Screening And Verification Of Susceptibility Genes For Dyslipidemia And Ischemic Cardio-cerebrovascular Disease

PartⅠScreening of Dyslipidemia Susceptibility Genes by Weighted Gene Co-Expression NetworkBackgrounds: In recent years,the prevalence of dyslipidemia in Chinese residents has been increasing continuously,but the awareness rate,treatment rate and control rate are still not ideal.Dyslipidemia is a kind of complex disease,which is affected by the interaction of environmental,genetic and environmentgenetic factors.Among them,about 40-60% of the incidence of dyslipidemia is related to genetic factors.Therefore,the influence of heredity on dyslipidemia cannot be ignored.With the completion of the human genome Project,the realization of high-throughput sequencing technology and the updating of data analysis methods of gene chips,it has become the mainstream research to explore the relationship between genes and diseases.Therefore,the active search for susceptibility genes and/or genetic markers closely related to the occurrence and development of dyslipidemia will help us to explore the pathogenesis of the disease from the genomics level.Objective: In this study,Weight Gene co-expression Network Analysis(WGCNA)was used to conduct in-depth mining and Analysis of the GSE3059 data set,so as to screen out susceptible genes or biological markers related to dyslipidemia.Methods: The GSE3059 data set used in this study was downloaded from the Gene Expression Omnibus(GEO)database established by the National Center for Biological Information(NCBI).The downloaded data were preprocessed,and the preprocessed data were used to construct gene modules and combine them with clinical phenotypes through WGCNA analysis.Co-expression relationship between gene modules was studied by protein-protein Interaction Networks Functional Enrichment Analysis(PPI).The susceptibility genes were then screened using Cytoscope.Finally,metabolic pathways and Gene functions of the susceptible Genes were enriched through the Kyoto Encyclopedia of Genes and Genomes(KEGG)and the Gene ontology Consortium(GO)database.Results: 1.Through WGCNA analysis,we successfully constructed 7 gene modules,namely blue,yellow,red,turquoise,green,brown and grey.After combining the gene modules with the clinical phenotype,it was found that brown and red modules were positively correlated with dyslipidemia,while blue,yellow,turquoise,green and grey modules were negatively correlated with dyslipidemia.2.Taking the correlation coefficient between module and gene > 0.85 as the inclusion criteria of candidate susceptibility genes for dyslipidemia,we obtained 213 candidate susceptibility genes.By applying candidate genes to protein-protein interaction network(PPI),we obtained 111 nodes and 67 edges.Subsequently,module mining was conducted by using the Cytoscope plug-in MOCDE,and it was found that the brown module was the susceptibility gene module in this study.The 31 genes in this module(RPL3,RPL12,RPL14,RPL15,RPL15,RPL22,RPL29,RPL30,RPL31,RPL32,RPL35,RPL36,RPS9,RPS10,RPS12,RPS15,RPS15,RPS28,RPS29,FAU,PABPC1,RPLP0,IMP3,RPL10 A,FBL,EIF3 F,EIF3K,EIF3 D and RPL23A)were mined for this project Out of the dyslipidemia susceptibility gene.3.By using R software cluster Profiler to enrich the KEGG and GO functions of susceptible genes,it was found that the functions of susceptible genes were mainly related to ribosomes.4.In comparison with the control group and the dyslipidemia group,it was found that the expression levels of 18 susceptible genes,RPS9,RPS10,RPS12,RPL14,RPL15,RPL19,RPL22,RPL29,RPL32,RPLP0,RPL10 A,IMP3,EIF3 D,EIF3K,FAU and PABPC1 were higher in the dyslipidemia group than in the control group,and the differences were statistically significant(all P < 0.05).Conclusion: 1.This study screened 31 dyslipidemia-susceptible genes(RPL3,RPL12,RPL14,RPL15,RPL18,RPL19,RPL22,RPL29,RPL30,RPL31,RPL32,RPL35,RPL36,RPS9,RPS10,RPS12,RPS15,RPS17,RPS21,RPS28,RPS29,FAU,PABPC1,RPLP0,IMP3,RPL10 A,FBL,EIF3 F,EIF3K,EIF3 D and RPL23A).2.The expression of 18 susceptible genes in the dyslipidemia group was significantly higher than the control group(RPS9,RPS10,RPS12,RPS17,RPL12,RPL14,RPL15,RPL19,RPL22,RPL29,RPL32,RPLP0,RPL10 A,IMP3,EIF3 D,EIF3K,FAU,and PABPC1).3.In this study,the functions of 31 dyslipidemia susceptibility genes