Functional Promoter Polymorphisms Govern Differential Expression of HMG-CoA Reductase Gene in Mouse Models of Essential Hypertension

3-Hydroxy-3-methylglutaryl-coenzyme A [HMG-CoA] reductase gene (Hmgcr) is a susceptibility gene for essential hypertension. Sequencing of the Hmgcr locus in genetically hypertensive BPH (blood pressure high), genetically hypotensive BPL (blood pressure low) and genetically normotensive BPN (blood pressure normal) mice yielded a number of single nucleotide polymorphisms (SNPs). BPH/BPL/BPN Hmgcr promoter-luciferase reporter constructs were generated and transfected into liver HepG2, ovarian CHO, kidney HEK-293 and neuronal N2A cells for functional characterization of the promoter SNPs. The BPH-Hmgcr promoter showed significantly less activity than the BPL-Hmgcr promoter under basal as well as nicotine/cholesterol-treated conditions. This finding was consistent with lower endogenous Hmgcr expression in liver and lower plasma cholesterol in BPH mice. Transfection experiments using 5′-promoter deletion constructs (strategically made to assess the functional significance of each promoter SNP) and computational analysis predicted lower binding affinities of transcription factors c-Fos, n-Myc and Max with the BPH-promoter as compared to the BPL-promoter. Corroboratively, the BPH promoter-luciferase reporter construct co-transfected with expression plasmids of these transcription factors displayed less pronounced augmentation of luciferase activity than the BPL construct, particularly at lower amounts of transcription factor plasmids. Electrophoretic mobility shift assays also showed diminished interactions of the BPH promoter with HepG2 nuclear proteins. Taken together, this study provides mechanistic basis for the differential Hmgcr expression in these mouse models of human essential hypertension and have implications for better understanding the role of this gene in regulation of blood pressure

Studies of common variations in two candidate genes of dyslipidemia and coronary artery disease

Ph.DDOCTOR OF PHILOSOPH

Metabolic syndrome, the leptin gene and kidney disease in non-diabetic black South Africans

Includes abstract.Includes bibliographical references (leaves 226-256).Obesity is a worldwide problem and is a factor in the pathogenesis of the metabolic syndrome and kidney disease through the development of obesity-related hypertension and neurohormonal mechanisms that include the action of leptin. As there appear to be no focussed studies that have looked at the association of the LEP gene with kidney disease phenotypes or cardiovascular disease markers like hypertension, the metabolic syndrome and obesity, and especially so in native black Africans, this study sought to establish an association between the obesity gene (LEP) and kidney disease phenotypes (independent of diabetes and hypertension) in a homogenous black African population

Genetic Heterogeneity of Familial Hypercholesterolemia: Repercussions for Molecular Diagnosis

: Genetics of Familial Hypercholesterolemia (FH) is ascribable to pathogenic variants in genes encoding proteins leading to an impaired LDL uptake by the LDL receptor (LDLR). Two forms of the disease are possible, heterozygous (HeFH) and homozygous (HoFH), caused by one or two pathogenic variants, respectively, in the three main genes that are responsible for the autosomal dominant disease: LDLR, APOB and PCSK9 genes. The HeFH is the most common genetic disease in humans, being the prevalence about 1:300. Variants in the LDLRAP1 gene causes FH with a recessive inheritance and a specific APOE variant was described as causative of FH, contributing to increase FH genetic heterogeneity. In addition, variants in genes causing other dyslipidemias showing phenotypes overlapping with FH may mimic FH in patients without causative variants (FH-phenocopies; ABCG5, ABCG8, CYP27A1 and LIPA genes) or act as phenotype modifiers in patients with a pathogenic variant in a causative gene. The presence of several common variants was also considered a genetic basis of FH and several polygenic risk scores (PRS) have been described. The presence of a variant in modifier genes or high PRS in HeFH further exacerbates the phenotype, partially justifying its variability among patients. This review aims to report the updates on the genetic and molecular bases of FH with their implication for molecular diagnosis

The APOA4 T347S variant is associated with reduced plasma TAOS in subjects with diabetes mellitus and cardiovascular disease

