Purpose of review
In this review, we will highlight recent advances in identifying genes and gene regions responsible for the variation in serum lipid levels. We will also consider the next directions for research based on these advances.
Large-scale genome-wide association studies have successfully screened common variants across the genome for association with serum lipids and have generated novel hypotheses about the causes of serum lipid variation.
Deep sequencing of GWA signals promises to expand the catalog of variants responsible for serum lipid variation and with a full catalog of variants, we may develop a panel of polymorphisms with clinical utility. In parallel, functional exploration of the GWA signals should expand our knowledge of lipoprotein metabolism and generate targets for pharmacologic intervention.
genetics; cholesterol; genome-wide association; lipids; triglycerides
Diabetes mellitus is a major risk factor for coronary heart disease (CHD), renal failure, retinopathy, and neuropathy. Lowering glycosylated hemoglobin (HbA1c) as well as low-density lipoprotein-cholesterol (LDL-C) has been associated with a decreased risk of these complications. We evaluated the utility of glycated albumin (GA) and direct LDL-C, 2 novel assays, as compared to HbA1c and calculated LDL-C, in evaluating diabetes control and lipid in a heterogeneous population and in specific subgroups of patients with type 2 diabetes mellitus.
We obtained fasting blood samples and measured HbA1c, GA, and direct LDL-C, as well as other parameters, in a multi-ethnic population of 616 male and female patients with type 2 diabetes and 895 non-diabetic controls.
HbA1c and GA levels, which measure different periods of glycemia, had a correlation of r=0.70 (p<0.001), and mean values in patients were 38.7% and 43.4% higher, respectively, than controls in men, and 41.1% and 40.1% higher, respectively, than controls, in women (both p<0.001). Calculated and direct LDL-C values correlated very highly (r=0.96, p<0.001). The correlations between HbA1c and GA, and between calculated and direct LDL-C were similar for subgroups defined by gender, race, age, and other factors.
Calculated LDL-C provides an accurate assessment of fasting LDL-C compared with a direct measurement in most subjects, except for those with hypertriglyceridemia, and GA correlates with HbA1c in diabetic and non-diabetic subjects and may serve as a reasonable marker of short term diabetic control.
glycated albumin; low density lipoprotein cholesterol; type 2 diabetes
Apo E mutants are associated with type III hyperlipoproteinemia characterized by high cholesterol and triglycerides levels. Autosomal Dominant Hypercholesterolemia (ADH), due to mutations in the LDLR, APOB or PCSK9 genes, is characterized by an isolated elevation of cholesterol due to high levels of low-density lipoproteins (LDL).
We now report an exceptionally large family including 14 members with ADH. Through genome wide mapping, analysis of regional/functional candidate genes and whole exome sequencing, we identified a mutation in the APOE gene, p.Leu167del previously reported associated with sea-blue histiocytosis and familial combined hyperlipidemia. We confirmed the involvement of the APOE p.Leu167del in ADH, with (1) a predicted destabilization of an alpha-helix in the binding domain; (2) a decreased apo E level in LDL; and (3) a decreased catabolism of LDL.
Our results show that mutations in the APOE gene can be associated with bona fide ADH.
Apolipoproteine E; Autosomal Dominant Hypercholesterolemia (ADH); mutation; low-density lipoproteins (LDL); linkage analysis
This study evaluated the association of timing of lipid levels and lipid genetic risk score (GRS) with subclinical atherosclerosis.
Atherosclerosis is a slowly progressive disorder influenced by suboptimal lipid levels. Long-term versus contemporary lipid levels may more strongly impact the development of coronary artery calcium (CAC).
Framingham Heart Study (FHS) Offspring Cohort participants (n=1156, 44%M, 63±9 years) underwent serial fasting lipids [low-density lipoprotein (LDL-C), high-density lipoprotein, and triglycerides], Exam 1 (1971–1975) – Exam 7 (1998–2001). FHS Third Generation Cohort participants (n=1954, 55%M, 45±6 years) had fasting lipid profiles assessed, 2002–2005. Computed tomography (2002–2005) measured CAC. Lipid GRSs were computed from significantly associated single nucleotide polymorphisms. The association between early, long-term average, and contemporary lipids, and lipid GRS, with elevated CAC was assessed using logistic regression.