were correlated with ribosomes.Part II Relationship Between RPLP0,KRS2 and HNF1 A Polymorphisms and Blood Lipid Levels in Maonan and Han Populations in GuangxiBackgrounds: Chromosome 12 is rich in genomes associated with human diseases.Studies have confirmed that region 2,band 4(12q24)of the long arm of chromosome 12 is the susceptible region of dyslipidemia.Ribosomal phosphoprotein large subunit P0(RPLP0),RAS2 kinase inhibitor(KSR2)and hepatocellular factor 1A gene(HNF1A)are all located in this region.Studies in European and American populations found that KSR2 and HNF1 A polymorphism were closely related to dyslipidemia.However,such studies have rarely been conducted in Chinese or even in Asian populations.In addition,the relationship between genotype frequency and lipid levels may vary among different races and populations,even with the same genes and mutation sites.Therefore,repeated verification of the relationship between known gene polymorphisms and lipid levels in specific populations will help us develop individualized treatment strategies for specific populations,so as to better control dyslipidemia and its possible adverse effects.As we know,Guangxi is a place where ethnic minorities live together,among which Maonan ethnic group is one of the ethnic minorities unique to Guangxi.Its geographical location,living environment,eating habits,intermarriage and genetic background are quite different from those of the Han population,making it a better group for us to study environmental factors and genetic traits.Objective: To investigate the effect of Single nucleotide polymorphism(SNP)of RPLP0,KRS2 and HNF1 A genes on serum lipid levels in Maonan and Han populations in Guangxi.Method:In the previous random stratified sample library,750 Maonan residents and 793 local Han residents were randomly selected as the research subjects in Huanjiang county,Guangxi.All subjects were tested for blood lipid level and epidemiology of cardiovascular disease risk factors.Seven SNPs(RPLP0 rs2074540,KRS2 rs10774926,KRS2 rs11068503,KRS2 rs11830157,HNF1 A rs2071190,HNF1 A rs2464196,HNF1 A rs2244608)were genotyped by polymerase chain react-restricted fragment length polymorphism(PCR-RFLP)combined with direct sequencing.To analyze and compare the distribution of genotypes and alleles of each gene locus in two ethnic groups and explore their correlation with environment and lipid level.Results: 1.Height,weight,body mass index(BMI),systolic blood pressure,pulse pressure,TC and TG levels in THE CHD group were all higher than those in the control group,while serum TC,HDL-C,Apo A1,Apo A1/Apo B ratio and percentage of drinkers were all lower than those in the control group(all P < 0.05).Height,weight,body mass index(BMI),systolic blood pressure,diastolic blood pressure,pulse pressure,TC and TG levels were all higher in THE IS group than in the control group,while serum TC,HDL-C,Apo A1 level,Apo A1/Apo B ratio and percentage of people drinking alcohol were all lower in the IS group than in the control group(all P < 0.05).2.The genotype and allele frequency of RPLP0 rs2074540 were statistically significant between Maonan and Han nationalities and between male and female Maonan nationalities.The genotype frequency of KSR2 rs11068503 was significantly different from that of the allele genotype.The difference in the frequency of KSR2 rs11830157 was statistically significant.The difference of HNF1 A rs2071190 genotype and allele frequency was statistically significant.There were statistically significant differences in the genotype and allele frequencies of rs2464196 between two ethnic groups,between male and female of Han nationality and between male and female of Maonan nationality(all P < 0.05).3.In the Han population,Apo B levels of different HNF1 A rs11830157 genotypes,TG,Apo B levels and Apo A1/Apo B ratios of different HNF1 A rs2071190 genotypes and TC levels of different rs2464196 genotypes were statistically significant(7 SNPs corrected by Bonferroni with P < 0.00714 was statistically significant).The HDL-C levels and Apo A1/Apo B ratios of different genotypes of RPLP0 rs2074540,the TG,LDL-C and Apo B levels of different genotypes of KRS2 rs10774926,and the Apo A1/Apo B ratios of different HNF1 A rs2244608 genotypes were statistically significant(P < 0.00714).4.In the