Apolipoprotein A-IV (apoA-IV) has been postulated to be antiatherogenic. Transgenic APOA4/Apoe−/− mice are protected against atherosclerosis, with plasma apoA-IV displaying antioxidant activity in vitro. In humans, there is an inverse relationship between apoA-IV levels and risk of coronary heart disease (CHD). Furthermore, the APOA4 T347S rare allele has been associated with increased risk of CHD and reduced apoA-IV levels. Reduced total antioxidant status (TAOS) due to increased oxidative stress is implicated in the process of atherogenesis. Thus, this study aimed to examine the association between the APOA4 T347S variant and TAOS in diabetic patients with (n = 196) or without (n = 509) cardiovascular disease (CVD). A higher percentage of CVD patients were present in the lowest quartile of TAOS, compared with the rest (P = 0.04). Overall, there was no association between genotype and TAOS. However, in patients with CVD, homozygotes for the S347 allele had significantly lower TAOS compared with TT and TS subjects (31.2 ± 9.89% and 42.5 ± 13.04% TAOS, respectively; P = 0.0024), an effect that was not seen in the patients without CVD. This study offers direct support for an antioxidant capacity of apoA-IV, thus providing some explanation for the antiatherogenic role of apoA-IV and the higher CVD risk in S347 homozygotes

Genomics to elucidate the molecular basis of calcific aortic valve disease

Le rétrécissement valvulaire aortique (RVA) est causé par une calcification et une fibrose progressive de la valve aortique. Le risque de développer la maladie augmente avec l’âge. À cause de l'augmentation de l'espérance de vie, le RVA est devenu un problème de santé publique. Le RVA est fatal en absence de traitement médical. Actuellement, la chirurgie est le seul traitement pour le stade sévère de la maladie, mais près de 50% des individus avec RVA n’y sont pas éligibles, principalement due à la présence de comorbidités. Plusieurs processus biologiques ont été associés à la maladie, mais les voies moléculaires spécifiques et les gènes impliqués dans le développement et la progression du RVA ne sont pas connus. Il est donc urgent de découvrir les gènes de susceptibilité pour le RVA afin d’identifier les personnes à risque ainsi que les biomarqueurs et les cibles thérapeutiques pouvant mener au développement de médicaments pour inverser ou limiter la progression de la maladie. L'objectif de cette thèse de doctorat était d'identifier la base moléculaire du RVA. Des approches modernes en génomique, incluant l’étude de gènes candidats et le criblage génomique par association (GWAS), ont été réalisées à l’aide de collections d’ADN provenant d’un grand nombre de patients bien caractérisés pour le RVA. Des études complémentaires en transciptomique ont comparé le profil d’expression global des gènes entre des valves calcifiées et non-calcifiées à l’aide de biopuces à ADN et de séquençage de l'ARN. Une première étude a identifié des variations dans le gène NOTCH1 et suggère pour la première fois la présence d'un polymorphisme commun dans ce gène conférant une susceptibilité au RVA. La deuxième étude a combiné par méta-analyse deux GWAS de patients provenant de la ville de Québec et Paris (France) aux données transcriptomiques. Cette étude de génomique intégrative a confirmé le rôle de RUNX2 dans le RVA et a permis l’identification d’un nouveau gène de susceptibilité, CACNA1C. Les troisième et quatrième études sur l’expression des gènes ont permis de mieux comprendre les bases moléculaires de la calcification des valves aortiques bicuspides et ainsi d’identifier de nouvelles cibles thérapeutiques pour le RVA. Les données générées par ce projet sont la base de futures découvertes importantes qui permettront d'améliorer les options de traitement et la qualité de vie des patients atteints du RVA.Calcific aortic valve disease (CAVD) is a common disease that causes the narrowing of the aortic valve due to fibrosis and calcification of the valve leaflets. The risk of CAVD increases with age. Due to the increase in life expectancy, CAVD is becoming a major public health problem. CAVD is fatal in the absence of medical treatment. Currently, surgery is the only treatment for severe stages of the disease, but nearly 50% of individual with CAVD are not eligible for surgery; mainly because of the presence of comorbidities. Several biological processes have been associated with the disease but the specific cell signaling pathways and genes implicated in CAVD development and progression are yet to be discovered. Thus, it is urgent to discover the susceptibility genes for CAVD, which will help identify individuals at risk as well as biomarkers and therapeutic targets for developing medication to reverse or limit disease progression. The objective of this thesis was to identify the molecular basis of CAVD. Modern genomic approaches including candidate gene and genome-wide association studies (GWAS) were performed with large DNA collections of patients well-characterized for CAVD. Whole-genome gene expression studies were also performed to compare calcified bicuspid and tricuspid valves with normal aortic valves using microarrays and RNA-Sequencing. A GWAS meta-analysis was performed using two cohorts of patients with CAVD from Quebec City and Paris. The integration of different whole-genome approaches revealed a new gene associated with CAVD called CACNA1C. This work also confirmed the potential role of NOTCH1 and RUNX2 in CAVD. In addition, this work identified new genes differentially expressed in calcified compared to normal aortic valves that are implicated in biological processes involved in the disease. These new developments are important to better understand the pathophysiological processes implicated in aortic valve calcification. Several genes differentially expressed in calcified compared to normal valves are targets for existing and emerging drugs. In general, this work has increased the knowledge about the etiology of CAVD in patients with bicuspid and tricuspid aortic valves and has identified new susceptibility genes for the development of this disease. The data generated by this project are the base of future important discoveries that will improve treatment options and the quality of life of patients with CAVD

