羧肽酶E基因多态性、胰岛素原水平与冠状动脉狭窄程度关系的流行病学研究
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摘要
心血管疾病最常见的形式是由于动脉粥样硬化引起的冠心病,预计到2020年冠心病将成为世界上最主要的死亡原因。我国冠心病的发病率与死亡率呈上升趋势,是全球上升较快的国家,也是当今严重危害我国人类健康、影响人们生活质量的最常见心血管疾病之一。
冠心病发病危险因素错综复杂,近年来胰岛素的前体物质-胰岛素原在冠心病中的作用渐受重视。国外群体研究结果认为,胰岛素原与胰岛素抵抗综合征的关系比胰岛素更密切,同时它比真胰岛素与冠心病的关系也更密切,因此胰岛素原在冠心病发生和发展中的重要性更大。正常生理情况下,只有少量的胰岛素原进入外周循环,但是在糖尿病、肥胖、冠心病等情况下胰岛素原呈现与胰岛素不呈比例的高分泌状态,外周循环中高胰岛素原血症被认为是胰岛β细胞功能缺陷的指标。横断面与前瞻性的流行病学研究显示,高胰岛素原水平为冠心病的独立危险因素。胰岛β细胞内,胰岛素原被酶解为胰岛素与C肽的过程中,激素原转化酶2、激素原转化酶3与羧肽酶E(carboxypeptidase E,CPE)被认为是三个关键性的酶。近年来,国际上研究热点集中在CPE基因突变与高胰岛素原水平关系上。CPE基因突变的老鼠发展成为较为显著的高胰岛素原血症与在人类CPE基因突变可导致高胰岛素原血症的结论相吻合。国外有研究表明,CPE基因突变可导致外周血中高胰岛素原血症。而前述有研究表明高胰岛素原血症为冠心病的独立危险因素。因此我们设想,羧肽酶E基因突变→高胰岛素原血症→冠心病这一假说是否存在?CPE基因突变是否为冠心病的分子生物学机制之一?高胰岛素原水平、胰岛素原代谢过程中的关键酶CPE基因突变与冠状动脉狭窄程度的关系如何?基因与高胰岛素原水平等之间是否存在交互作用?国际上仅有的几项关于CPE基因突变的研究多集中在糖尿病与肥胖的分子生物学机制,迄今未见有关CPE基因多态性与冠心病/冠状动脉硬化特征关系的研究。
为验证以上假设,本研究以医院为研究现场,采用国际上公认的Gensini评分(Gensini's score,GS)系统对冠状动脉造影特征进行系统的定量评价,并应用大样本的分子流行病学研究探讨胰岛素原水平及相关环境因素在冠状动脉粥样硬化发生发展中的作用;探讨胰岛素原代谢过程中羧肽酶E基因多态性与胰岛素原水平的关系,以及CPE基因多态性与高胰岛素原水平的交互作用及其与冠状动脉硬化程度的关系,对于进一步阐明冠状动脉粥样硬化发生的分子遗传学机制及可能存在的CPE基因-环境交互作用等具有重要的理论意义。此外,还可发现与我国人群冠心病相关的易感性基因型,并将其作为分子标志物用于筛选高危人群或易感个体,对冠心病的早期诊断与治疗,以及实施有效和目标明确的个体预防与干预等均具有重要的现实意义。
第一部分胰岛素原水平及相关环境因素与冠状动脉狭窄程度关系的流行病学研究
胰岛素原为胰岛素的前体物质,在糖耐量低减、糖尿病、肥胖、冠心病等情况下胰岛素原呈现与胰岛素不呈比例的高分泌状态。国际上横断面与前瞻性的流行病学研究显示,胰岛素原的浓度而非真胰岛素或免疫反应性胰岛素浓度是冠心病的独立危险因素。然而,也有研究报道胰岛素原水平独立于糖尿病状态而与冠心病没有关联。因此胰岛素原水平与冠心病之间的关系目前仍不太明确。本研究采用国际上认可的灵敏度好、特异性强的双单克隆抗体酶免疫方法测定研究对象外周循环中的真胰岛素、胰岛素原水平,以探讨中国人群胰岛素原水平及相关环境因素与冠状动脉狭窄程度的关系。
目的:研究胰岛素原水平及相关环境因素与冠状动脉狭窄程度的关系。
方法:选择行冠状动脉造影检查的1044例(男性777人,女性267人)疑诊与确诊冠心病患者作为研究对象;对研究对象进行体格检查及胰岛素原等血生化指标的检测;采用Gensini评分系统对冠状动脉造影特征进行系统的定量评价;采用协方差、偏相关、多元线性回归、单因素和多因素有序分类变量Logistic回归分析等方法评价胰岛素原水平与冠状动脉狭窄程度的相关性。
结果:
1.研究对象的—般情况与生化指标的比较
以Gensini评分四分位间距作为分组变量,研究对象的平均年龄(P＜0.001)、高密度脂蛋白胆固醇(P＜0.001)、低密度脂蛋白胆固醇(P=0.042)、空腹血糖(P＜0.001)、胰岛素(P=0.005)、HOMA胰岛素抵抗指数(P＜0.001)、胰岛素原水平(P＜0.001)以及性别(P＜0.001)、吸烟(P＜0.001)构成在各组间的分布均存在显著性差异。控制了年龄、性别与BMI的协方差分析结果显示,总胆固醇(P=0.041)、低密度脂蛋白胆固醇(P=0.001)、甘油三酯(P=0.015)、空腹血糖(P＜0.001)、胰岛素(P=0.024)、HOMA胰岛素抵抗指数(P=0.001)与胰岛素原水平(P＜0.001)等在各组间的差异也均具有统计学意义。
2.胰岛素原水平及各测量指标与Gensini评分的偏相关分析
以年龄、性别与BMI为控制变量,以Gensini评分为应变量的偏相关分析结果显示,胰岛素原(P＜0.001)、胰岛素(P=0.011)、空腹血糖(P＜0.001)、HOMA胰岛素抵抗指数(P=0.001)、总胆固醇(P=0.028)及低密度脂蛋白胆固醇水平(P=0.001)均与Gensini评分呈显著的正相关。
3.胰岛素原水平及各测量指标与Gensini评分的多元线性回归分析
以Gensini评分为应变量的多元线性回归分析结果显示,胰岛素原(P=0.002)、高密度脂蛋白胆固醇(P=0.024)、低密度脂蛋白胆固醇(P=0.040)、空腹血糖(P＜0.001)水平以及年龄(P＜0.001)均与Gensini评分呈独立的显著正相关;同时,男性(P＜0.001)与吸烟(P=0.002)均是冠状动脉狭窄增高的独立的危险因素。
4.胰岛素原水平与冠状动脉狭窄程度关系的Logistic回归分析
以Gensini评分(四分位间距)分组变量作为有序分类应变量,胰岛素原浓度(按照其四分位间距界值将研究对象分为Group1、2、3和4组)分组变量作为自变量的单因素Ordinal Logistic回归分析结果显示,胰岛素原的水平每增加一个等级(25百分位数),冠状动脉狭窄程度增高的OR值亦增加(Group2 vs Groupl:OR=1.00,95%CI=0.74-1.37);Group3 vs Groupl:OR=1.31,95%CI=0.96-1.78;Group4vs Group1:OR=2.23,95%CI=1.63-3.04)。多因素Logistic回归分析结果显示,调整了年龄、性别、BMI、血糖及吸烟等因素后,与胰岛素原水平较低的第一组研究对象相比,胰岛素原浓度最高的第四组研究对象的冠状动脉狭窄程度增高的风险显著增加了61%(校正OR=1.61,95%CI=1.12-2.30)。
结论:高胰岛素原水平可能与冠状动脉狭窄程度增高有显著关联,确切的生物学机制有待于进一步的研究及不同种族人群的验证。
第二部分冠状动脉粥样硬化症人群中羧肽酶E基因突变的分子扫描与筛查研究
胰岛素原被酶解为胰岛素和C肽的过程中,羧肽酶E被认为是一个关键性的酶。有研究表明,CPE基因突变的老鼠发展成为较为显著的高胰岛素原血症与人类CPE基因突变可导致高胰岛素原血症的结论相吻合。国际上大量研究和本文第一部分的研究结果均表明,高胰岛素原血症为冠心病/冠状动脉硬化的独立危险因素。但未见有关该基因多态性与冠心病/冠状动脉硬化特征关系的研究报道。本研究采用目前最理想的基因检测分型技术即聚合酶链反应-测序分型法(PCR-SBT)对冠状动脉硬化症人群的CPE基因进行全面扫描与初步分析,为进一步探讨CPE基因多态性与冠状动脉硬化特征的关系奠定基础。
目的:初步探讨引起高胰岛素原血症的CPE基因的突变及其与冠心病的关系。
方法:随机选择51例(男性34人,女性17人)疑诊与确诊冠心病患者作为研究对象,并根据冠状动脉造影检查与调查问卷结果将研究对象分为冠心病组与对照组;采用聚合酶链反应.测序分型法与序列比对分析法对研究对象的CPE基因进行全面扫描与鉴定,初步分析各SNPs位点的分布及其与冠心病等临床特征的关系。
结果:
1.研究对象的—般情况与生化指标的比较
研究对象的收缩压(P=0.015)、舒张压(P=0.026)、高密度脂蛋白胆固醇(P=0.013)和胰岛素水平(P=0.035)在冠心病病例组与对照组中的分布均有显著性差异。
2.CPE基因的测序及其比对分析
GenBank数据库公布的CPE基因(accession numbers.AB006890-AB006898)第2、4、7、9内含子和第7外显子中的某些序列与本研究结果可能存在不一致的地方。
3.CPE基因多态性位点的鉴定与分布情况
通过对研究对象CPE基因8个外显子及其外显子-内含子连接处区域的测序与鉴定分析,在其第3、4、5外显子和相应内含子区域共发现了15个突变位点。其中,位于非编码区的T1951C、T1969A、T1951G、T1977C(第2内含子区),T2681C(第3内含子区)以及T4367C、A4350T、A4309T、T4587A(第7内含子区)共9个位点的突变频率极低;A2925G(第4内含子区)、T3609C(第5内含子区)和A4545G(第7内含子区)位点的突变频率均较高。另外,在编码区发现了三个点突变位点:第3外显子区的G2294T、第4外显子区的G2855A与第5外显子区的C3164T突变均为同义突变,不引起编码氨基酸的改变,其中G2294T和G2855A位点先前没有被报道过,G2294T位点的突变频率很低。
4.CPE基因多态性与研究对象临床特征的关系
对5个突变频率较高的位点的进一步分析表明,这些多态性均不显著影响血糖、胰岛素与胰岛素原的水平,但A4545G位点的变异型基因频率在冠心病病例组与对照组中的分布有显著性差异(χ~2=5.436,P=0.020)。
结论:本研究人群中共发现5个突变频率较高的多态性位点,这些多态性位点可能均不影响胰岛素原、胰岛素及血糖水平,CPE基因多态性在冠心病发生发展中的作用需要大规模的流行病学和功能学研究进一步验证。
第三部分羧肽酶E第五外显子区基因多态性与胰岛素原水平、冠状动脉狭窄程度关系的研究