Han male subgroup,the serum Apo B levels of different genotypes of RPLP0 rs2074540 and KSR2 rs11830157,the serum Apo A1/Apo B ratios of different genotypes of KSR2 rs2071190,and the TC levels of different genotypes of rs2464196 were statistically significant(Bonferroni corrected P < 0.00714).In the Han female subgroup,the differences in Apo A1/Apo B ratios of RPLP0 rs2074540 genotypes,serum TG levels of KSR2 rs2071190 genotypes and TG and TC levels of HNF1 A rs2244608 genotypes were statistically significant(Bonferroni corrected P < 0.00714).5.In the Maonan male subgroup,the difference in the Apo A1/Apo B ratio of HNF1 A rs2244608 genotypes and the levels of LDL-C and Apo B of rs2464196 genotypes was statistically significant.In the Maonan female subgroup,the HDLC and Apo A1 levels of different genotypes of RPLP0 rs2074540,the HDL-C levels of different genotypes of KSR2 rs11068503 and the Apo A1/Apo B ratio of different genotypes of KSR2 rs11068503 and the TG levels of different HNF1 A rs2244608 genotypes were statistically significant(Bonferroni correction P < 0.00714).6.The results of multi-factor regression analysis showed that the lipid levels of the two ethnic groups were also closely related to environmental factors such as age,gender,waist circumference,alcohol consumption,smoking,blood sugar,height and genotype.Conclusion: 1.The difference in blood lipid levels between Maonan and local Han populations in Guangxi was statistically significant.2.The effects of RPLP0,KSR2 and HNF1 A polymorphisms on blood lipid levels of the two ethnic groups are different,which are ethnic-specific and genderspecific.3.Blood lipid levels of the Han and Maonan nationalities in Guangxi are also correlated with environmental factors such as age,gender,waist circumference,smoking and drinking.Part III Relationship Between RPLP0,KRS2 and HNF1 A Polymorphisms and Ischemic Cardio-cerebrovascular DiseasesBackgrounds: In recent decades,the morbidity and mortality of ischemic cardiovascular and cerebrovascular diseases in China have been increasing year by year,which has caused heavy mental and economic burden to the country and the people.Two diseases,Coronary heart disease(CHD)and Ischemic stroke(IS),are common in Ischemic cerebrovascular diseases.Dyslipidemia IS an independent risk factor and common pathological basis for CHD and IS.Previous studies have confirmed that the occurrence and development of CHD and IS are jointly affected by genetic factors,environmental factors and geneticenvironmental factors.The second part of this study found that polymorphisms of RPLP0,HNF1 A and KSR2 located in chromosome 12q24 in Maannan and local Han populations in Guangxi were correlated with lipid levels.Therefore,we speculated that the polymorphisms of RPLP0,HNF1 A and KSR2 were also correlated with CHD and IS.Objective: To investigate the relationship between RPLP0,HNF1 A and KRS2 single nucleotides polymorphism(RPLP0 rs2074540,KSR2 rs10774926,rs11068503,rs11830157,HNF1 A rs2071190,rs2244608,rs2464196)and the risk of CHD and IS,as well as the gene-gene and gene-environment interactions.Methods: A case-control study was performed in the Han population in Guangxi,of which 523 were in the normal control group,549 were diagnosed with coronary heart disease after coronary angiography,and 511 were clinically diagnosed with ischemic stroke.Seven polymerase chain reaction-restriction fragment length polymorphisms(PCR-RFLP)combined with direct sequencing were used to genotype 7 SNPs including RPLP0 rs2074540,KRS2 rs10774926,rs11068503,rs11830157,HNF1 A rs2071190,rs2464196,and rs2244608.Analyze and compare the distribution of genotypes and alleles in control and case groups at each site,and explore the impact of their interaction with the environment and genes on the risk of ischemic cardiovascular and cerebrovascular disease.Results: 1.Height,weight,body mass index(BMI),systolic blood pressure,pulse pressure,TC and TG levels in THE CHD group were all higher than those in the control group,while serum TC,HDL-C,Apo A1,Apo A1/Apo B ratio and percentage of people drinking alcohol were all lower than those in the control group(all P < 0.05).Height,weight,body mass index(BMI),systolic blood pressure,diastolic blood pressure,pulse pressure,TC and TG levels were all higher in THE IS group than