Identification of novel biomarkers and candidate genes associated to lipid traits : improving the lipid metabolism knowledge base

FH, the most common monogenic dyslipidaemia, is characterised by increased circulating LDL-C levels leading to premature cardiovascular disease when undiagnosed or untreated. Current guidelines support genetic testing in patients fulfilling clinical diagnostic criteria and cascade screening of their family members. However, about half of clinical FH patients do not present pathogenic variants in the known disease genes (LDLR, APOB, PCSK9), and these most likely suffer from polygenic hypercholesterolaemia, which translates into a relatively low yield of genetic screening programs. This project aimed to identify new biomarkers able to improve the distinction between monogenic and polygenic profiles. Using a machine-learning approach in a paediatric dataset, tested for disease causative genes and investigated with an extended lipid profile, we developed new models that classify FH patients with higher specificity than currently used methods. The best performing models incorporated parameters absent from the common FH clinical criteria, which rely only on TC and LDL-C. A hierarchical clustering analysis of the same dataset showed that the study population can be clearly divided in three groups of dyslipidaemic individuals, showing the complexity of the dyslipidaemic biological context and the need of an integrative and multidisciplinary approach for biomarker selection. Both clustering and modelling analysis have revealed that the extended lipid profile contains important biomarkers. The exploration of lipid metabolic pathways associated with the identified biomarkers allowed us to identify a set of related genes. Using additional information from public databases, including gene expression data, associated GWAS and GO terms, we defined a universe of lipid-related genes and molecular interactions relevant for the dyslipidaemic context and future genetic studies. All this information was used to establish a new lipid knowledge base available online. The obtained results can be applied to improve the yield of genetic screening programs and decrease the associated costs, and also provide novel contributions to our understanding of dyslipidaemias

An assessment of gene polymorphisms in young South African Indians with coronary artery disease and the effect of atorvastan in vitro.