国际上和本文第一部分的研究结果表明,高胰岛素原血症为冠心病/冠状动脉硬化的独立危险因素。胰岛素原被酶解的过程中,关键性的酶CPE基因的功能性位点如果发生改变,将可能影响胰岛素的生物合成过程。国外有研究表明CPE基因突变的肥胖老鼠未来可能发展成为较为显著的高胰岛素原血症和晚期肥胖与糖尿病,这与人类CPE基因突变在糖尿病与肥胖发生发展中的作用研究结论相吻合。Chen等在德系犹太人2型糖尿病家系人群CPE基因第5外显子编码区发现两个显著的突变位点(R283W,c.847C＞T和H267H,c.801C＞T),同时本课题第二部分的全基因扫描结果也表明801C＞T位点的突变频率比较高。因此,本研究挑选了以上2个单核苷酸多态性位点,探讨CPE相关基因多态性与胰岛素原水平及冠状动脉狭窄程度的关系。
目的:研究CPE第5外显子区801C＞T和847C＞T基因多态性与胰岛素原水平及冠状动脉狭窄程度的关系。
方法:选择行冠状动脉造影检查的1044例(男性775人,女性267人)疑诊与确诊冠心病患者作为研究对象;对研究对象进行体格检查以及胰岛素原等血生化指标的检测;采用Gensini评分系统对冠状动脉造影特征进行系统的定量评价;采用聚合酶链反应-测序分型法检测2个位点的基因型;采用单因素和多因素有序分类变量Logistic回归模型计算各不同基因型人群其冠状动脉狭窄程度增高的风险(采用OR及其95%CI表示)。
结果:
1.研究对象的—般情况、生化指标及各位点等位基因在不同胰岛素原水平组间的比较
研究对象的BMI(P＜0.001)、高密度脂蛋白胆固醇(P＜0.001)、低密度脂蛋白胆固醇(P=0.050)、甘油三酯(P＜0.001)、空腹血糖(P＜0.001)、胰岛素(P=0.005)、HOMA胰岛素抵抗指数(P＜0.001)与Gensini评分(P＜0.001)以及性别(P=0.050)、饮酒(P=0.009)构成在不同胰岛素原浓度组间(四分位间距分组)的分布均存在统计学差异。CPE exon5 801C＞T等位基因频率在各组中的分布无显著性差异(P=0.760),CPE exon5 847C＞T位点没有发现变异基因型。
2.胰岛素原水平等相关指标在CPE exon5 801C＞T不同基因型组中的比较
以年龄、性别与BMI为协变量的协方差分析结果显示,空腹血糖(P=0.157)、胰岛素(P=0.539)、HOMA胰岛素抵抗指数(P=0.608)与胰岛素原水平(P=0.975)在CPE exon5 801C＞T不同基因型组间的差异均无统计学意义。
3.CPE exon5 801C＞T基因多态性与胰岛素原关系的Logistic回归分析
CPE exon5 801C＞T的三种基因型频率在不同胰岛素原水平组间(四分位间距分组)的分布不存在显著性差异(χ~2=2.877,P=0.824)。以胰岛素原水平(四分位间距)分组变量为有序分类应变量的单因素Ordinal Logistic回归分析与调整了年龄、性别、BMI、血糖及吸烟等因素的多因素Logistic回归分析结果均显示,CPE exon5 801C＞T位点的基因多态性与胰岛素原水平可能不存在关联。
4.CPE exon5 801C＞T各基因型与冠状动脉狭窄程度关系的Logistic回归分析
CPE exon5 801C＞T的三种基因型频率在不同Gensini评分组间(四分位间距分组)的分布存在显著性差异(χ~2=13.745,P=0.033);CPE exon5 847C＞T位点没有发现变异基因型。以Gensini评分(四分位间距)分组变量作为有序分类应变量的多因素Ordinal Logistic回归分析结果表明,调整了年龄、性别、BMI、血糖及吸烟等因素后,携带801TT的个体与携带801CC者比较,其冠状动脉狭窄程度增高的风险增加了2.13倍(校正OR=3.13,95%CI=1.18-8.28);将两个变异的基因型联合分析表明,携带801CT+TT的个体其冠状动脉狭窄程度增高的风险则增加了30%(校正OR=1.30,95%CI=0.99-1.70),亦接近统计学显著性水平。
5.CPE exon5 801C＞T基因多态性与冠状动脉狭窄程度关系的分层分析
携带CPE exon5 801C＞T变异基因型的个体与携带CPE exon5801CC基因型者相比,其冠状动脉狭窄程度增高的风险在年龄≥60岁的个体(801TT基因型的校正OR=3.86,95%CI=1.07-13.90)、男性个体(801TT基因型的校正OR=3.43,95%CI=1.20-9.87;801CT+TT基因型的校正OR=1.37,95%CI=1.00-1.87)以及吸烟个体(801 CT基因型的校正OR=1.69,95%CI=1.12-2.56;801TT基因型的校正OR=5.73,95%CI=1.56-21.12;801CT+TT基因型的校正OR=1.85,95%CI=1.24-2.76)中增加得更为显著。
结论:中国汉族人群CPE基因第5外显子区的801C＞T多态性可能与冠状动脉狭窄程度有关联,有待于进一步的功能性研究及不同种族人群研究的验证。
PartⅠAn epidemiological study on the relationship between proinsulin level and angiographical characteristics of coronary atherosclerosis
Proinsulin,the propeptide of insulin and C-peptide,is increased in patients with impaired glucose tolerance,type 2 diabetes,obesity,or coronary heart disease.As a matter of fact,an increase in proinsulin level is probably due to a processing failure of the insulin peptide within the beta cell allowing disproportionately high amounts of proinsulin to enter the blood stream.In addition,in contrast to specific insulin,proinsulin level was recently shown to be an independent risk factor of coronary heart disease in the cross-sectional and prospective studies.However, there were no association between the proinsulin level and coronary heart disease independent of diabetes status in other epidemiological studies. Thus,the evidence linking proinsulin level with coronary heart disease is not entirely clear at present.In the present cross-sectional study,we evaluated the association between the angiographical characteristics of coronary atherosclerosis and proinsulin level measured by highly sensitive and specific 2-monoclonal antibody-based sandwich enzyme immunoassay in Chinese.
Objective:To study the associations between proinsulin level,relating environmental factors and angiographical characteristics of coronary atherosclerosis defined by Gensini's score(GS)system in Chinese.
Methods:In this hospital-based study,the study population consisted of 1044 consecutive patients(777 males and 267 females)who underwent coronary angiography for suspected or known coronary atherosclerosis. The patients' anthropometric and plasma measurements including body mass index,blood pressure,blood lipid,fasting blood glucose,and proinsulin level were performed.The angiographical characteristics of coronary atherosclerosis(i.e.the severity of coronary heart disease)were defined by the GS system.Analysis of covariance,partial correlation, multiple linear regression,univariate and multivariate ordinal logistic regression were used to discover the relationship between proinsulin level and the severity of coronary atherosclerosis.
Results:
1.Anthropometric and biochemical characteristics in patients grouped according to the quartile values of GS
