Association Of G82S Polymorphism In The RAGE Gene With Coronary Heart Disease In The Chinese Population

Background: A large body of evidence suggests that the nonezymatic reation between protein, lipids, ribonucleotide and aldoses produces unstable compound termed a Schiff base, which in turn undergoes rearrangement to form the more stable Amadori-type products-advanced glycation end products (AGEs). An emerging view is that a critical property of AGEs is their ability to bind and activate receptor for advanced glycation end products (RAGE), a signal transduction receptor of the immunoglobulin superfamily. And the engagement of RAGE by AGEs in multiple settings triggers rapid generation of reactive oxygen species(ROS), amplifies immune/inflammatory reaction, perturbs endothelium properties, upregulate irreversible formation of cross-linked collagen and induces cardiac myocytes apoptosis and the like, thereby imparting a potential impact in cardiovascular system. For example, our previous studies found that AGEs could significantly increase the number of apoptosis body/nucleus on CMs in a time- and dose-dependent manner, and by virtue of their engagement of RAGE increase the cytosolic free calcium concentration in cultured neonatal rat cardiac myocytes, all of which could be inhibited by antibody of RAGE. In addition, subsequent evidence has been presented that in human diabetic atherosclerotic plaques RAGE expression is highly upregulated. These considerations, together with our findings, highlight RAGE as the vital receptors linked to chronic cardiovascular perturbation, a key target for drug intervention studies and a candidate for genetic susceptibility in the development of cardiovascular disease. The gene encoding for RAGE is located on chromosome 6p21.3 in the major histocompatibility complex(MHC), and so far more than 30 polymorphisms of RAGE gene have been identified. Since Hudson et al. detected a functional amino acid change in 1998--a glycine-to-serine polymorphism at codon 82 in exon 3 of the RAGE gene--that leaded to the formation of an Alul restriction site(AG↓CT), experimental studies have elucidated that the 82S isoform displayed a higher affinity for RAGE ligands and led to increased ligand-stimulated activation of proinflammatory mediators in transfected cells and human monocytes compared to the common RAGE G isoform suggesting that S allelic variation in the RAGE gene could potentially enhance the development of coronary heart disease(CHD).Objective: This study aims to assess the distribution frequency of the AGE gene G82S polymorphism in a case-control study of 700 unrelated Chinese Han subjects participating in the Department of Cardiology of Nan Fang Hospital from July 2004 to December 2005 and investigate its association with the incidence of hypertension(HP), diabetes mellitus(DM) and CHD in a Chinese population. Meanwhile, in order to assess any possible characteristic/significance of 82S allelic variation in the RAGE gene, we compare its distribution frequency between different countries and ethnic groups, thereby providing powerful epidemiology data which elucidate the importance of SNPs such as G82S polymorphism in the pathogenesis of a complex polygenic disorder including cardiovascular disease and diabetes in different ethnic groups.Methods: The genomic DNA was extracted from peripheral venous blood leukocytes from all the subjects using standard extraction method. Exon 3 of RAGE gene was amplified by PCR and the polymerase chain reaction--restriction fragment length polymorphism (PCR-RFLP) was used for detection of genotype variants. Firstly, the allele frequencies and genotype distribution of the G82S polymorphism in the RAGE gene were compared in a case-control study of 700 subjects primarily divided into four groups--355 cases of HP(with presence/absence of DM or CHD), 170 cases of DM(with presence/absence of HP or CHD), 330 cases of CHD(with presence/absence of HP or DM) and 170 control subjects. Secondly, all the recruited subiects were then divided into four groups (200 cases of HP, 155 cases of CHD&HP, 175 cases of CHD, and 170 control subjects) to investigate the association of G82S polymorphism with the incidence of HP, CHD&HP and CHD in a Chinese population. Thirdly, to assess any possible influence of diabetes in the association of G82S genetic variant with the incidence of HP and CHD in a Chinese population, the 700 study subjects were further divided into seven groups (126 cases of HP, 74 cases of HP&DM, 59 cases of CHD&HP&DM, 37 cases of CHD&DM, 96 cases of CHD&HP, 138 cases of CHD and 170 cases of CTRL) based on the group division above. The allele frequencies and genotype distribution of the G82S polymorphism were analyzed by the same methods mentioned above. At last, according to a number of studies performed to assess the prevalence of the G82S polymorphism in vascular disease of both diabetes and non-diabetes, combinding with our current analysis, distribution frequency of the 82S allelic variation was compared between different countries and ethnic groups, thereby testing whether RAGE gene containing G82S polymorphism exert potent influences on cardiovascular disease progression and outcome in terms of ethnical diffenence in different populations.Results:1.Influence of RAGE gene G82S polymorphism to the susceptibility of HP, DM and CHD in a Chinese population. Significantly higher frequencies of mutation homozygote(SS) of G82S polymorphism were observed in CHD patients than in the control subjects( x~2=18.119, P=0.000, according to Bonferroni method significance level:α'=0. 