodeling in transgenic mice. Arterioscler Thromb Vasc Biol. 2000;20:459-66.
57. Peelman F, Vanloo B, Perez-Mendez O, et al. Characterization of functional residues in the interfacial recognition domain of lecithin cholesterol acyltransferase(LCAT). Protein Eng. 1999;12:71-8.
58. Francone OL, Fielding CJ. Structure-function relationships in human lecithin:cholesterol acyltransferase.Site-directed mutagenesis at serine residues 181and 216.Biochemistry.1991;30:10074-7.
59.Hoeq Jm,Santamarina-Fojo S,Berard AM.Overexpression of lecithin:cholesterol acyltransferase in transgenic rabbits prevents diet-induced atherosclerosis.Proc Nat1Acad Sci USA.1996;93:11448-53.
60.Santamarina-Fojo S,Lambert G.Heq JM,et al.Lecithin-cholesterol acyltmnsferase:role in lipoprotein metabolism,reverse cholesterol transport and atherosclerosis.Curr Opin Lipidol.2000;11:267-75
61.Cargill M,Altshuler D,Ireland J,et al.Characterization of single-nucleotide polymorphisms in coding regions of human genes.Nat Genet.1999;22:231-8.
62.张克兰,张思仲,郑克勤等人.卵磷脂胆固醇酰基转移酶基因单核苷酸多态性与冠心病脂代谢易感性的关联研究.中华医学遗传学杂志.2003;20:135-7.
63.Zhang K,Zheng K,He Y,et al.Novel P143L polymorphism of the LCAT gene is associated with dyslipidemia in Chinese patients who have coronary atherosclerotic heart disease.Biochem Biophys Res Commun,2004,318:4-10.