The high-density lipoprotein cholesterol(HDL-C,P＜0.001), low-density lipoprotein cholesterol(LDL-C,P=0.042),fasting blood glucose(FBG,P＜0.001),insulin(P=0.005),HOMA insulin resistance index(HOMA IRI,P＜0.001),and proinsulin(P＜0.001)levels showed statistically significant differences among the patients of the 4 groups who were classified by quartile values of GS.Compared with the patients with a lower GS,the patients with a higher GS were older(P＜0.001)and more likely to be smokers(P＜0.001).Also,being male appeared to be a risk factor(P＜0.001).Furthermore,there were significant differences between the 4 groups in the levels of total cholesterol(TCH,P=0.041), LDL-C(P=0.001),triglyceride(TG,P=0.015),FBG(P＜0.001),insulin (P=0.024),HOMA IRI(P=0.001),and proinsulin(P＜0.001)by an analysis of variance covariance controlling for age,sex,and body mass index(BMI).
2.Partial correlations between proinsulin level,biochemical characteristics and GS(as dependent variable)in patients
A partial correlation analysis controlling for age,sex,and BMI indicated that the levels of proinsulin(P＜0.001),insulin(P=0.011),FBG (P＜0.001),HOME IRI(P=0.001),TCH(P=0.028),and LDL-C(P=0.001) were significantly positively correlated with the GS.
3.Multiple linear regression analysis with Gensini's score as dependent variable
Multiple linear regression analysis indicated that the proinsulin (P=0.002),HDL-C(P=0.024),LDL-C(P=0.040),FBG(P＜0.001)levels, and age(P＜0.001)were significantly independently associated with the GS.
4.Logistic regression analysis for the association between proinsulin level and angiographical characteristics of coronary atherosclerosis
All patients were classified according to the quartile values of proinsulin level in this analysis.Patients in quartileⅠ,Ⅱ,Ⅲ,andⅣ(groups 1,2,3,and 4,respectively)had an increasingly high proinsulin concentration.The univariate ordinal logistic regression analysis with quartile values of GS as dependent variable revealed that the concentration of proinsulin associated with significantly increased risk of angiographical characteristics of coronary atherosclerosis[OR=1.00 (95%CI=0.74-1.37)for Group 2,1.31(95%CI=0.96-1.78)for Group 3 and 2.23(95%CI=1.63-3.04)for Group4,respectively,compared with Group 1].And the multivariate ordinal logistic regression indicated that compared with Group 1,elevated risk for the angiographical characteristics of coronary atherosclerosis was associated with Group 4 (adjusted OR=1.61,95%CI=1.12-2.30)after adjustment for age,sex,BMI, fasting blood glucose,smoking status,and other confounding factors.
Conclusion:Proinsulin level may be significantly positively associated with the severity of coronary atherosclerosis,as measured by Gensini's score.And the exact biological mechanisms need further study in different ethnic populations.
PartⅡMolecular scanning of the human carboxypeptidase E gene for mutations in Chinese subjects with coronary atherosclerosis
Carboxypeptidase E(CPE)is the major carboxypeptidase in proinsulin processing,and suggesting that functional DNA variants of CPE might cause type 2 diabetes mellitus(T2DM)as a result of deficient proinsulin processing.Recent studies suggest that mutations in CPE might be responsible for the hyperproinsulinemia,insulin deficiency and no-insulin-dependent diabetes mellitus(NIDDM).A mutation in CPE, Ser202Pro,is responsible for the phenotype of the fat/fat mouse,which is characterized by marked hyperproinsulinemia and develops late-onset obesity and diabetes.In addition,high proinsulin level was recently shown to be an independent risk factor of the severity of coronary atherosclerosis in the above-mentioned study.However,the evidence linking the mutations in CPE gene,hyperproinsulinaemia with cardiovascular disease is not clearly at present.Thus,we identified the mutations of the human CPE gene by polymerase chain reaction-sequence based typing(PCR-SBT)in Chinese subjects with coronary atherosclerosis.
Objective:To test the hypothesis that the identification of mutation in the CPE gene which leads to marked hyperproinsulinaemia is consistent with a possible role for mutations in CPE in the development of coronary heart disease.
Methods:The study subjects consisted of 51 consecutive patients(34 males and 17 females)who will coronary angiography for suspected or known coronary atherosclerosis.Coronary heart disease(CHD)was defined as having a luminal diameter stenosis≥50%in at least one of three major coronary arteries by coronary angiography or based on the Rose Questionnaire.Screening for mutations of all promoters and exons of the CPE gene was performed by polymerase chain reaction followed by bidirectional sequencing.