Thesis (M.Med.)-University of KwaZulu-Natal, Durban, 2012.The global burden of heart disease increases every year. It has been estimated that by the year 2020, coronary artery disease (CAD) will be the number one cause of death worldwide. Indian populations throughout the world have the highest prevalence of CAD and early onset of the disease compared to other ethnic groups. Glutathione S-transferases (GSTs) detoxify environmental agents which influence the onset and progression of disease. Dysfunctional detoxification enzymes are responsible for prolonged exposure to reactive molecules and can contribute to endothelial damage, an underlying factor in CAD. Uncoupling proteins (UCPs) 2 and 3 play an important role in the regulation of oxidative stress which contributes to chronic inflammation. Coronary artery disease is a chronic inflammatory disorder characterized by elevated levels of C-reactive protein (CRP) and pro-inflammatory cytokines such as interleukin 6 (IL-6). Polymorphisms of these genes have been linked to CAD and other chronic diseases. Statins, metabolised in the liver, are the most commonly used drug to control atherosclerosis progression in CAD patients. The pleiotropic effects of statins have been attributed to both favourable and adverse outcomes in CAD patients particularly related to myopathy and hepatotoxicity. All patients (n=102) recruited into this study were South African Indian males. A corresponding age-, gender- and ethnicity-matched control group (n=100) was also recruited. The frequency of the GSTM1 +/0, GSTP1 A105/G105, IL6 -174G/C and CRP -390C/A/T genotypes was assessed by polymerase chain reaction (PCR) and PCR restriction fragment length polymorphism (PCR-RFLP). For the in vitro study, the biological effect of atorvastatin on HepG2 cells was assessed. The metabolic activity, cytotoxicity, oxidative stress and nitric oxide production was assessed by the ATP, lactate dehydrogenase (LDH), thiobarbituric acid reactive substance (TBARS) and Griess assays, respectively. The profile of 84 microRNA (miRNA) species was evaluated using the miRNA Pathway Finder PCR SuperArray. The predicted targets of up-regulated miRNAs were determined using the online software, Targetscan. The mRNA levels of guanidinoacetoacetate (GAMT), arginine glycine aminotransferase (AGAT) and spermine oxidase (SMO) were determined using quantitative PCR. Western blotting was used to determine GAMT and phosphorylated p53 levels in treated cells. The GSTM1 0/0 and GSTP1 A105/A105 genotypes occurred at higher frequencies in CAD patients compared with the control group (36% vs. 18% and 65% vs. 48%, respectively). A significant association with CAD was observed in GSTM1 0/0 (odds ratio (OR)=2.593; 95% confidence interval (CI) 1.353 - 4.971; p=0.0043) and GSTP1 A105/A105 OR=0.6011; 95% CI 0.3803 - 0.9503; p=0.0377). We found a significant association between smoking and CAD; the presence of either of the respective genotypes together with smoking increased the CAD risk (GSTP1 A105 relative risk (RR)=1.382; 95% CI 0.958 - 1.994; p=0.0987 and GSTM1 null RR=1.725; 95% CI 1.044 - 2.851; p=0.0221). The UCP2 -866G/A and UCP3 -55C/C genotypes occurred at highest frequency in CAD patients (59% vs. 52% and 66% vs. controls: 63% respectively) and did not influence the risk of CAD. Homozygous UCP3 -55T/T genotype was associated with highest fasting glucose (11.87±3.7mmol/L vs. C/C:6.11±0.27mmol/L and C/T:6.48±0.57mmol/L, p=0.0025), HbA1c (10.05±2.57% vs. C/C:6.44±0.21% and C/T:6.76±0.35%, p=0.0006) and triglycerides (6.47±1.7mmol/Lvs. C/C:2.33±0.17mmol/L and C/T:2.06±0.25mmol/L, p<0.0001) in CAD patients. A significant association between the G allele of the IL6 -174 polymorphism and non-diabetic CAD patients was found (p=0.0431 odds ratio: 1.307, 95% CI: 1.047-1.632). A significant association with the C allele of the -390 CRP triallelic variants and CAD (p=0.021 odds ratio: 1.75, 95% CI: 1.109-2.778) was also found using a contingency of the C allele vs. the minor A and T allele frequencies. The strength of the association of the C allele with non- diabetic CAD subjects was much higher (p=0.0048 odds ratio: 2.634, 95% CI: 1.350-5.138). Circulating median levels of IL-6 (0.9 (0.90, 0.91) pg/ml and 0.9 (0.87, 0.92) pg/ml) and CRP (5.65 (1.9, 8.2) mg/l and 2.90 (1.93, 8.35) mg/l) were similar between CAD patients and controls, respectively. A similar finding was observed between controls and non-diabetic CAD subjects. Levels of IL-6 and CRP in CAD subjects were not significantly influenced by polymorphic variants of IL-6 and CRP. In the control group, the level of IL-6 was significantly influenced by the IL6 -174 G allele (p=0.0002) and the CRP -390 C allele (p=0.0416), where subjects with the homozygous GG (0.9 (0.9, 1,78) pg/ml) and CC (0.9 (0.9, 0.95) pg/ml) genotype had higher levels than the C allele carriers (0.9 (0.64, 0.91) pg/ml) or A and T carriers (0.9 (0.69, 0.91) pg/ml) combined. The lowest measure of proliferation/metabolism in HepG2 cells was observed at 20μM atorvastatin, with 82±9.8% viability. The level of cytotoxicity was increased in statin treated cells from 0.95±0.02 units to 1.11±0.03 units (p=0.001) and malondialdehyde levels was reduced from 0.133±0.003 units to 0.126±0.005 units (p=0.009) whilst nitrite levels were elevated (0.0312±0.003 units vs. control: 0.027±0.001 units, p=0.044). MicroRNAs most significantly upregulated by atorvastatin included miR-302a-3p (3.05-fold), miR-302c-3p (3.61-fold), miR-124-3p (3.90-fold) and miR-222-3p (4.4-fold); miR-19a-3p, miR-101-3p and let-7g were downregulated (3.63-fold, 2.92-fold, 2.81-fold, respectively). A list of miRNA targets identified included those with a role in metabolism and inflammation. The miR-124a specifically targets the mRNA of GAMT and SMO

Genetics and genomics of cholesterol and polyunsaturated fatty acid metabolism in relation to coronary heart disease risk