In FHS Offspring, Exam 1 and long-term average versus Exam 7 lipid measurements, including untreated lipid levels, were strongly associated with elevated CAC. In the FHS Third Generation, contemporary lipids were associated with CAC. The LDL-C GRS was associated with CAC (age/sex-adjusted OR 1.14, 95%CI 1.00–1.29, p=0.04). However, addition of the GRS to the lipid models did not result in a significant increase in the OR or C-statistic for any lipid measure.
Early and long-term average lipid levels, as compared with contemporary measures, are more strongly associated with elevated CAC. Lipid GRS was associated with lipid levels but did not predict elevated CAC. Adult early and long-term average lipid levels provide important information when assessing subclinical atherosclerosis and cardiovascular risk.
Lipids; Genetic risk score; Coronary artery calcium
Diminished serum paraoxonase and arylesterase activities (measures of paraoxonase-1 [PON-1] function) in humans have been linked to heightened systemic oxidative stress and atherosclerosis risk. The clinical prognostic utility of measuring distinct PON1 activities has not been established, and the genetic determinants of PON-1 activities are not known.
Methods and Results
We established analytically robust high throughput assays for serum paraoxonase and arylesterase activities and measured these in 3,668 stable subjects undergoing elective coronary angiography without acute coronary syndrome, and were prospectively followed for major adverse cardiac events (MACE = death, myocardial infarction, stroke) over 3 years. Low serum arylesterase and paraoxonase activities were both associated with increased risk for MACE, with arylesterase activity showing greatest prognostic value (Q4 versus Q1, Hazard Ratio [HR] 2.63, 95%CI 1.97–3.50, p<0.01). Arylesterase remained significant after adjusting for traditional risk factors, C-reactive protein, and creatinine clearance (HR 2.20, 95%CI 1.60–3.02, p<0.01), predicted future development of MACE in both primary and secondary prevention populations, and reclassified risk categories incrementally to traditional clinical variables. A genome-wide association study (GWAS) identified distinct SNPs within the PON-1 gene that were highly significantly associated with serum paraoxonase (1.18×10−303) or arylesterase (4.99×10−116) activity but these variants were not associated with either 3-year MACE risk in an angiographic cohort (n=2,136) or history of either coronary artery disease or myocardial infarction in the CARDIoGRAM consortium (n~80,000 subjects).
Diminished serum arylesterase activity, but not the genetic determinants of PON-1 functional measures, provides incremental prognostic value and clinical reclassification of stable subjects at risk of developing MACE.
paraoxonase 1 gene; coronary artery disease; oxidative stress; arylesterase activity
Limited information is available regarding genetic contributions to valvular calcification, which is an important precursor of clinical valve disease.
We determined genomewide associations with the presence of aorticvalve calcification (among 6942 participants) and mitral annular calcification (among 3795 participants), as detected by computed tomographic (CT) scanning; the study population for this analysis included persons of white European ancestry from three cohorts participating in the Cohorts for Heart and Aging Research in Genomic Epidemiology consortium (discovery population). Findings were replicated in independent cohorts of persons with either CT-detected valvular calcification or clinical aortic stenosis.
One SNP in the lipoprotein(a) (LPA) locus (rs10455872) reached genomewide significance for the presence of aorticvalve calcification (odds ratio per allele, 2.05; P = 9.0×10−10), a finding that was replicated in additional white European, African-American, and Hispanic-American cohorts (P<0.05 for all comparisons). Genetically determined Lp(a) levels, as predicted by LPA genotype, were also associated with aorticvalve calcification, supporting a causal role for Lp(a). In prospective analyses, LPA genotype was associated with incident aortic stenosis (hazard ratio per allele, 1.68; 95% confidence interval [CI], 1.32 to 2.15) and aortic-valve replacement (hazard ratio, 1.54; 95% CI, 1.05 to 2.27) in a large Swedish cohort; the association with incident aortic stenosis was also replicated in an independent Danish cohort. Two SNPs (rs17659543 and rs13415097) near the proinflammatory gene IL1F9 achieved genomewide significance for mitral annular calcification (P = 1.5×10−8 and P = 1.8×10−8, respectively), but the findings were not replicated consistently.
Genetic variation in the LPA locus, mediated by Lp(a) levels, is associated with aorticvalve calcification across multiple ethnic groups and with incident clinical aortic stenosis. (Funded by the National Heart, Lung, and Blood Institute and others.)