Results:
1.Clinical and biochemical characteristics in subjects grouped according to coronary heart disease status
The levels of systolic blood pressure(SBP,P=0.015),diastolic blood pressure(DBP,P=0.026),HDL-C(P=0.013),and insulin(P=0.035) differed significantly between the coronary heart disease cases and controls.
2.Sequencing and characterization of the human CPE gene
The results of sequencing and basic local alignment search tool (BLAST)analysis indicated that the sequence of the human CPE gene which has been deposited in the GenBank data base with accession numbers AB006890-AB006898 maybe inconsistent with the sequence of CPE gene in our present study.
3.Identification and distribution of CPE polymorphisms
We scanned eight exons and exon-intron junctional region.Overall, we found 15 genetic variants in exons 3,4,5,and intron regions.Among them,the polymorphisms including T1951C,T1969A,T1951G,T1977C in intron 2,T2681C in intron 3,T4367C,A4350T,A4309T,T4587A in intron7 are rare.Three variants were presented in the coding region: G2294T in exon 3,G2855A in exon 4,C3164T in exon 5.Among the above three variants,the polymorphisms of G2294T and G2855A have not been reported previously,the frequency of G2294T mutant is rare. The other three polymorphisms including A2925G in intron 4,T3609C in intron 5,and A4545G in intron 7 are not rare.
4.Association of CPE polymorphisms with the characteristics of patients
The further explored study revealed that the above five non-rare variants would not affect the levels of glucose,insulin,and proinsulin.
However,the A4545G genetype frequencies were 60.53%(AA),39.47% (AG+GG)in the cases and 23.08%(AA),76.92%(AG+GG)in the controls,and the difference was statistically significant(χ~2=5.436, P=0.020).
Conclusion:In the present study,the five non-rare mutations identified in the CPE gene would not affect the levels of glucose,insulin,and proinsulin.The hypothesis of a possible role for mutations in CPE in the development of coronary heart disease needs further epidemiological and functional studies.
PartⅢAssociations of human carboxypeptidase E exon5 gene polymorphisms with proinsulin level and angiographical characteristics of coronary atherosclerosis in a Chinese population
The above-mentioned study and many prospective studies indicated that high proinsulin level was an independent risk factor of the severity of coronary atherosclerosis.CPE is the major carboxypeptidase in proinsulin processing,and suggesting that functional DNA variants of CPE might cause T2DM as a result of deficient proinsulin processing.A single amino acid change resulted in significantly reduced level of CPE enzyme activity and elevated level of proinsulin.As a result,the identification of a mutation in the CPE gene of the fat/fat mouse,which led to marked hyperproinsulinemia,late-onset obesity,and diabetes,was consistent with a possible role in mutations in CPE during the development of diabetes and obesity in humans.Chen et al identified two novel single nucleotide polymorphisms(SNPs)in the coding region of CPE exon 5(R283W,c. 847C＞T and H267H,c.801C＞T)in a collection of Ashkenazi T2DM families and suggested that the 847C＞T mutant could cause hyperproinsulinism and diabetes in the homozygous state by altering enzymatic properties.In addition,the frequency of 801C＞T mutant is not rare in the study of part 2.Thus,we selected the two SNPs and explored the association between the 2 polymorphisms of the human CPE gene exon5 and proinsulin level,angiographical characteristics of coronary atherosclerosis.
Objective:The aim of this study is to test the hypothesis that 801C＞T and 847C＞T polymorphisms of the human CPE gene exon5 which could cause hyperproinsulinemia are associated with the proinsulin level and the angiographical characteristics of coronary atherosclerosis.
Methods:In total,1044 consecutive patients who underwent coronary angiography for suspected or known coronary atherosclerosis were examined with respect to their genotypes,insulin,proinsulin levels,and other risk factors of coronary atherosclerosis.The angiographical characteristics of coronary atherosclerosis(i.e.the severity of coronary heart disease)were measured by GS system.Associations between genotypes and proinsulin level,angiographical characteristics of coronary atherosclerosis risk(OR and 95%CI)were estimated by univariate and multivariate ordinal logistic regression analyses.
Results:
1.Distribution of selected variables and CPE variant alleles in patients grouped according to quartile values of proinsulin level
The frequency distribution of sex(P=0.050)and alcohol consumption differed significantly among the patients of the 4 groups who were classified by quartile values of proinsulin concentration. Moreover,the BMI(P＜0.001),HDL-C(P＜0.001),LDL-C(P=0.050),TG (P＜0.001),FBG(P＜0.001),insulin(P=0.005),HOMA IRI(P＜0.001) levels,and GS showed statistically significant differences among the 4 groups.However,the frequency of the CPE exon5 801T allele was 0.137, 0.115,0.123,and 0.127,respectively,in the 4 groups,and the differences were not statistically significant(P=0.760).And the frequency of the 847T allele was 0 for all of the patients.
2.Effect of CPE exon5 801C＞T genotypes on fasting blood glucose, insulin,proinsulin concentrations,and insulin resistance
Differences between genotype groups were assessed by an analysis of covariance controlling for age,sex,and BMI.The CPE exon5 801C＞T polymorphism was not significantly associated with FBG(P=0.157), insulin(P=0.539),HOMA IRI(P=0.608),and proinsulin(P=0.975) levels.
3.Logistic regression analysis for the associations between variant genotypes of CPE exon5 801C＞T and proinsulin level
The frequency distribution of the CPE exon5 801C＞T showed no statistically significant differences among the patients of the 4 groups who were classified by quartile values of proinsulin concentration (χ~2=2.877,P=0.824).And there were no associations between the CPE exon5 801C＞T genotypes and hyperproinsulinemia risk in the Chinese by univariate and multivariate ordinal logistic regression analysis.
4.Genotype distribution of the human CPE gene exon5 polymorphisms in grouped patients and their associations with angiographical characteristics of coronary atherosclerosis risk
The results showed that the genotype frequencies of CC,CT,and TT at 801C＞T locus were significantly different among the patients of the 4 groups who were classified by quartile values of GS(χ~2=13.745, P=0.033).However,we did not find any mutations at 847C＞T locus in this study.The ordinal logistic regression analysis with adjustment for age,sex,BMI,fasting blood glucose,smoking status,and other confounding factors revealed that compared with the 801CC genotype, elevated risks for the angiographical characteristics of coronary atherosclerosis were associated with 801CT(adjusted OR=1.23,95% CI=0.93-1.63),801TT(3.13,1.18-8.28),and their combined genotypes 801CT+TT(1.30,0.99-1.70).
5.Stratification analysis of the association between the CPE exon5 polymorphism and the angiographicai characteristics of coronary atherosclerosis risk
The association of the CPE exon5 801C＞T variant genotypes with the angiographical characteristics of coronary atherosclerosis risk was further stratified by selected variables.A significantly increased risk of the severity of coronary atherosclerosis associated with the CPE exon5 801CT/TT genotype was more evident among older people(≥60 years, adjusted OR=3.86,95%CI=1.07-13.90 for 801TT),males(adjusted OR=3.43,95%CI=1.20-9.87 for 801TT;adjusted OR=1.37,95% CI=1.00-1.87 for 801CT+TT),and smokers(adjusted OR=1.69,95% CI=1.12-2.56 for 801CT;adjusted OR=5.73,95%CI=1.56-21.12 for 801TT;adjusted OR=1.85,95%CI=1.24-2.76 for 801CT+TT),compared with the 801CC genotype.
