Study On Influencing Factors Of Total Cholesterol Level And Genome-wide Association Study In Rural Population Of Guangxi Counties

BackgroundWith the progress of society,the development of health,the improvement of people's living standard and the increasing life expectancy of the population,China has gradually entered an aging society,as a result,cardiovascular disease ranks first in the disease spectrum,becoming the main cause of death and the main factor affecting disability adjusted life year for Chinese residents.Higher and abnormal blood lipids are important risk factors for cardiovascular disease,which is closely related to the human nutritional status,metabolic level,occurrence and development of diseases,aging,etc.Except for cardiovascular disease,total cholesterol is also related to the occurrence and development of various diseases and dangerous behaviors such as Alzheimer's disease,cancer,and suicide.Hence,it is of great significance to study the influencing factors of abnormal total cholesterol level in the population.Currently,it is generally accepted that total cholesterol level is affected by environment,gene and their interactions,which means total cholesterol level in different populations may be affected by different risk factors.In terms of genes,some scholars believed that the effects of genetic variation found so far on total cholesterol level is only 18.8%,while the estimated value should be 51%.It suggested that there were still many unknown environmental and genetic factors to be explored.In addition,in most previous studies about the blood lipid that recruited participants from urban or rural population of all ages,and few studies were specifically aimed at rural elderly population involved genetic factors and geneenvironment interactions,among them there were fewer studies on rural elderly in Guangxi,and further exploration is urgently needed.This study intended to analyze the status and influencing factors of abnormal total cholesterol level in the elderly population of Donglan County,Hechi City,Guangxi.Genome-wide association study were used to screen genetic variants affecting total cholesterol level,which were replicated in the population of Gongcheng County,Guilin City,Guangxi,and to explore the association of these single nucleotide polymorphism with total cholesterol in the population of Gongcheng County.The purpose of this study was to understand the status of abnormal total cholesterol level in the population and provide new clues for exploring the environmental and genetic factors affecting the total cholesterol level in rural population of Guangxi,provide the basis for revealing the regulation mechanism of cholesterol level.PART ? Study on the Status of Total Cholesterol Level and Its Influencing Factors in the Rural Elderly from a County of GuangxiObjective: To understand the distribution and influencing factors of abnormal total cholesterol(TC)level among rural elderly people in a county of Guangxi,and to provide some basis for the prevention of abnormal TC level in the elderly.Methods: Cluster sampling was used to sample the elderly who aged 60 and above in three towns.The baseline data which including questionnaire survey,physical examination and blood test results were analyzed by SPSS 21.0.Results: 1.Socio-demographic characteristics of subjects: in this research,the total number of subjects was 4230,which included 1681 males(accounting for 39.74%)and 2549 females(accounting for 60.26%).Meanwhile,the subjects were grouped on whether they had abnormal TC levels or not,1898(accounting for 44.86%)and 2332(accounting for 55.13%)subjects in normal and abnormal TC level groups respectively.2.Comparison of living habits and general health status between normal and abnormal TC level groups: smoking,drinking,staple food at each meal,self-perceived physical condition,body mass index(BMI),waist-to-hip ratio(WHR),and hypertension showed statistically significant differences between this two groups.On the contrary,differences in family smoking,number of meals per day,and cooking methods were not statistically significant.3.Comparison of blood test results between normal and abnormal TC level groups: the TC level of total subjects was 5.42±1.10 mmol/L,which was 4.48±0.54 mmol/L in normal TC level group and 6.17±0.81 mmol/L in abnormal TC level group.The levels of high-density lipoprotein cholesterol(HDL-C),lowdensity lipoprotein cholesterol(LDL-C),triglyceride(TG)and alanine aminotransferase in the abnormal TC level group were higher than those in the normal group,but the total bilirubin level in the abnormal TC level group was lower,the differences were statistically significant.Between the two groups,there were no statistically significant differences in creatinine,uric acid,fasting blood glucose and glutamate transaminase.4.Multi-factor analysis: gender,age,staple food at each meal,BMI,WHR,and total bilirubin might be affecting factors to the abnormal TC level.Among them,female(OR=1.405;95% CI=1.232~1.602),BMI?24 kg/m2(OR=1.369;95% CI=1.172~1.599)and WHR?0.9(OR=1.221;95% CI=1.067~1.397)might be risk factors.Older age,staple food at each meal,and higher total bilirubin might be protective factors,the differences were statistically significant.Conclusions: Compared to other elderly populations,the rate of abnormal TC level of the elderly in this county was higher,which might be related to gender,age,staple food at each meal,BMI,WHR,total bilirubin.The results suggested that the elderly should pay attention to avoid obesity,and lay emphasis on the control of TC level especially women and people aged 60-69.PART ? Study on Screening the Genes and SNPs Associated with Total Cholesterol Level in the Rural Elderly from a County of GuangxiObjective: To screen the genes and SNPs by performing the genome-wide association study(GWAS)for exploring the genetic factors that affecting TC level.Methods: Elderly people in a county of Guangxi were selected as research subjects,after quality control and genome-wide population,PLINK 1.90 software was used for genome-wide association analysis after genotype imputation.During the analysis,single nucleotide polymorphism(SNP)that had a P value <10-4 were selected and their potential functions were assessed.Moreover,the susceptibility gene were further analyzed by using Versatile gene-based Association study-2(VEGAS2).According to the analysis,the biological process,cell composition,molecular function and signaling pathway that mainly involved in the selected genes were obtained through Gene Ontology(GO)functional annotation and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis.Results: 1.Socio-demographic characteristics of subjects: 928 subjects were left after quality control that included 324 males(34.91%)and 604 females(65.09%).Average TC level was 5.40±1.13 mmol/L.2.Genome-wide association analysis: 69 sites were screened,rs7412,which located in APOE was the most significant SNP.Other SNPs were locate in NECTIN2,CYP19A1,DISP2,CCDC9 B,Linc02274,APOC1,SORL1,INSIG2,NMB,TMEM18 etc.3.VEGAS2 gene-based analysis: APOE,DISP2,CYP19A1,and NECTIN2 were related to TC level.4.GO functional annotation and KEGG pathway enrichment analysis: in GO functional annotation,biological process included positive and negative regulation of cholesterol esterification,cholesterol metabolic process,phospholipid efflux,cholesterol efflux,regulation of lipid transport,etc.Cellular component included plasma lipoprotein particle,lipoprotein particle,protein-lipid complex,chylomicron,etc.Molecular function included lipoprotein particle binding,lipase inhibitor activity,phospholipase inhibitor activity,cholesterol transporter activity,etc.The KEGG pathway was Cholesterol metabolism(hsa04979).Conclusions: According to GWAS,the results showed that APOE,NECTIN2,CYP19A1,DISP2,CCDC9 B,Linc02274,APOC1,SORL1,INSIG2,NMB,TMEM18 might be related to TC level.Among them,DISP2,CCDC9 B,Linc02274,NMB and other genes had not been reported.PART ? Replication of Genes and SNPs Related to Total Cholesterol LevelObjective: To replicate SNPs that screened from GWAS and explore the association between these SNPs and TC level in rural population of Guangxi.Methods: SNPs including Linc02274 rs12100961,CCDC9 B rs4143838,DISP2 rs1898883,DISP2 rs12911691,CYP19A1 rs10459592,NECTIN2 rs7254892,APOE rs72654473,APOE rs7412 were replicated in subjects from Gongcheng County,Guangxi.we analyzed association between SNP and abnormal TC level,and then performed meta-analyses of the results from the discovery GWAS and replication.The multi-factor dimensionality reduction analysis and logistic regression analysis were used to investigate the effects of gene-gene and gene-environment interactions on abnormal TC level.DualLuciferase Reporter assay system was used to study the function of CYP19A1 rs10459592.Results: 1.Socio-demographic characteristics of subjects: the total number of subjects was 2068 in this research,which including 796 males(38.49%)and 1272 females(61.51%).2.The basic information of genotype: all genotypes were consistent with Hardy-Weinberg equilibrium(PHWE> 0.05).Minor allele frequency>0.05,sample call rate>0.95.3.Alleles and genotype frequency: in Gongcheng County,distributions of alleles in NECTIN2 rs7254892,APOE rs72654473 were significant differences between the normal group and the abnormal group.Significant differences were observed in the frequencies of DISP2 rs12911691,NECTIN2 rs7254892,APOE rs72654473 genotypes between the two groups.4.Association of genetic models with abnormal TC level: in Gongcheng County,the dominant and recessive models of NECTIN2 rs7254892,APOE rs72654473 showed protective effects on abnormal TC level.Compared with people with APOE rs72654473 CC genotype,the risk of abnormal TC level to people who with AA genotype was the least(OR=0.254;95% CI=0.112~0.576).While the recessive models of DISP2 rs12911691 showed increased morbidity.5.Association of genetic models with TC level: in Gongcheng County,DISP2 rs1898883,DISP2 rs12911691,NECTIN2 rs7254892 and APOE rs72654473 were associated to TC level.6.The combined analysis: Linc02274 rs12100961,DISP2 rs1898883,DISP2 rs12911691,NECTIN2 rs7254892 and APOE rs72654473 were related to the morbidity of abnormal TC level or TC level.7.The effects of gene-gene and gene-environment interactions: in Gongcheng County,the effects of interactions in NECTIN2 rs7254892 and APOE rs72654473,APOE rs72654473 and age?60 were the mostly significant antagonism.The mutations of NECTIN2 rs7254892,APOE rs72654473 might reduce the risk of abnormal TC level when interacted with other SNPs except for the rs10459592 TT genotype.The effects of interactions in the mutations of NECTIN2 rs7254892,APOE rs72654473 and environmental factors mainly showed to reduce the risk of abnormal TC level,while BMI?24 kg/m2 and age?60 in environmental factors mainly showed to increase the risk.BMI?24 kg/m2,age?60,smoke might counteract the protective effects of NECTIN2 rs7254892,APOE rs72654473 mutations.8.Result of Dual-Luciferase Reporter assay: CYP19A1 rs10459592 G allele compared with the T allele,the relative luciferase activity was up-regulated by 58.67%,the difference was statistically significant.Conclusions: Linc02274 rs12100961,DISP2 rs12911691,NECTIN2 rs7254892,APOE rs72654473 might be related to the abnormal TC level according to the result of combined analysis.DISP2 rs12911691,NECTIN2 rs7254892,APOE rs72654473 might be related to abnormal TC level in Gongcheng County.Gene-gene,gene-environment interactions had different effects to the abnormal TC level,the interactions between NECTIN2 rs7254892,APOE rs72654473 and other factors were mainly related to the risk reduction of abnormal TC level.BMI ? 24 kg/m2 and age ? 60 years were contributed significantly to the risk of abnormal TC level.