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Genetic Susceptibility Of Lipid Levels And Coronary Heart Disease In The Chinese Han Population

Posted on:2012-11-14Degree:DoctorType:Dissertation
Country:ChinaCandidate:L ZhouFull Text:PDF
GTID:1114330335455184Subject:Occupational and Environmental Health
Abstract/Summary:PDF Full Text Request
Coronary heart disease (CHD) is the most common chronic disease with increasing incidence and is the leading cause of morbidity and mortality worldwide. In China, with the rapid development of social economy, the change of lifestyle and the population aging, the incidence of CHD is rapidly increasing.The World Health Organization estimated that more than 1,000,000 people died from CHD each year in China. CHD will be a serious threat to people's health and become the heavy burden of society and family. To improve health and health care of diseases such as CHD, Chinese government has recently announced Healthy China 2020 programme with an overarching goal of strengthening public health and medicine. Reducing chronic disease risk is also a key to construct the harmonious society.The development of CHD is a complex process. It is caused by multiple genetic and environmental factors, and interactions among these factors. Many risk factors have been identified for CHD, such as smoking, advanced age, male gender, diabetes mellitus, lipid levels, high blood pressure and family history of CHD. However, the traditional risk factors can only explain 50%-60% causes of CHD, and 40%-50% will be attributed to hereditability.Among these factors, family history is one of the most significant independent risk factor for CHD. Twin studies also suggested that genetic factors contribute to the development of CHD. All these support the hypothesis that genetic factors contribute to the development of CHD. A main underlying pathology of CHD is atherosclerosis, a process of cumulative deposition of low-density lipoprotein cholesterol (LDL) in the arteries supplying blood to the heart that eventually leads to impaired or absent blood supply and CHD. Consistent and compelling evidence has demonstrated association between lipoprotein-associated lipid levels and CHD incidence worldwide.After completing human genome sequencing and hapmap project, genome-wide association studies (GWAS) which provide high throughput approach to detect whole genome have been localized common DNA sequence variants that contribute to many human phenotypes and diseases. In the past 5 years, GWAS have identified single nucleotide polymorphisms (SNPs) implicating hundreds of genetic loci for complex diseases in Caucasians, such as cancers, diabetes, CHD. Nearly 600 GWAS covering 150 distinct diseases and traits have been published, with nearly 800 SNP-trait associations reported as significant. In 2007, SCIENCE firstly reported the genetic variants were associated with CHD risk in GWAS. Until now, GWAS have identified SNPs on chromosomes 1p13,6p24,9p21, and so on, that are associated with risk of CHD or its major complication, myocardial infarction (MI). Recent GWAS have also identified several new loci that influence levels of blood lipids. Kathiresan et al. found some SNPs were not only associated with lipid leves, but also associated with CHD.These associations need to be confirmed by further replication studies, particularly in other ethnic groups. However, most GWAS were performed in populations of European ancestry, and few GWAS has been reported in the Chinese population when we started this project. In this study, we carried out a GWAS for lipid levels in the Chinese Han population. We also assessed the association between the SNPs at lipid loci and risk of CHD in large case-control studies in Chinese Han population. Furthermore, we validated the novel genetic variants in chromosome 9p21 which were associated with CHD in GWAS in Caucasions. We also aimed to identify the causal variants and explore the mechanistic changes, as well as to investigate the gene-environment interactions in relation to CHD risk. Blood lipids are important determinants of CHD and are related to morbidity. The lipids include total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL), high-density lipoprotein cholesterol (HDL). Whereas high levels of LDL are associated with increased risk of CHD, high levels of HDL are associated with decreased risk of CHD. Age, gender, diet and physical activity all have roles in determining individual lipid profiles. Still, family studies suggest that in many populations, about half of the variation in these traits is genetically determined, and it is clear that TC, TG, LDL and HDL are strongly influenced by the genetic constitution of each individual.We constructed a cohort called Tongji-Dongfeng cohort in 2008. The participants