Multiple genome-wide association studies (GWAS) have been performed in HIV-1 infected individuals, identifying common genetic influences on viral control and disease course. Similarly, common genetic correlates of acquisition of HIV-1 after exposure have been interrogated using GWAS, although in generally small samples. Under the auspices of the International Collaboration for the Genomics of HIV, we have combined the genome-wide single nucleotide polymorphism (SNP) data collected by 25 cohorts, studies, or institutions on HIV-1 infected individuals and compared them to carefully matched population-level data sets (a list of all collaborators appears in Note S1 in Text S1). After imputation using the 1,000 Genomes Project reference panel, we tested approximately 8 million common DNA variants (SNPs and indels) for association with HIV-1 acquisition in 6,334 infected patients and 7,247 population samples of European ancestry. Initial association testing identified the SNP rs4418214, the C allele of which is known to tag the HLA-B*57:01 and B*27:05 alleles, as genome-wide significant (p = 3.6×10−11). However, restricting analysis to individuals with a known date of seroconversion suggested that this association was due to the frailty bias in studies of lethal diseases. Further analyses including testing recessive genetic models, testing for bulk effects of non-genome-wide significant variants, stratifying by sexual or parenteral transmission risk and testing previously reported associations showed no evidence for genetic influence on HIV-1 acquisition (with the exception of CCR5Δ32 homozygosity). Thus, these data suggest that genetic influences on HIV acquisition are either rare or have smaller effects than can be detected by this sample size.
Comparing the frequency differences between common DNA variants in disease-affected cases and in unaffected controls has been successful in uncovering the genetic component of multiple diseases. This approach is most effective when large samples of cases and controls are available. Here we combine information from multiple studies of HIV infected patients, including more than 6,300 HIV+ individuals, with data from 7,200 general population samples of European ancestry to test nearly 8 million common DNA variants for an impact on HIV acquisition. With this large sample we did not observe any single common genetic variant that significantly associated with HIV acquisition. We further tested 22 variants previously identified by smaller studies as influencing HIV acquisition. With the exception of a deletion polymorphism in the CCR5 gene (CCR5Δ32) we found no convincing evidence to support these previous associations. Taken together these data suggest that genetic influences on HIV acquisition are either rare or have smaller effects than can be detected by this sample size.
Genotyping arrays are a cost effective approach when typing previously-identified genetic polymorphisms in large numbers of samples. One limitation of genotyping arrays with rare variants (e.g., minor allele frequency [MAF] <0.01) is the difficulty that automated clustering algorithms have to accurately detect and assign genotype calls. Combining intensity data from large numbers of samples may increase the ability to accurately call the genotypes of rare variants. Approximately 62,000 ethnically diverse samples from eleven Cohorts for Heart and Aging Research in Genomic Epidemiology (CHARGE) Consortium cohorts were genotyped with the Illumina HumanExome BeadChip across seven genotyping centers. The raw data files for the samples were assembled into a single project for joint calling. To assess the quality of the joint calling, concordance of genotypes in a subset of individuals having both exome chip and exome sequence data was analyzed. After exclusion of low performing SNPs on the exome chip and non-overlap of SNPs derived from sequence data, genotypes of 185,119 variants (11,356 were monomorphic) were compared in 530 individuals that had whole exome sequence data. A total of 98,113,070 pairs of genotypes were tested and 99.77% were concordant, 0.14% had missing data, and 0.09% were discordant. We report that joint calling allows the ability to accurately genotype rare variation using array technology when large sample sizes are available and best practices are followed. The cluster file from this experiment is available at www.chargeconsortium.com/main/exomechip.
High levels of cardiac troponin T measured by a highly sensitive assay (hs-cTnT) are strongly associated with incident coronary heart disease (CHD) and heart failure (HF). No large-scale genome-wide association study (GWAS) of hs-cTnT has been reported to date. We sought to identify novel genetic variants that are associated with hs-cTnT levels.
Methods and Results
We performed a GWAS in 9,491 European-Americans and 2,053 African-Americans free of CHD and HF from 2 prospective cohorts: the Atherosclerosis Risk in Communities Study (ARIC) and the Cardiovascular Health Study (CHS). GWASs were conducted in each study and race stratum. Fixed-effect meta-analyses combined the results of linear regression from 2 cohorts within each race stratum, and then across race strata to produce overall estimates and p-values. The meta-analysis identified a significant association at chromosome 8q13 (rs10091374, p = 9.06 × 10−9) near the nuclear receptor coactivator 2 (NCOA2) gene. Over-expression of NCOA2 can be detected in myoblasts An additional analysis using logistic regression and the clinically motivated 99th percentile cut-point detected a significant association at 1q32 (rs10091374, p = 9.06 × 10−8) in the gene TNNT2, which encodes the cardiac troponin T protein itself. The hs-cTnT-associated SNPs were not associated with CHD in a large case-control study, but rs12564445 was significantly associated with incident HF in ARIC European-Americans (HR = 1.16, p-value = 0.004).