Conclusion:These findings indicated that the 801C＞T polymorphism in the CPE exon5 gene may contribute to the etiology of angiographical characteristics of coronary atherosclerosis in the Chinese population. More functional data for this specific association are needed to illustrate this biological mechanism in different ethnic populations.
冠心病发病危险因素错综复杂,近年来胰岛素的前体物质-胰岛素原在冠心病中的作用渐受重视。国外群体研究结果认为,胰岛素原与胰岛素抵抗综合征的关系比胰岛素更密切,同时它比真胰岛素与冠心病的关系也更密切,因此胰岛素原在冠心病发生和发展中的重要性更大。正常生理情况下,只有少量的胰岛素原进入外周循环,但是在糖尿病、肥胖、冠心病等情况下胰岛素原呈现与胰岛素不呈比例的高分泌状态,外周循环中高胰岛素原血症被认为是胰岛β细胞功能缺陷的指标。横断面与前瞻性的流行病学研究显示,高胰岛素原水平为冠心病的独立危险因素。胰岛β细胞内,胰岛素原被酶解为胰岛素与C肽的过程中,激素原转化酶2、激素原转化酶3与羧肽酶E(carboxypeptidase E,CPE)被认为是三个关键性的酶。近年来,国际上研究热点集中在CPE基因突变与高胰岛素原水平关系上。CPE基因突变的老鼠发展成为较为显著的高胰岛素原血症与在人类CPE基因突变可导致高胰岛素原血症的结论相吻合。国外有研究表明,CPE基因突变可导致外周血中高胰岛素原血症。而前述有研究表明高胰岛素原血症为冠心病的独立危险因素。因此我们设想,羧肽酶E基因突变→高胰岛素原血症→冠心病这一假说是否存在?CPE基因突变是否为冠心病的分子生物学机制之一?高胰岛素原水平、胰岛素原代谢过程中的关键酶CPE基因突变与冠状动脉狭窄程度的关系如何?基因与高胰岛素原水平等之间是否存在交互作用?国际上仅有的几项关于CPE基因突变的研究多集中在糖尿病与肥胖的分子生物学机制,迄今未见有关CPE基因多态性与冠心病/冠状动脉硬化特征关系的研究。
为验证以上假设,本研究以医院为研究现场,采用国际上公认的Gensini评分(Gensini's score,GS)系统对冠状动脉造影特征进行系统的定量评价,并应用大样本的分子流行病学研究探讨胰岛素原水平及相关环境因素在冠状动脉粥样硬化发生发展中的作用;探讨胰岛素原代谢过程中羧肽酶E基因多态性与胰岛素原水平的关系,以及CPE基因多态性与高胰岛素原水平的交互作用及其与冠状动脉硬化程度的关系,对于进一步阐明冠状动脉粥样硬化发生的分子遗传学机制及可能存在的CPE基因-环境交互作用等具有重要的理论意义。此外,还可发现与我国人群冠心病相关的易感性基因型,并将其作为分子标志物用于筛选高危人群或易感个体,对冠心病的早期诊断与治疗,以及实施有效和目标明确的个体预防与干预等均具有重要的现实意义。
第一部分胰岛素原水平及相关环境因素与冠状动脉狭窄程度关系的流行病学研究
胰岛素原为胰岛素的前体物质,在糖耐量低减、糖尿病、肥胖、冠心病等情况下胰岛素原呈现与胰岛素不呈比例的高分泌状态。国际上横断面与前瞻性的流行病学研究显示,胰岛素原的浓度而非真胰岛素或免疫反应性胰岛素浓度是冠心病的独立危险因素。然而,也有研究报道胰岛素原水平独立于糖尿病状态而与冠心病没有关联。因此胰岛素原水平与冠心病之间的关系目前仍不太明确。本研究采用国际上认可的灵敏度好、特异性强的双单克隆抗体酶免疫方法测定研究对象外周循环中的真胰岛素、胰岛素原水平,以探讨中国人群胰岛素原水平及相关环境因素与冠状动脉狭窄程度的关系。
目的:研究胰岛素原水平及相关环境因素与冠状动脉狭窄程度的关系。
方法:选择行冠状动脉造影检查的1044例(男性777人,女性267人)疑诊与确诊冠心病患者作为研究对象;对研究对象进行体格检查及胰岛素原等血生化指标的检测;采用Gensini评分系统对冠状动脉造影特征进行系统的定量评价;采用协方差、偏相关、多元线性回归、单因素和多因素有序分类变量Logistic回归分析等方法评价胰岛素原水平与冠状动脉狭窄程度的相关性。
结果:
1.研究对象的—般情况与生化指标的比较
以Gensini评分四分位间距作为分组变量,研究对象的平均年龄(P＜0.001)、高密度脂蛋白胆固醇(P＜0.001)、低密度脂蛋白胆固醇(P=0.042)、空腹血糖(P＜0.001)、胰岛素(P=0.005)、HOMA胰岛素抵抗指数(P＜0.001)、胰岛素原水平(P＜0.001)以及性别(P＜0.001)、吸烟(P＜0.001)构成在各组间的分布均存在显著性差异。控制了年龄、性别与BMI的协方差分析结果显示,总胆固醇(P=0.041)、低密度脂蛋白胆固醇(P=0.001)、甘油三酯(P=0.015)、空腹血糖(P＜0.001)、胰岛素(P=0.024)、HOMA胰岛素抵抗指数(P=0.001)与胰岛素原水平(P＜0.001)等在各组间的差异也均具有统计学意义。
2.胰岛素原水平及各测量指标与Gensini评分的偏相关分析
以年龄、性别与BMI为控制变量,以Gensini评分为应变量的偏相关分析结果显示,胰岛素原(P＜0.001)、胰岛素(P=0.011)、空腹血糖(P＜0.001)、HOMA胰岛素抵抗指数(P=0.001)、总胆固醇(P=0.028)及低密度脂蛋白胆固醇水平(P=0.001)均与Gensini评分呈显著的正相关。
3.胰岛素原水平及各测量指标与Gensini评分的多元线性回归分析
以Gensini评分为应变量的多元线性回归分析结果显示,胰岛素原(P=0.002)、高密度脂蛋白胆固醇(P=0.024)、低密度脂蛋白胆固醇(P=0.040)、空腹血糖(P＜0.001)水平以及年龄(P＜0.001)均与Gensini评分呈独立的显著正相关;同时,男性(P＜0.001)与吸烟(P=0.002)均是冠状动脉狭窄增高的独立的危险因素。
4.胰岛素原水平与冠状动脉狭窄程度关系的Logistic回归分析
以Gensini评分(四分位间距)分组变量作为有序分类应变量,胰岛素原浓度(按照其四分位间距界值将研究对象分为Group1、2、3和4组)分组变量作为自变量的单因素Ordinal Logistic回归分析结果显示,胰岛素原的水平每增加一个等级(25百分位数),冠状动脉狭窄程度增高的OR值亦增加(Group2 vs Groupl:OR=1.00,95%CI=0.74-1.37);Group3 vs Groupl:OR=1.31,95%CI=0.96-1.78;Group4vs Group1:OR=2.23,95%CI=1.63-3.04)。多因素Logistic回归分析结果显示,调整了年龄、性别、BMI、血糖及吸烟等因素后,与胰岛素原水平较低的第一组研究对象相比,胰岛素原浓度最高的第四组研究对象的冠状动脉狭窄程度增高的风险显著增加了61%(校正OR=1.61,95%CI=1.12-2.30)。
结论:高胰岛素原水平可能与冠状动脉狭窄程度增高有显著关联,确切的生物学机制有待于进一步的研究及不同种族人群的验证。
第二部分冠状动脉粥样硬化症人群中羧肽酶E基因突变的分子扫描与筛查研究
胰岛素原被酶解为胰岛素和C肽的过程中,羧肽酶E被认为是一个关键性的酶。有研究表明,CPE基因突变的老鼠发展成为较为显著的高胰岛素原血症与人类CPE基因突变可导致高胰岛素原血症的结论相吻合。国际上大量研究和本文第一部分的研究结果均表明,高胰岛素原血症为冠心病/冠状动脉硬化的独立危险因素。但未见有关该基因多态性与冠心病/冠状动脉硬化特征关系的研究报道。本研究采用目前最理想的基因检测分型技术即聚合酶链反应-测序分型法(PCR-SBT)对冠状动脉硬化症人群的CPE基因进行全面扫描与初步分析,为进一步探讨CPE基因多态性与冠状动脉硬化特征的关系奠定基础。
目的:初步探讨引起高胰岛素原血症的CPE基因的突变及其与冠心病的关系。
方法:随机选择51例(男性34人,女性17人)疑诊与确诊冠心病患者作为研究对象,并根据冠状动脉造影检查与调查问卷结果将研究对象分为冠心病组与对照组;采用聚合酶链反应.测序分型法与序列比对分析法对研究对象的CPE基因进行全面扫描与鉴定,初步分析各SNPs位点的分布及其与冠心病等临床特征的关系。
结果:
1.研究对象的—般情况与生化指标的比较
研究对象的收缩压(P=0.015)、舒张压(P=0.026)、高密度脂蛋白胆固醇(P=0.013)和胰岛素水平(P=0.035)在冠心病病例组与对照组中的分布均有显著性差异。
2.CPE基因的测序及其比对分析
GenBank数据库公布的CPE基因(accession numbers.AB006890-AB006898)第2、4、7、9内含子和第7外显子中的某些序列与本研究结果可能存在不一致的地方。
3.CPE基因多态性位点的鉴定与分布情况