in this cohort are up to 27,000 now. All the participants provided questionnaire information and blood samples. In this study, we carried out GWAS for the lipid levels in the Chinese Han population. At the discovery stage, we selected 1461 healthy subjects from the cohort. All enrolled subjects were unrelated ethnic Han Chinese. We used the Affymetrix GeneChip Human Mapping 6.0 to test the associations between the lipid levels (TC, TG, LDL and HDL) and SNPs, which simultaneously types approximately 666,141 SNPs. Only SNPs showing nominal P<10-5 in the discovery stage were seleted for further validation studies.At the validation studies, we constructed a two-stage validation study consisting of 3840 healthy subjects in the first validation stage and 5374 healthy subjects in the second validation stage in Chinese Han population from the cohort. We used this independent replication population to replicate the positive SNPs from the GWAS. In the first stage, we validated them in 3840 independent subjects. The first validation study showed that 12 SNPs at 6 loci were significantly associatied with the lipid levels in Chinese Han population (P<0.05). We then validated all the positive SNPs from the first validation stage in another group of 5374 independent subjects and the results from the second validation stage showed that all of the SNPs were significantly associated with lipid levels. Of the 12 identified SNPs,5 SNPs on DOCK? were associated with TC (rs11207995, rs1168114, rs7518497, rs10889332 and rs4350231),6 SNPs were associated with TG (rs7396835 on APOA4, rs651821 on APOA5, rs6589566, rs964184 and rs4417316 on ZNF259 and rs17119975 on BUD13), one SNP on TOMM40 was associated with LDL (rs1160985). However, there was no significant association between the SNP and HDL level. With increasing genotype score, the levels of TC and TG increased significantly. Notably, we identified 10 new SNPs (rs11207995, rs1168114, rs7518497, rs10889332 and rs4350231 on DOCK7; rs7396835, rs6589566, rs4417316 and rs17119975 on APOPA4-ZNF259-BUD13 gene cluster and rs1160985 on TOMM40) associated with lipid levels in Chinese Han population.Part II The associations between lipid-associated SNPs and risk of coronary heart disease in Chinese Han populationCompelling evidence has demonstrated that blood lipid levels including increased levels of TC, TG, LDL, and decreased levels of HDL are key modifiable risk factors for CHD. Previous studies suggested that both of the lipid levels and risk of CHD have been associated with genetic variants. These observations suggested a DNA sequence variant that is related to blood lipid levels may influence the risk of CHD. Thus, we aimed to demonstrated not only that genetic variants account for a substantial fraction of individual variation in lipid levels, but also that lipid levels are associated with the risk of CHD. Recent GWAS in the Chinese Han population and in European ancestry have identified several loci that influence levels of blood lipids. We combined the results from these large cohorts to identify loci affecting blood levels of lipids across different populations. We further examined whether the associated lipid loci showed any impact on susceptibility of CHD in the Chinese Han population.We identified two novel SNPs (rs599839 in CELSR2-PSRC1-SORT1 and rs16996148 in NCAN-CILP2) that were significantly associated with CHD risk in Chinese [OR (95% CI) in the dominant model:0.76 (0.65-0.89; P=0.001),0.66 (0.57-0.76; P=3.3×10-8), respectively]. Multiple linear regression analyses using dominant model showed that rs599839 was significantly associated with decreased LDL levels (P=0.022) and rs16996148 was significantly associated with increased LDL and HDL levels (P=2.9×10-4 and 0.001, respectively).Recent GWAS have also mapped a CHD susceptibility locus to the SLC22A3-LPAL2-LPA gene cluster on chromosome 6q26-27 in Caucasians. This gene cluster includes the LPA gene, which encodes the Lipoprotein(a) [Lp(a)]. Lp(a) has many properties in common with low-density lipoprotein (LDL), which is known to cause atherosclerosis and CHD. We therefore conducted a large case-control study (2308 CHD patients and 2332 control subjects) to identify the genetic variants on SLC22A3-LPAL2-LPA gene cluster associated with CHD susceptibility in Chinese. We genotyped five risk SNPs and found that three of them were significantly associated with CHD risk in Chinese (rs10755578, rs3088442 and rs7767084) after adjusting for conventional risk factors. The minor allele of the SNPs rs10755578 and rs3088442 occurred more frequently in control subjects compared to CHD patients [OR (95% CI):0.91(0.84-0.99; P=0.033) and 0.92(0.85-0.99); P=0.045, respectively]. Forward analysis of lipids levels and SNPs indicated that SNP rs3088442 was associated