We identified 2 loci, near NCOA2 and in the TNNT2 gene, at which variation was significantly associated with hs-cTnT levels. Further use of the new assay should enable replication of these results.
genetics; genome-wide association study; troponin
Cardiovascular disease encompasses a range of conditions extending from myocardial infarction to congenital heart disease most of which are heritable. Enormous effort has been invested in understanding the genes and specific DNA sequence variants responsible for this heritability. Here, we review the lessons learned for monogenic and common, complex forms of cardiovascular disease. We also discuss key challenges that remain for gene discovery and for moving from genomic localization to mechanistic insights with an emphasis on the impact of next generation sequencing and the use of pluripotent human cells to understand the mechanism by which genetic variation contributes to disease.
Earlier studies have suggested that a common genetic architecture underlies the clinically heterogeneous polygenic Fredrickson hyperlipoproteinemia (HLP) phenotypes defined by hypertriglyceridemia (HTG). Here, we comprehensively analyzed 504 HLP-HTG patients and 1213 normotriglyceridemic controls and confirmed that a spectrum of common and rare lipid-associated variants underlies this heterogeneity.
Methods and Results
First, we demonstrated that genetic determinants of plasma lipids and lipoproteins, including common variants associated with plasma triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) from the Global Lipids Genetics Consortium were associated with multiple HLP-HTG phenotypes. Second, we demonstrated that weighted risk scores composed of common TG-associated variants were distinctly increased across all HLP-HTG phenotypes compared with controls; weighted HDL-C and LDL-C risk scores were also increased, although to a less pronounced degree with some HLP-HTG phenotypes. Interestingly, decomposition of HDL-C and LDL-C risk scores revealed that pleiotropic variants (those jointly associated with TG) accounted for the greatest difference in HDL-C and LDL-C risk scores. The APOE E2/E2 genotype was significantly overrepresented in HLP type 3 versus other phenotypes. Finally, rare variants in 4 genes accumulated equally across HLP-HTG phenotypes.
HTG susceptibility and phenotypic heterogeneity are both influenced by accumulation of common and rare TG-associated variants.
lipoproteins; genetic risk scores; genetic variation; hypertriglyceridemia; pleiotropy
Rare variant accumulation studies can implicate genes in disease susceptibility when a significant burden is observed in patients versus controls. Such analyses might be particularly useful for candidate genes that are selected based on experiments other than genome-wide association studies (GWAS). We sought to determine whether rare variants in non-GWAS candidate genes identified from mouse models and human Mendelian syndromes of hypertriglyceridemia (HTG) accumulate in patients with polygenic adult-onset HTG.
Methods and Results
We resequenced protein coding regions of 3 genes with established roles (APOC2, GPIHBP1, LMF1) and 2 genes recently implicated (CREB3L3 and ZHX3) in TG metabolism. We identified 41 distinct heterozygous rare variants, including 29 singleton variants, in the combined sample; in total, we observed 47 rare variants in 413 HTG patients versus 16 in 324 controls (OR=2.3; P=0.0050). Post hoc assessment of genetic burden in individual genes using three different tests suggested that the genetic burden was most prominent in the established genes LMF1 and APOC2, and also in the recently identified CREB3L3 gene.
These extensive resequencing studies show a significant accumulation of rare genetic variants in non-GWAS candidate genes among patients with polygenic HTG, and indicate the importance of testing specific hypotheses in large-scale resequencing studies.
hyperlipoproteinemia; genetics; apolipoproteins; lipoproteins; cardiovascular diseases
Using the genome-wide association approach in individuals of European ancestry, we and others recently identified single nucleotide polymorphisms (SNPs) at 19 loci as associated with blood lipids; eight of these loci were novel. Whether these same SNPs associate with lipids in a broader range of ethnicities is unknown.
Methods and Results
We genotyped index SNPs at 19 loci in the Third United States National Health and Nutrition Examination Survey (n=7159), a population-based probability sample of the U.S. comprised primarily of non-Hispanic blacks, Mexican Americans, and non-Hispanic whites. We constructed ethnic-specific residual blood lipid levels after adjusting for age and gender. Ethnic-specific linear regression was used to test the association of genotype with blood lipids. To summarize the statistical evidence across three racial groups, we conducted a fixed-effects variance-weighted meta-analysis.