Background Coronary heart disease (CHD) continues to be a leading cause of morbidity and mortality among adults worldwide. Deregulated lipid metabolism (dyslipidemia) that manifests as hypercholesterolemia, hypertriglyceridemia, low high-density-lipoprotein (HDL) cholesterol levels or a combination of those, is an established risk factor for CHD among other established risk factors. Linoleic acid (LA, C18:2n-6) and alpha-linolenic acid (ALA, C18:3n-3) are polyunsaturated fatty acids (PUFAs) that cannot be synthesized de novo by human or animal cells, and therefore must be obtained from the diet. From these two PUFAs, two series of long-chain PUFAs are formed; the omega-6 series that are synthesized from LA, and the omega-3 series that are from ALA. Formation of these long-chain PUFAs involves a series of alternate desaturation and elongation processes. These PUFAs, especially, omega-3 PUFAs, have long been observed to reduce CHD risk. In contrast to the consistently observed cardiovascular protective effects of omega-3 PUFAs, accumulating evidence suggests a potential pro-atherogenic effects of omega-6 PUFAs, which is now still under debate. It has been estimated that genetic factors account for 26%-69% of inter-individual variation in CHD risk. These genetic factors are thought to influence CHD risk both directly and through effects on known CHD risk factors such as plasma lipid levels. The heritability of plasma lipid levels (total cholesterol, LDL cholesterol, HDL cholesterol, and triglycerides (TG)) is estimated to be about 50% (ranging from 28%-78%). With the success of recent genome-wide association studies (GWAS), many genetic variants underlying intermediate risk factors of CHD (including plasma lipid levels) and CHD itself have been identified. Whether this new genetic information could be used to improve CHD risk prediction is still marginally explored, and for some variants, the underlying mechanisms for their mediated effects on CHD risk are still unknown. The aim of this research is to investigate common genetic determinants of plasma lipid levels (cholesterol and polyunsaturated fatty acid levels) using a pathway-driven approach, and to explore whether such common genetic variants could be used to improve CHD prediction using a population based genetic approach. An additional aim was to explore the underlying mechanisms of cardiovascular protective effects of PUFAs using a genomic approach. Methods In order to explore whether common genetic variants are involved in determining plasma cholesterol levels, we used data from 3575 men and women from the Doetinchem cohort, which was examined thrice over 11 years. They were genotyped on 384 single nucleotide polymorphisms (SNPs) across 251 genes in regulatory pathways that control fatty acid, glucose, cholesterol and bile salt homeostasis. In order to explore whether common genetic variants could be used to predict future CHD risk,we used the data from CAREMA cohort that involved 15,236 middle-aged subjects and was followed up for a median of 12.1 years. 179 SNPs associated with CHD or its risk factors in GWAS published up to May 2, 2011 were genotyped in the 2221 subcohort members and 742 incident CHD cases. In addition, fatty acids from plasma cholesteryl esters were quantified in 1323 subcohort members and 537 CHD cases. They were used to explore whether δ-5 and δ-6 desaturase activities were associated with CHD risk. In order to perform a comparative analysis of the effects of fenofibrate and fish oil at transcriptome and metabolome level, 34 mice were randomized by weight-matching into three groups (n = 10 in control group, and n = 12 in fenofibrate or fish oil intervention group), and fed a research diet supplemented with sunflower oil (containing 81.3% oleic acid, 7% energy intake) in control group, sunflower oil (7% energy intake) and fenofibrate (0.03% w/w) in fenofibrate group, and fish oil (Marinol C-38 fish oil: 23.1% EPA and 21.1% DHA, 7% energy intake) in fish oil group for 2 weeks. At the end of treatment, mice were fasted with drinking water available, and were subsequently sacrificed by cervical dislocation under isoflurane anesthesia. Blood was collected via orbital puncture. Livers were dissected, directly frozen in liquid nitrogen and stored at −80°C until further analysis. Microarray analysis was performed on individual mouse livers. The LC-MS method was used for measuring plasma lipids and non-esterified free fatty acids, and the GC-MS method was used for measuring a broad range of metabolites. Results In chapter 2, 3, and 4, common genetic variants in the genes along known cholesterol metabolic pathways, such as bile acid and bile metabolic pathways, the HDL cholesterol metabolic pathway, and the plasma total cholesterol metabolic pathway, are involved in determining plasma cholesterol levels. The modest effect associated with each individual variant, however, caused the amount of heritability explained by them in aggregate to be relatively small: 13 single nucleotide polymorphisms (SNPs) explained 4% of