通过对研究对象CPE基因8个外显子及其外显子-内含子连接处区域的测序与鉴定分析,在其第3、4、5外显子和相应内含子区域共发现了15个突变位点。其中,位于非编码区的T1951C、T1969A、T1951G、T1977C(第2内含子区),T2681C(第3内含子区)以及T4367C、A4350T、A4309T、T4587A(第7内含子区)共9个位点的突变频率极低;A2925G(第4内含子区)、T3609C(第5内含子区)和A4545G(第7内含子区)位点的突变频率均较高。另外,在编码区发现了三个点突变位点:第3外显子区的G2294T、第4外显子区的G2855A与第5外显子区的C3164T突变均为同义突变,不引起编码氨基酸的改变,其中G2294T和G2855A位点先前没有被报道过,G2294T位点的突变频率很低。
4.CPE基因多态性与研究对象临床特征的关系
对5个突变频率较高的位点的进一步分析表明,这些多态性均不显著影响血糖、胰岛素与胰岛素原的水平,但A4545G位点的变异型基因频率在冠心病病例组与对照组中的分布有显著性差异(χ~2=5.436,P=0.020)。
结论:本研究人群中共发现5个突变频率较高的多态性位点,这些多态性位点可能均不影响胰岛素原、胰岛素及血糖水平,CPE基因多态性在冠心病发生发展中的作用需要大规模的流行病学和功能学研究进一步验证。
第三部分羧肽酶E第五外显子区基因多态性与胰岛素原水平、冠状动脉狭窄程度关系的研究
国际上和本文第一部分的研究结果表明,高胰岛素原血症为冠心病/冠状动脉硬化的独立危险因素。胰岛素原被酶解的过程中,关键性的酶CPE基因的功能性位点如果发生改变,将可能影响胰岛素的生物合成过程。国外有研究表明CPE基因突变的肥胖老鼠未来可能发展成为较为显著的高胰岛素原血症和晚期肥胖与糖尿病,这与人类CPE基因突变在糖尿病与肥胖发生发展中的作用研究结论相吻合。Chen等在德系犹太人2型糖尿病家系人群CPE基因第5外显子编码区发现两个显著的突变位点(R283W,c.847C＞T和H267H,c.801C＞T),同时本课题第二部分的全基因扫描结果也表明801C＞T位点的突变频率比较高。因此,本研究挑选了以上2个单核苷酸多态性位点,探讨CPE相关基因多态性与胰岛素原水平及冠状动脉狭窄程度的关系。
目的:研究CPE第5外显子区801C＞T和847C＞T基因多态性与胰岛素原水平及冠状动脉狭窄程度的关系。
方法:选择行冠状动脉造影检查的1044例(男性775人,女性267人)疑诊与确诊冠心病患者作为研究对象;对研究对象进行体格检查以及胰岛素原等血生化指标的检测;采用Gensini评分系统对冠状动脉造影特征进行系统的定量评价;采用聚合酶链反应-测序分型法检测2个位点的基因型;采用单因素和多因素有序分类变量Logistic回归模型计算各不同基因型人群其冠状动脉狭窄程度增高的风险(采用OR及其95%CI表示)。
结果:
1.研究对象的—般情况、生化指标及各位点等位基因在不同胰岛素原水平组间的比较
研究对象的BMI(P＜0.001)、高密度脂蛋白胆固醇(P＜0.001)、低密度脂蛋白胆固醇(P=0.050)、甘油三酯(P＜0.001)、空腹血糖(P＜0.001)、胰岛素(P=0.005)、HOMA胰岛素抵抗指数(P＜0.001)与Gensini评分(P＜0.001)以及性别(P=0.050)、饮酒(P=0.009)构成在不同胰岛素原浓度组间(四分位间距分组)的分布均存在统计学差异。CPE exon5 801C＞T等位基因频率在各组中的分布无显著性差异(P=0.760),CPE exon5 847C＞T位点没有发现变异基因型。
2.胰岛素原水平等相关指标在CPE exon5 801C＞T不同基因型组中的比较
以年龄、性别与BMI为协变量的协方差分析结果显示,空腹血糖(P=0.157)、胰岛素(P=0.539)、HOMA胰岛素抵抗指数(P=0.608)与胰岛素原水平(P=0.975)在CPE exon5 801C＞T不同基因型组间的差异均无统计学意义。
3.CPE exon5 801C＞T基因多态性与胰岛素原关系的Logistic回归分析
CPE exon5 801C＞T的三种基因型频率在不同胰岛素原水平组间(四分位间距分组)的分布不存在显著性差异(χ~2=2.877,P=0.824)。以胰岛素原水平(四分位间距)分组变量为有序分类应变量的单因素Ordinal Logistic回归分析与调整了年龄、性别、BMI、血糖及吸烟等因素的多因素Logistic回归分析结果均显示,CPE exon5 801C＞T位点的基因多态性与胰岛素原水平可能不存在关联。
4.CPE exon5 801C＞T各基因型与冠状动脉狭窄程度关系的Logistic回归分析
CPE exon5 801C＞T的三种基因型频率在不同Gensini评分组间(四分位间距分组)的分布存在显著性差异(χ~2=13.745,P=0.033);CPE exon5 847C＞T位点没有发现变异基因型。以Gensini评分(四分位间距)分组变量作为有序分类应变量的多因素Ordinal Logistic回归分析结果表明,调整了年龄、性别、BMI、血糖及吸烟等因素后,携带801TT的个体与携带801CC者比较,其冠状动脉狭窄程度增高的风险增加了2.13倍(校正OR=3.13,95%CI=1.18-8.28);将两个变异的基因型联合分析表明,携带801CT+TT的个体其冠状动脉狭窄程度增高的风险则增加了30%(校正OR=1.30,95%CI=0.99-1.70),亦接近统计学显著性水平。
5.CPE exon5 801C＞T基因多态性与冠状动脉狭窄程度关系的分层分析
携带CPE exon5 801C＞T变异基因型的个体与携带CPE exon5801CC基因型者相比,其冠状动脉狭窄程度增高的风险在年龄≥60岁的个体(801TT基因型的校正OR=3.86,95%CI=1.07-13.90)、男性个体(801TT基因型的校正OR=3.43,95%CI=1.20-9.87;801CT+TT基因型的校正OR=1.37,95%CI=1.00-1.87)以及吸烟个体(801 CT基因型的校正OR=1.69,95%CI=1.12-2.56;801TT基因型的校正OR=5.73,95%CI=1.56-21.12;801CT+TT基因型的校正OR=1.85,95%CI=1.24-2.76)中增加得更为显著。
结论:中国汉族人群CPE基因第5外显子区的801C＞T多态性可能与冠状动脉狭窄程度有关联,有待于进一步的功能性研究及不同种族人群研究的验证。
PartⅠAn epidemiological study on the relationship between proinsulin level and angiographical characteristics of coronary atherosclerosis
Proinsulin,the propeptide of insulin and C-peptide,is increased in patients with impaired glucose tolerance,type 2 diabetes,obesity,or coronary heart disease.As a matter of fact,an increase in proinsulin level is probably due to a processing failure of the insulin peptide within the beta cell allowing disproportionately high amounts of proinsulin to enter the blood stream.In addition,in contrast to specific insulin,proinsulin level was recently shown to be an independent risk factor of coronary heart disease in the cross-sectional and prospective studies.However, there were no association between the proinsulin level and coronary heart disease independent of diabetes status in other epidemiological studies. Thus,the evidence linking proinsulin level with coronary heart disease is not entirely clear at present.In the present cross-sectional study,we evaluated the association between the angiographical characteristics of coronary atherosclerosis and proinsulin level measured by highly sensitive and specific 2-monoclonal antibody-based sandwich enzyme immunoassay in Chinese.