After exclusions, there were 1627 non-Hispanic blacks, 1659 Mexican Americans, and 2230 non-Hispanic whites. At five loci (1p13 near CELSR2/PSRC1/SORT1, HMGCR, CETP, LPL, and APOA5), the index SNP was associated with low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, or triglycerides in all three ethnic groups. At the remaining loci, there was mixed evidence by ethnic group. In meta-analysis, we found that, at 14 of the 19 loci, SNPs exceeded a nominal P < 0.05.
At five loci including the recently-discovered region on 1p13 near CELSR2/PSRC1/SORT1, the same SNP discovered in whites associates with blood lipids in non-Hispanic blacks and Mexican Americans. For the remaining loci, fine-mapping and resequencing will be required to definitively evaluate the relevance of each locus in individuals of African and Hispanic ancestries.
lipids; genetics; epidemiology; risk factors
Coronary artery calcification (CAC) detected by computed tomography is a non-invasive measure of coronary atherosclerosis, that underlies most cases of myocardial infarction (MI). We aimed to identify common genetic variants associated with CAC and further investigate their associations with MI.
Methods and Results
Computed tomography was used to assess quantity of CAC. A meta-analysis of genome-wide association studies for CAC was carried out in 9,961 men and women from five independent community-based cohorts, with replication in three additional independent cohorts (n=6,032). We examined the top single nucleotide polymorphisms (SNPs) associated with CAC quantity for association with MI in multiple large genome-wide association studies of MI. Genome-wide significant associations with CAC for SNPs on chromosome 9p21 near CDKN2A and CDKN2B (top SNP: rs1333049, P=7.58×10−19) and 6p24 (top SNP: rs9349379, within the PHACTR1 gene, P=2.65×10−11) replicated for CAC and for MI. Additionally, there is evidence for concordance of SNP associations with both CAC and with MI at a number of other loci, including 3q22 (MRAS gene), 13q34 (COL4A1/COL4A2 genes), and 1p13 (SORT1 gene).
SNPs in the 9p21 and PHACTR1 gene loci were strongly associated with CAC and MI, and there are suggestive associations with both CAC and MI of SNPs in additional loci. Multiple genetic loci are associated with development of both underlying coronary atherosclerosis and clinical events.
cardiac computed tomography; coronary artery calcification; coronary atherosclerosis; genome-wide association studies; myocardial infarction
We examined the association of common variants at the NPPA-NPPB locus with circulating concentrations of the natriuretic peptides, which have blood pressure–lowering properties. We genotyped SNPs at the NPPA-NPPB locus in 14,743 individuals of European ancestry, and identified associations of plasma atrial natriuretic peptide with rs5068 (P = 8 × 10−70), rs198358 (P = 8 × 10−30) and rs632793 (P = 2 × 10−10), and of plasma B-type natriuretic peptide with rs5068 (P = 3 × 10−12), rs198358 (P = 1 × 10−25) and rs632793 (P = 2 × 10−68). In 29,717 individuals, the alleles of rs5068 and rs198358 that showed association with increased circulating natriuretic peptide concentrations were also found to be associated with lower systolic (P = 2 × 10−6 and 6 × 10−5, respectively) and diastolic blood pressure (P = 1 × 10−6 and 5 × 10−5), as well as reduced odds of hypertension (OR = 0.85, 95% CI = 0.79–0.92, P = 4 × 10−5; OR = 0.90, 95% CI = 0.85–0.95, P = 2 × 10−4, respectively). Common genetic variants at the NPPA-NPPB locus found to be associated with circulating natriuretic peptide concentrations contribute to interindividual variation in blood pressure and hypertension.
Genetic association and linkage studies can provide insights into complex disease biology, guiding the development of new diagnostic and therapeutic strategies. Over the past decade, genetic association studies have largely focused on common, easy to measure genetic variants shared between many individuals. These common variants typically have subtle functional consequence and translating the resulting association signals into biological insights can be challenging. In the last few years, exome sequencing has emerged as a cost-effective strategy for extending these studies to include rare coding variants, which often have more marked functional consequences. Here, we provide practical guidance in the design and analysis of complex trait association studies focused on rare, coding variants.
