Establishment Of Multiplexed SNPs Genotyping And Genes Expression Methods And Their Application In Predicting Genetic Susceptibility And Diagnoses To CAD

[Objective]Coronary artery disease(CAD) is the leading cause of morbidity and mortality in recent years in China.It is a multifactorial disease caused by various genetic and environmental factors involved in accumulation of atherosclerosis in the walls of the coronary arteries.In order to find single nucleotide polymorphisms(SNP) and genes that were associated with CAD, multiplexed SNPs genotyping and genes expression methods were established in the current work.With the multiplexed methods,lots of tag SNPs and genes in peripheral blood white cells from CAD patients and normal control individuals were investigated.[Methods](1) The method of multiplexed SNPs genotyping:DNA samples were extracted from peripheral blood white cells and 44 tag SNPs of 13 genes (ADD1,PECAM-1,CRP,ecNOS,PC-1,SELL,GNB3,ACE,AT1R,AGT, ICAM-1,MTHFR and HL) that were listed in the International Haplotype Map Project Database for CHB(Chinese Han nationality in Beijing) were selected in the current work.Three primers were designed for each SNP(two for polymerase chain reaction(PCR) and one for extension reaction).After PCR, extension and hybridization,genotypes of SNPs were investigated with the GenomeLab SNPstream genotyping system.(2) The method of multiplexed genes expression:RNA samples were extracted from peripheral blood white cells and two primers(one for reverse transcription and one for PCR) were designed for each of the 14 target genes(MTHFR,IL-8,ecNOS,TLR4,FOS, ID2,ADD1,ICAM-1,PECAM-1,CRP,LDLR,vWF,SELL and TNF-α) and 3 house keeping genes(β-actin,SRP14 and 18S-rRNA).After reverse transcription and PCR,expression levels of the 17 genes were detected with high-throughput GenomeLab GeXP Genetic Analysis System.(3) The levels of serum lipid,CRP,ACE and Hcy were detected with automatic biochemical analysis system.[Result](1) Thirty two SNPs were genotyped and the rate of succeed SNP genotyping was 72.7%(32/44).The average genotyped rate of the 32 SNPs was 98.3%in the test of 583 DNA samples.With the 32 plexes SNPs genotyping method,DNA samples of CAD patients and normal control individuals from the local ethnic Han Chinese population were investigated.The results showed that the genotype and/or allele frequencies of 6 tag SNPs in CRP,ecNOS,PC-1 and ACE genes were significantly different in both groups.According the linkage-disequilibrium levels of SNPs,the haplotypes were constructed and the results showed that the frequencies of the constructed haplotypes of ecNOS, PC-1 and ACE genes were associated with CAD.Genotpyes of CRP,ACE and MTHFR genes were associated with serum CRP,ACE and Hcy level.(2) Confirmed by single plex PCR and capillary electrophoresis,the size of each gene's PCR production was consistent with original designed size.The specific of each target gene was confirmed according the capillary electrophoresis size of each gene's PCR production,control gene and size standard.The appropriate primer concentration of genes which were expressed abundant in peripheral blood white cells were confirmed by twofold serial dilutions in multiplexed PCR. In the current work we found the house keeping genes expression ofβ-actin and SRP14 were more stable than 18S-rRNA between the CAD and normal control groups,and the geometry average ofβ-actin and SRP14 could be regarded as normalization factor for quantification of target genes in the current work.We detected 14 genes expression profile in CAD patients and normal control subjects with the method described as above,and found expressions of ecNOS, SELL,ADD1 and MTHFR in the peripheral blood white cells were significantly different in both groups.The expression levels of these four genes were associated with genotypes of some SNPs.[Conclusion]Multiplexed SNPs genotyping and genes expression methods established in the current work were quick,high-throughput,easy operation and microanalyse.Six SNPs and 4 genes discovered in the work that were associated with CAD may help in explaining the molecular bases of the disorder and the susceptibility to coronary atherosclerosis.It also can guide early predicting, preventing and therapy for CAD.