Objective:To study the associations between proinsulin level,relating environmental factors and angiographical characteristics of coronary atherosclerosis defined by Gensini's score(GS)system in Chinese.
Methods:In this hospital-based study,the study population consisted of 1044 consecutive patients(777 males and 267 females)who underwent coronary angiography for suspected or known coronary atherosclerosis. The patients' anthropometric and plasma measurements including body mass index,blood pressure,blood lipid,fasting blood glucose,and proinsulin level were performed.The angiographical characteristics of coronary atherosclerosis(i.e.the severity of coronary heart disease)were defined by the GS system.Analysis of covariance,partial correlation, multiple linear regression,univariate and multivariate ordinal logistic regression were used to discover the relationship between proinsulin level and the severity of coronary atherosclerosis.
Results:
1.Anthropometric and biochemical characteristics in patients grouped according to the quartile values of GS
The high-density lipoprotein cholesterol(HDL-C,P＜0.001), low-density lipoprotein cholesterol(LDL-C,P=0.042),fasting blood glucose(FBG,P＜0.001),insulin(P=0.005),HOMA insulin resistance index(HOMA IRI,P＜0.001),and proinsulin(P＜0.001)levels showed statistically significant differences among the patients of the 4 groups who were classified by quartile values of GS.Compared with the patients with a lower GS,the patients with a higher GS were older(P＜0.001)and more likely to be smokers(P＜0.001).Also,being male appeared to be a risk factor(P＜0.001).Furthermore,there were significant differences between the 4 groups in the levels of total cholesterol(TCH,P=0.041), LDL-C(P=0.001),triglyceride(TG,P=0.015),FBG(P＜0.001),insulin (P=0.024),HOMA IRI(P=0.001),and proinsulin(P＜0.001)by an analysis of variance covariance controlling for age,sex,and body mass index(BMI).
2.Partial correlations between proinsulin level,biochemical characteristics and GS(as dependent variable)in patients
A partial correlation analysis controlling for age,sex,and BMI indicated that the levels of proinsulin(P＜0.001),insulin(P=0.011),FBG (P＜0.001),HOME IRI(P=0.001),TCH(P=0.028),and LDL-C(P=0.001) were significantly positively correlated with the GS.
3.Multiple linear regression analysis with Gensini's score as dependent variable
Multiple linear regression analysis indicated that the proinsulin (P=0.002),HDL-C(P=0.024),LDL-C(P=0.040),FBG(P＜0.001)levels, and age(P＜0.001)were significantly independently associated with the GS.
4.Logistic regression analysis for the association between proinsulin level and angiographical characteristics of coronary atherosclerosis
All patients were classified according to the quartile values of proinsulin level in this analysis.Patients in quartileⅠ,Ⅱ,Ⅲ,andⅣ(groups 1,2,3,and 4,respectively)had an increasingly high proinsulin concentration.The univariate ordinal logistic regression analysis with quartile values of GS as dependent variable revealed that the concentration of proinsulin associated with significantly increased risk of angiographical characteristics of coronary atherosclerosis[OR=1.00 (95%CI=0.74-1.37)for Group 2,1.31(95%CI=0.96-1.78)for Group 3 and 2.23(95%CI=1.63-3.04)for Group4,respectively,compared with Group 1].And the multivariate ordinal logistic regression indicated that compared with Group 1,elevated risk for the angiographical characteristics of coronary atherosclerosis was associated with Group 4 (adjusted OR=1.61,95%CI=1.12-2.30)after adjustment for age,sex,BMI, fasting blood glucose,smoking status,and other confounding factors.
Conclusion:Proinsulin level may be significantly positively associated with the severity of coronary atherosclerosis,as measured by Gensini's score.And the exact biological mechanisms need further study in different ethnic populations.
PartⅡMolecular scanning of the human carboxypeptidase E gene for mutations in Chinese subjects with coronary atherosclerosis
Carboxypeptidase E(CPE)is the major carboxypeptidase in proinsulin processing,and suggesting that functional DNA variants of CPE might cause type 2 diabetes mellitus(T2DM)as a result of deficient proinsulin processing.Recent studies suggest that mutations in CPE might be responsible for the hyperproinsulinemia,insulin deficiency and no-insulin-dependent diabetes mellitus(NIDDM).A mutation in CPE, Ser202Pro,is responsible for the phenotype of the fat/fat mouse,which is characterized by marked hyperproinsulinemia and develops late-onset obesity and diabetes.In addition,high proinsulin level was recently shown to be an independent risk factor of the severity of coronary atherosclerosis in the above-mentioned study.However,the evidence linking the mutations in CPE gene,hyperproinsulinaemia with cardiovascular disease is not clearly at present.Thus,we identified the mutations of the human CPE gene by polymerase chain reaction-sequence based typing(PCR-SBT)in Chinese subjects with coronary atherosclerosis.
Objective:To test the hypothesis that the identification of mutation in the CPE gene which leads to marked hyperproinsulinaemia is consistent with a possible role for mutations in CPE in the development of coronary heart disease.
Methods:The study subjects consisted of 51 consecutive patients(34 males and 17 females)who will coronary angiography for suspected or known coronary atherosclerosis.Coronary heart disease(CHD)was defined as having a luminal diameter stenosis≥50%in at least one of three major coronary arteries by coronary angiography or based on the Rose Questionnaire.Screening for mutations of all promoters and exons of the CPE gene was performed by polymerase chain reaction followed by bidirectional sequencing.