inter-individual variation in HDL cholesterol levels (Chapter 3), whereas 12 SNPs explained 6.9% of inter-individual variation in total cholesterol levels (Chapter 4). In chapter 5, we found that genetic variants in the FADS1 gene potentially interact with dietary PUFA intake to affect plasma cholesterol levels. A high intake of omega-3 PUFAs was associated with increased plasma non-HDL cholesterol levels, consistent with increased plasma LDL cholesterol levels observed in fish oil intervention studies. Increased LDL cholesterol levels could be due to hepatic downregulation of the LDL receptor gene (LDLR) in subjects with high omega-3 PUFA intakes. This is further confirmed by the findings described in Chapter 6 that the hepatic LDLR gene was significantly downregulated in fish oil treated mice. This study also confirmed PUFAs to be weak PPAR ligands. The increased plasma HDL cholesterol levels in the subjects with high PUFA intakes in Chapter 5 could be due to PPARs-mediated genes that are directly involved in HDL lipoprotein metabolism. All these may explain the changes in blood cholesterol levels upon PUFA intake observed in human studies. In Chapter 6, we found that not only downregulation in the hepatic lipogenic pathway but also upregulation in hepatic fatty acid oxidation pathways are involved in lowering plasma TG levels upon fish oil treatment. The striking parallel between fenofibrate and fish oil in hepatic downregulation of blood coagulation and fibrinolysis pathways suggest that hepatic activation of PPARα is potentially one of the mechanisms responsible for anticoagulation effects of fish oil treatment observed in humans. In Chapter 7, with confirmed effects of rs174547 in FADS1 on PUFA levels and δ-5 desaturase activities and also protective effects of DHA on CHD, we observed a reduced CHD risk of increased δ-5 desaturase activity. Increased δ-5 desaturase activity could contribute to the intracellular increase of EPA and especially arachidonic acid (C20:4n-6) levels. Despite the potential pro-coagulant and pro-inflammatory effects of increased exposures of arachidonic acid and its derived eicosanoid metabolites, there is no evidence of increased CHD risk with increased habitual arachidonic acid intake so far. Some of the oxygenated metabolites of arachidonic acid were found to have anti-inflammatory and pro-resolving actions. High dietary n-6 PUFA intakes or high plasma n-6 PUFA levels are associated with increased blood HDL cholesterol levels and reduced TG (or VLDL particle) levels. All these point to a potential cardiovascular protective effect of n-6 PUFAs. The fact that increased EPA and/or DHA levels associated with increased δ-5 desaturase activity protect against CHD is consistent with the current established cardiovascular protective effect of increased n-3 PUFA exposure, especially EPA and DHA. In Chapter 8, the current known common genetic variants associated with CHD risk factors (blood pressure, obesity, blood lipid levels, and type 2 diabetes) and CHD itself from published GWAS are examined to see whether they provide additional value in CHD risk prediction beyond established traditional CHD risk factors. We constructed several gene risk scores (GRS) for CHD that consisted of SNPs directly associated with CHD or intermediate CHD risk factors in GWAS, and tested their relationship to incident CHD and their potential to improve risk prediction. The weighted GRS based on 29 CHD SNPs predicted future CHD independently from established traditional risk factors. However, the GRS based on 153 SNPs associated with intermediate risk factors and the GRS based on the total 179 SNPs did not. None of them improved risk discrimination. Risk classification of CHD, measured by the net reclassification index, improved only when the GRS based on the 29 CHD SNPs was used. These results are generally consistent with the results from other recent studies that took a similar approach as ours. However, the final conclusions on GRS application could not be drawn at this early stage. With a great understanding of the genetic architecture of CHD in the future, more research should be done on this topic. Conclusions Our studies in this thesis demonstrated that common genetic variants along the known candidate cholesterol metabolic pathways are involved in determining the plasma cholesterol levels. PUFAs are not only weak PPARα ligands, but also inhibit SREBPs’ activities. All these could explain part of the cardiovascular protective effects (increased HDL cholesterol levels and reduced TG levels) of PUFAs, increased LDL cholesterol levels upon fish oil treatment in humans, and potentially reduced CHD risk of high δ-5 desaturase activities. At present, many questions remain about the feasibility of genetic risk prediction of CHD. Clinicians should continue to inquire about family history of CHD for risk prediction, because this represents a simple, cheap, and useful risk factor for CHD that likely represents the net integrated effects from hundreds of genetic risk variants. </p