with Lp(a) levels (P for trend=0.026). These results support that the genetic variants on the SLC22A3-LPAL2-LPA gene cluster might influence risk of CHD through affect on the circulating Lp(a) levels in subjects. Using PHASE 2.0 software to reconstruct haplotype of rs10755578, rs3088442 and rs7767084, we observed that compared with GTC haplotype, the ACG haplotype had a significant decreasing risk of CHD (OR=0.84,95%CI:0.74-0.96, P=0.01).The results of this part indicated that five lipid-associated genetic variants identified in GWAS were associated with CHD risk in the Chinese Han population. rs599839 and rs16996148 were novel SNPs which functions were unclear. The SNPs rs10755578, rs3088442 and rs7767084 were on the SLC22A3-LPAL2-LPA gene cluster. These studies can provide new insights into the biological mechanisms regulating lipid metabolism and identify potentially novel therapeutic targets for CHD.Part III Replication analysis of SNPs on 9p21 associated with CHD risk in the Chinese Han populationThe development of CHD is a complex process. It is caused by multiple genetic and environmental factors, and interactions among these factors. In 2007, SCIENCE firstly reported the genetic variants were associated with CHD risk in GWAS. Until now, GWAS have identified SNPs on chromosomes 1p13,9p21, and so on, that are associated with risk of CHD. Notably, the effect on risk of 9p21 was independent of all known risk factors, including elevated lipid levels. Therefore, we carried out a large case-control association study including 1,360 CHD patients and 1,360 age-and sex-frequency matched controls in an unrelated Chinese Han population. We tested the gene-environment interaction and aimed to find out a new biologic pathway that was relevant to CHD.In GWAS, a susceptibility locus for CHD has been mapped to chromosome 9p21, adjacent to the tumor suppressor genes CDKN2A and CDKN2B. Helgadottir et al.found that the variant rs2383207 was associated with MI. Meanwhile, in another GWAS, McPherson et al. found that the homozygotes of the risk alleles of rs2383206 were associated with an increased risk of CHD. We selected two SNPs (rs2383206 and rs2383207) in the interesting locus and genotyped the two SNPs on chromosome 9p21 in 1,360 paired Chinese Han CHD cases and controls. The multivariated logistic regression after adjusting for conventional CHD risk factors such as age, gender, smoking, BMI, hypertension, diabetes, and family history of CHD reveal that, when compare with the rs2383207 AA genotype, subjects with the rs2383207 GG genotype had an increased risk of CHD (OR=1.52, 95%CI=1.13-2.04, P=0.015). For the risk allele G of the two SNPs, the univariate ORs were 1.14 (95%CI 1.03-1.27) and 1.26 (95%CI 1.12-1.41), respectively.The large sample size allowed investigation of further subgroups based on the underlying risk profile. We conducted stratified analysis for the two SNPs. GG carriers of two SNPs (rs2383206 and rs2383207) had higher risk in males (OR=1.57,95%CI 1.14-2.16 and OR=1.96,95%CI 1.32-2.90, respectively), less than 60 years old subjects (OR-1.35,95%CI 1.00-1.84 and OR-1.58,95%CI 1.02-2.47, respectively), smokers (OR=1.55,95%CI 1.10-2.20 and OR=2.03,95%CI 1.34-3.08, respectively), and BMI> 25 kg/m2 subjects (OR=1.52,95%CI 1.00-2.33 and OR=2.39,95%CI 1.41-4.05, respectively). Furthermore, When multiplicative interaction was tested for each possible pair of these two SNPs, we found significant interactions between rs2383207 and gender (P=0.018) and smoking (P=0.037).We evaluated cumulative effects of risk allele G of the rs2383207 and family history of CHD. Subjects who carried all risk factors, including two risk allele G and family history of CHD had an OR of 4.59 (95%CI 2.52-8.37) for CHD, as compared with subjects who carried none of the risk factors, after adjusting for other conventional risk factors. The P for trend of this cumulative effect was 1.0×10-6.Our large case-control study not only replicated the findings of the SNPs on chromosome 9p21 that were associated with CHD established previously by genome-wide association studies, but also provided several novel findings that were relevant to this locus. Our results showed that the genotype GG of the SNP rs2383207 was associated with increased overall risk of CHD. Especially among males, young subjects (age≤60), smokers, and overweight patients (BMI≥25 kg/m2), the genotype GG of the two SNPs rs2383206 and rs2383207 were associated with higher risk of CHD. The interaction was found between the traditional CHD risk factors (gender and smoking) and rs2383207 in our study.In this part, we succesully replicated the association between the genetic variants on 9p21 and CHD risk in the Chinese Han population. We found the rs2383207 GG gentotype was associated with increased CHD