017, P＜0.017). The 82S allele frequency of G82S polymorphism was much higher in CHD group( x~2= 16. 285, P=0.000, P＜0. 017, odd ratio 2.081, 95% CI 1. 450～2.985 compared with the control group, while analysis of the genotype distribution in subjects with prevlent HP or prevalent DM revealed no significant differencs compared to the control group (P＞0. 017). Stratification Chi-Square test showed that mutation genotype (GS&SS) was associated with male CHD patients.2.Influence of RAGE gene G82S polymorphism to the susceptibility of HP, CHD&HP and CHD in a Chinese population independent of type 2 diabetes.There were significantly higher frequencies of mutation homozygote(SS) of G82S polymorphism in CHD patients and CHD&HP patients than in the control subjects( x~2 19. 637, 9: 126 respectively, P 0.000, 0.010 respectively, according to Bonferroni method significance level:α'=0.017, P＜0.017). The 82S allele frequency of G82S polymorphism was much higher in CHD group (x~2=17.757 P=0.000, P＜0. 017, odd ratio 2.303, 95%CI 1.553～3.416) and CHD&HP group( x~2=8. 546, P=0.003, P＜0.017, odd ratio 1.842, 95%CI 1.219～2.785) compared with the control group, while no statistically significant difference of genotype or allele frequency distributions was observed in the HP group(P＞0.017). Stratification Chi-Square test showed that mutation genotype (GS&SS) was associated with male CHD patients and male CHD&HP patients aged≥60 years. Furthermore, Binary Logistic Regression indicated that mutation genotype(GS&SS) was identified as an important risk factor for CHD patients and marginally related to CHD&HP patients.3.Influence of RAGE gene G82S polymorphism to the susceptibility of HP, CHD&HP and CHD in a Chinese population linked to type 2 diabetes. Significantly higher frequencies of mutation homozygote(SS) of G82S polymorphism were observed in CHD patients than in the control subjects (x~2=20.716, P＜0.001, according to Bonferroni method significance level:α'=0. 008, P＜0. 008). The 82S allele frequency of G82S polymorphism was much higher in CHD group (x~2=18.869, P=0.000, P＜0. 008, odd ratio 2.446, 95%CI 1.622～3.689) and CHD&HP group( x~2=8.256, P=0.004, P＜0. 008, odd ratio 1.949, 95%CI 1.230～3.087) compared with the control group, while no statistically significant difference of genotype or allele frequency distributions was observed in the HP group, HP&DM group, CHD&HP&DM group and CHD&DM group (P＞0. 008). Stratification Chi-Square test showed that mutation genotype (GS&SS) was associated with male CHD patients. Furthermore, Binary Logistic Regression indicated that mutation genotype(GS&SS) was identified as an important risk factor for CHD patients and marginally related to CHD&HP and CHD&HP&DM patients.4.Comparison of distribution frequency of the S allelic variation between different countries and ethnic groups4.1 The mutation genotype(GS&SS) frequency and the S allele frequency of G82S polymorphism in Chinese population were lower than what Liu LM reported (P＜0.01), similar to those in Asian Japanase and Koreans(P＞0.05), while statistically higher than those in Caucasian-English population, American-Caucasian population, Finnish population, Brazilian population, Asian-Indian population and Cancasian-Czech population respectively (P＜0.01).4.2 The mutation genotype(GS&SS) frequency and the S allele frequency of G82S polymorphism in Chinese type 2 diabetic patients were similar to what Liu LM reported (P＞0.05), while statistically higher than those in Asian-Indian DM patients (P＜0.05), and statistically higher than those in Asian Japanase DM patients, Finnish DM patients, Cancasian-Czech DM patients, American-Caucasian DM patients, Caucasian-English DM patients and Brazilian patients with type 2 DM (P＜0.01).4.3 Statistically higher distribution frequencies of G82S polymorphism in Chinese CHD patients were observed compared to those in Asian Korean CHD patients, Finnish CHD patients and American-Caucasian CHD patients respectively (P＜0.001).Conclusions:1.A significant association of RAGE G82S polymorphism with increased incidence of coronary heart disease in a Chinese population highlights S variant of RAGE as an important risk factor for CHD.2.The influence of RAGE G82S genetic susceptibility to CHD is more obvious in Chinese male CHD individuals.3.RAGE Gly82Ser polymorphism did not demonstrate any association with the prevalence of hypertension or diabetes mellitus in a Chinese population.4.The disfribution frequencies of G82S polymorphism were statistically higher in Chinese population, type 2 diabetic Chinese patients and Chinese CHD patients than those in other countries and ethnic groups, which supporting the concept that individuals belonging to the same race shared a common biological inheritance.