Epidemiological Studies On The Associations Between Carboxypeptidase E Gene Polymorphisms, Proinsulin Level And Angiographical Characteristics Of Coronary Atherosclerosis In A Chinese Population

PartⅠAn epidemiological study on the relationship between proinsulin level and angiographical characteristics of coronary atherosclerosisProinsulin,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 GSThe 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 variableMultiple 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 atherosclerosisAll 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 atherosclerosisCarboxypeptidase 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 statusThe 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 geneThe 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 polymorphismsWe 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 patientsThe 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 populationThe 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 levelThe 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 resistanceDifferences 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 levelThe 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 riskThe 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 riskThe 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.