susceptibility and the risk alleles of rs2383206 and rs2383207 plus family history of CHD had a cumulative, significant association with CHD.Part IV Functional analysis of the CHD risk associated SNP on SLC22A3-LPAL2-LPA gene clusterThe 3'untranslated region (3'UTR) was located downstream of the gene translation terminator.3' UTR may contain sequences that regulate translation efficiency, mRNA stability, and polyadenylation signals and have microRNA binding sites. It may change the regulation mechanism and affect the expression level of the target gene. In Part II, we found that the SNP rs3088442 in 3'UTR of SLC22A3-LPAL2-LPA gene cluster was associated with susceptibility of CHD. We will analyze the biological function of this SNP in this part.Because the SNP rs3088442 was located within the 3'UTR of SLC22A3-LPAL2-LPA gene cluster and computational analysis showed that it might be microRNA binding sites, we therefore examined whether the SNP has an impact on the mRNA stability by a set of luciferase reporter gene constructs, which were used to transfect transiently human HepG2 cell line. Intriguingly, we observed a significantly higher luciferase activity for constructs rs3088442-G in the two types of cell lines (P<0.05). These data suggested that the rs3088442-G in 3'UTR of SLC22A3-LPAL2-LPA gene cluster may enhance mRNA stability and mRNA expression level.In conclusion, we investigated the associations between the genetic variants and lipid levels in the Chinese Han population in GWAS. We also identified the relations between the lipid-associated genetic variants and CHD risk in Chinese Han population. We validated the SNPs on chromosome 9p21 had strongly association with risk of CHD. Furthermore, we examed the potential functions of SNP in the 3'UTR. Our results suggested:1. The GWAS identified 12 SNPs in 6 loci were associated with lipid levels in the Chinese Han population.10 SNPs were newly identified in our GWAS.2. The newly identified lipid-associated SNPs rs599839 and rs16996148 were associated with risk of CHD. Thay may influence the CHD risk through increasing the lipids levels.3. Three SNPs rs3088442, rs7767084 and rs10755578 on SLC22A3-LPAL2-LPA gene cluster were associated with risk of CHD. The risk allele of rs3088442 in the 3'UTR of SLC22A3-LPAL2-LPA gene cluster was associated with increased luciferase expression and activity, suggested it may be a causal variant.4. The SNPs on chromosome 9p21 were strongly associated with risk of CHD in the Chinese Han population. Especially the risk alleles plus family history of CHD had a cumulative, significant association with CHD. rs2383207 had gene-environment interactions with smoking.This study has some advantages and innovations:1. It is the first GWAS for lipid levels in the Chinese Han population. In this study, we not only replicated the GWAS results from European population, we also identified some novel genetic variants and loci in the Chinese Han population.2. We identified two novel SNPs at newly identified, lipid-associated loci that were significantly associated with CHD susceptibility in Chinese Han population. We used the "genotype-phenotype-disease" model to investigate the associations among the genetic variants, lipid levels and susceptibility of CHD. This design was better to search for causal SNPs and potential therapeutic targets.3. We firstly validated the association between the genetic variants on 9p21 and CHD risk in the Chinese Han population. In addition, we demonstrated the gene-environment interactions on 9p21 and cumulative effects of risk factors.4. The epidemiological studies had large sample size and they were multicenter studies.However, there were several potential limitations in the present study that need to be addressed.1. Some genetic variants and loci identified in our study were in gene "desert". There were non-coding sequence and the biological mechanisms were unknown. They need Fine-Mapping or long-range regulation experiments to reveal the functions and biological mechanisms.2. In the lipid related GWAS, the sample size of the discovery stage was small. So the statistic power was not enough to find out the genetic variants which were modest or lower frequencies.3. Because of the high costs of GWAS, the GWAS of CHD risk in the Chinese Han population did not conduct until now. Further analyze the association between genetic variants and risk of CHD still needs GWAS.4. Corhort study is a long-term study and need several decades. Because the time was limited in this study, we could not perform the prospective study of the CHD and occupational-related diseases.
Keywords/Search Tags:Genome-wide association study, Lipid levels, Coronary heart disease, Single nucleotide polymorphism, Susceptibility
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