Results:
1.Clinical and biochemical characteristics in subjects grouped according to coronary heart disease status
The levels of systolic blood pressure(SBP,P=0.015),diastolic blood pressure(DBP,P=0.026),HDL-C(P=0.013),and insulin(P=0.035) differed significantly between the coronary heart disease cases and controls.
2.Sequencing and characterization of the human CPE gene
The results of sequencing and basic local alignment search tool (BLAST)analysis indicated that the sequence of the human CPE gene which has been deposited in the GenBank data base with accession numbers AB006890-AB006898 maybe inconsistent with the sequence of CPE gene in our present study.
3.Identification and distribution of CPE polymorphisms
We scanned eight exons and exon-intron junctional region.Overall, we found 15 genetic variants in exons 3,4,5,and intron regions.Among them,the polymorphisms including T1951C,T1969A,T1951G,T1977C in intron 2,T2681C in intron 3,T4367C,A4350T,A4309T,T4587A in intron7 are rare.Three variants were presented in the coding region: G2294T in exon 3,G2855A in exon 4,C3164T in exon 5.Among the above three variants,the polymorphisms of G2294T and G2855A have not been reported previously,the frequency of G2294T mutant is rare. The other three polymorphisms including A2925G in intron 4,T3609C in intron 5,and A4545G in intron 7 are not rare.
4.Association of CPE polymorphisms with the characteristics of patients
The further explored study revealed that the above five non-rare variants would not affect the levels of glucose,insulin,and proinsulin.
However,the A4545G genetype frequencies were 60.53%(AA),39.47% (AG+GG)in the cases and 23.08%(AA),76.92%(AG+GG)in the controls,and the difference was statistically significant(χ~2=5.436, P=0.020).
Conclusion:In the present study,the five non-rare mutations identified in the CPE gene would not affect the levels of glucose,insulin,and proinsulin.The hypothesis of a possible role for mutations in CPE in the development of coronary heart disease needs further epidemiological and functional studies.
PartⅢAssociations of human carboxypeptidase E exon5 gene polymorphisms with proinsulin level and angiographical characteristics of coronary atherosclerosis in a Chinese population
The above-mentioned study and many prospective studies indicated that high proinsulin level was an independent risk factor of the severity of coronary atherosclerosis.CPE is the major carboxypeptidase in proinsulin processing,and suggesting that functional DNA variants of CPE might cause T2DM as a result of deficient proinsulin processing.A single amino acid change resulted in significantly reduced level of CPE enzyme activity and elevated level of proinsulin.As a result,the identification of a mutation in the CPE gene of the fat/fat mouse,which led to marked hyperproinsulinemia,late-onset obesity,and diabetes,was consistent with a possible role in mutations in CPE during the development of diabetes and obesity in humans.Chen et al identified two novel single nucleotide polymorphisms(SNPs)in the coding region of CPE exon 5(R283W,c. 847C＞T and H267H,c.801C＞T)in a collection of Ashkenazi T2DM families and suggested that the 847C＞T mutant could cause hyperproinsulinism and diabetes in the homozygous state by altering enzymatic properties.In addition,the frequency of 801C＞T mutant is not rare in the study of part 2.Thus,we selected the two SNPs and explored the association between the 2 polymorphisms of the human CPE gene exon5 and proinsulin level,angiographical characteristics of coronary atherosclerosis.
Objective:The aim of this study is to test the hypothesis that 801C＞T and 847C＞T polymorphisms of the human CPE gene exon5 which could cause hyperproinsulinemia are associated with the proinsulin level and the angiographical characteristics of coronary atherosclerosis.
Methods:In total,1044 consecutive patients who underwent coronary angiography for suspected or known coronary atherosclerosis were examined with respect to their genotypes,insulin,proinsulin levels,and other risk factors of coronary atherosclerosis.The angiographical characteristics of coronary atherosclerosis(i.e.the severity of coronary heart disease)were measured by GS system.Associations between genotypes and proinsulin level,angiographical characteristics of coronary atherosclerosis risk(OR and 95%CI)were estimated by univariate and multivariate ordinal logistic regression analyses.
Results:
1.Distribution of selected variables and CPE variant alleles in patients grouped according to quartile values of proinsulin level
The frequency distribution of sex(P=0.050)and alcohol consumption differed significantly among the patients of the 4 groups who were classified by quartile values of proinsulin concentration. Moreover,the BMI(P＜0.001),HDL-C(P＜0.001),LDL-C(P=0.050),TG (P＜0.001),FBG(P＜0.001),insulin(P=0.005),HOMA IRI(P＜0.001) levels,and GS showed statistically significant differences among the 4 groups.However,the frequency of the CPE exon5 801T allele was 0.137, 0.115,0.123,and 0.127,respectively,in the 4 groups,and the differences were not statistically significant(P=0.760).And the frequency of the 847T allele was 0 for all of the patients.
2.Effect of CPE exon5 801C＞T genotypes on fasting blood glucose, insulin,proinsulin concentrations,and insulin resistance
Differences between genotype groups were assessed by an analysis of covariance controlling for age,sex,and BMI.The CPE exon5 801C＞T polymorphism was not significantly associated with FBG(P=0.157), insulin(P=0.539),HOMA IRI(P=0.608),and proinsulin(P=0.975) levels.
3.Logistic regression analysis for the associations between variant genotypes of CPE exon5 801C＞T and proinsulin level
The frequency distribution of the CPE exon5 801C＞T showed no statistically significant differences among the patients of the 4 groups who were classified by quartile values of proinsulin concentration (χ~2=2.877,P=0.824).And there were no associations between the CPE exon5 801C＞T genotypes and hyperproinsulinemia risk in the Chinese by univariate and multivariate ordinal logistic regression analysis.
4.Genotype distribution of the human CPE gene exon5 polymorphisms in grouped patients and their associations with angiographical characteristics of coronary atherosclerosis risk
The results showed that the genotype frequencies of CC,CT,and TT at 801C＞T locus were significantly different among the patients of the 4 groups who were classified by quartile values of GS(χ~2=13.745, P=0.033).However,we did not find any mutations at 847C＞T locus in this study.The ordinal logistic regression analysis with adjustment for age,sex,BMI,fasting blood glucose,smoking status,and other confounding factors revealed that compared with the 801CC genotype, elevated risks for the angiographical characteristics of coronary atherosclerosis were associated with 801CT(adjusted OR=1.23,95% CI=0.93-1.63),801TT(3.13,1.18-8.28),and their combined genotypes 801CT+TT(1.30,0.99-1.70).
5.Stratification analysis of the association between the CPE exon5 polymorphism and the angiographicai characteristics of coronary atherosclerosis risk
The association of the CPE exon5 801C＞T variant genotypes with the angiographical characteristics of coronary atherosclerosis risk was further stratified by selected variables.A significantly increased risk of the severity of coronary atherosclerosis associated with the CPE exon5 801CT/TT genotype was more evident among older people(≥60 years, adjusted OR=3.86,95%CI=1.07-13.90 for 801TT),males(adjusted OR=3.43,95%CI=1.20-9.87 for 801TT;adjusted OR=1.37,95% CI=1.00-1.87 for 801CT+TT),and smokers(adjusted OR=1.69,95% CI=1.12-2.56 for 801CT;adjusted OR=5.73,95%CI=1.56-21.12 for 801TT;adjusted OR=1.85,95%CI=1.24-2.76 for 801CT+TT),compared with the 801CC genotype.
Conclusion:These findings indicated that the 801C＞T polymorphism in the CPE exon5 gene may contribute to the etiology of angiographical characteristics of coronary atherosclerosis in the Chinese population. More functional data for this specific association are needed to illustrate this biological mechanism in different ethnic populations.
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