in the control group,while serum TC,HDL-C,Apo A1 level,Apo A1/Apo B ratio and percentage of people drinking alcohol were all lower in the IS group than in the control group(all P < 0.05).2.In the CHD group,the genotypes and alleles of KSR2 rs11830157,HNF1 A rs2244608 and HNF1 A rs2464196 SNPs were significantly different from those of the control group.In the ischemic stroke group,the frequency of KSR2 rs11830157 allele was significantly different from that of the control group.The genotype and allele frequency of HNF1 A rs2244608 were significantly different from that of the control group(all P < 0.05).3.Five genetic models of RPLP0,KSR2 and HNF1 A in the CHD group showed that KSR2 rs11830157 increased the risk of CHD in the dominant model TT vs GT+GG(OR = 1.40,95%CI = 1.10-1.78,P = 0.006).HNF1 A rs2244608 reduced the risk of CHD in dominant model CC vs.CT+TT(OR = 0.59,95%CI= 0.44-0.81,P = 7E-04)and in addition model C vs.T(OR = 0.70,95%CI = 0.58-0.85,P =3E-04).HNF1 A rs2464196 reduced CHD risk in codominant model TT vs.CC(OR=0.58,95%CI= 0.39-0.87,P = 0.00706)and in recessive model TT+CT vs.CC(OR=0.58,95%CI=0.41-0.84),P = 0.0018.The remaining sites were not found to be associated with CHD risk.4.Five genetic models of RPLP0,KSR2 and HNF1 A SNPs were established in the ischemic stroke group,and KSR2 rs11830157 increased the risk of ischemic stroke in the dominant model TT vs.GT+GG(OR = 1.36,95%CI = 1.25-2.52,P < 0.0001).HNF1 A rs2244608 reduced the risk of ischemic stroke in the dominant model CC vs.CT+TT(OR = 0.38,95%CI = 0.27-0.54,P < 0.0001).rs2464196 reduced the risk of ischemic stroke in the additive model C vs.T(OR = 0.74,95%CI = 0.60-0.91,P = 0.0041).The remaining loci were not found to be related to the risk of ischemic stroke.5.In the linkage imbalance analysis of RPLP0,KSR2 and HNF1 A sites,it was found that there was a linkage imbalance between KSR2 rs10774926,rs11068503 and rs11830157 in the coronary heart disease group and the ischemic stroke group.In the CHD group,the most common haploid was A-C-T,and the risk of CHD was reduced in those with A-T-T(OR = 0.71,95%CI = 0.52-0.98,P = 0.039)and A-T-G(OR = 0.59,95%CI = 0.99-0.91,P = 0.017)compared with those with A-C-T.However,A-C-G(OR = 1.50,95%CI= 1.08-2.08,P = 0.017)and G-T-G(OR = 2.37,95%CI = 1.35-4.19,P = 0.0029)haplotype carriers had an increased risk of coronary heart disease.In the ischemic stroke group,the most common haploid was A-C-T.Compared with A-C-T,haploid individuals with AC-G(OR = 1.66,95%CI = 1.01-2.28,P = 0.005)had an increased risk of ischemic stroke,while haploid individuals with A-T-G(OR = 0.61,95%CI = 0.42-0.89,P = 0.009)had reduced risk of ischemic stroke.6.Through the generalized multi-factor dimensional reduction(GMDR)analysis of RPLP0,KSR2 and HNF1 A it was found that gene-gene interaction rs2074540-rs10774926-rs11830157-rs2464196 plays an important role in the pathogenesis of coronary heart disease.Gene-environment interaction Hypertension-rs2074540-rs10774926-rs11068503-rs11830157 also plays an important role in the pathogenesis of coronary heart disease.Gene-gene interaction rs2074540-rs11068503-rs2071190-rs2244608 plays an important role in the pathogenesis of ischemic stroke.Gene-environment interaction AgeHypertension-rs10774926-rs11068503-rs11830157-rs2464196 also plays an important role in the pathogenesis of ischemic stroke.Conclusions: 1.There were statistically significant differences in general conditions and lipid levels between the coronary heart disease group and the ischemic stroke group and the control group.2.The frequency of KSR2 rs11830157,HNF1 A rs2244608,and rs2464196 genotypes or/and alleles in CHD and IS groups were significantly different from those in the control group.3.In genetic model studies,KSR2 rs11830157 increased the risk of coronary heart disease and ischemic stroke,while HNF1 A rs2244608 and HNF1 A rs2464196 reduced the risk of coronary heart disease and ischemic stroke.4.KSR2 haploid has an impact on the risk of coronary heart disease and ischemic stroke.5.Rs2074540-rs10774926-rs11830157-rs2464196 and hypertensionrs2074540-rs11068503-rs11830157 interact to influence the risk of CHD.The interaction of rs2074540-rs11068503-rs2071190-rs2244608 and agehypertension rs10774926-rs11068503-rs11830157-rs2464196 affected the risk of IS.