Genome-wide association studies have identified hundreds of loci for type 2 diabetes, coronary artery disease and myocardial infarction, as well as for related traits such as body mass index, glucose and insulin levels, lipid levels, and blood pressure. These studies also have pointed to thousands of loci with promising but not yet compelling association evidence. To establish association at additional loci and to characterize the genome-wide significant loci by fine-mapping, we designed the “Metabochip,” a custom genotyping array that assays nearly 200,000 SNP markers. Here, we describe the Metabochip and its component SNP sets, evaluate its performance in capturing variation across the allele-frequency spectrum, describe solutions to methodological challenges commonly encountered in its analysis, and evaluate its performance as a platform for genotype imputation. The metabochip achieves dramatic cost efficiencies compared to designing single-trait follow-up reagents, and provides the opportunity to compare results across a range of related traits. The metabochip and similar custom genotyping arrays offer a powerful and cost-effective approach to follow-up large-scale genotyping and sequencing studies and advance our understanding of the genetic basis of complex human diseases and traits.
Recent genetic studies have identified hundreds of regions of the human genome that contribute to risk for type 2 diabetes, coronary artery disease and myocardial infarction, and to related quantitative traits such as body mass index, glucose and insulin levels, blood lipid levels, and blood pressure. These results motivate two central questions: (1) can further genetic investigation identify additional associated regions?; and (2) can more detailed genetic investigation help us identify the causal variants (or variants more strongly correlated with the causal variants) in the regions identified so far? Addressing these questions requires assaying many genetic variants in DNA samples from thousands of individuals, which is expensive and timeconsuming when done a few SNPs at a time. To facilitate these investigations, we designed the “Metabochip,” a custom genotyping array that assays variation in nearly 200,000 sites in the human genome. Here we describe the Metabochip, evaluate its performance in assaying human genetic variation, and describe solutions to methodological challenges commonly encountered in its analysis.
The genetic loci that have been found by genome-wide association studies to modulate risk of coronary heart disease explain only a fraction of its total variance, and gene-gene interactions have been proposed as a potential source of the remaining heritability. Given the potentially large testing burden, we sought to enrich our search space with real interactions by analyzing variants that may be more likely to interact on the basis of two distinct hypotheses: a biological hypothesis, under which MI risk is modulated by interactions between variants that are known to be relevant for its risk factors; and a statistical hypothesis, under which interacting variants individually show weak marginal association with MI. In a discovery sample of 2,967 cases of early-onset myocardial infarction (MI) and 3,075 controls from the MIGen study, we performed pair-wise SNP interaction testing using a logistic regression framework. Despite having reasonable power to detect interaction effects of plausible magnitudes, we observed no statistically significant evidence of interaction under these hypotheses, and no clear consistency between the top results in our discovery sample and those in a large validation sample of 1,766 cases of coronary heart disease and 2,938 controls from the Wellcome Trust Case-Control Consortium. Our results do not support the existence of strong interaction effects as a common risk factor for MI. Within the scope of the hypotheses we have explored, this study places a modest upper limit on the magnitude that epistatic risk effects are likely to have at the population level (odds ratio for MI risk 1.3–2.0, depending on allele frequency and interaction model).
Genome-wide association studies (GWAS) have identified a genetic variant at a locus on chromosome 1p13 that is associated with reduced risk of myocardial infarction, reduced plasma levels of LDL cholesterol (LDL-C), and markedly increased expression of the gene sortilin-1 (SORT1) in liver. Sortilin is a lysosomal sorting protein that binds ligands both in the Golgi apparatus and at the plasma membrane and traffics them to the lysosome. We previously reported that increased hepatic sortilin expression in mice reduced plasma LDL-C levels. Here we show that increased hepatic sortilin not only reduced hepatic apolipoprotein B (APOB) secretion, but also increased LDL catabolism, and that both effects were dependent on intact lysosomal targeting. Loss-of-function studies demonstrated that sortilin serves as a bona fide receptor for LDL in vivo in mice. Our data are consistent with a model in which increased hepatic sortilin binds intracellular APOB-containing particles in the Golgi apparatus as well as extracellular LDL at the plasma membrane and traffics them to the lysosome for degradation. We thus provide functional evidence that genetically increased hepatic sortilin expression both reduces hepatic APOB secretion and increases LDL catabolism, providing dual mechanisms for the very strong association between increased hepatic sortilin expression and reduced plasma LDL-C levels in humans.
Whereas it is well established that plasma lipid levels have substantial heritability within populations, it remains unclear how many of the genetic determinants reported in previous studies (largely performed in European American cohorts) are relevant in different ethnicities.
We tested a set of ∼50,000 polymorphisms from ∼2,000 candidate genes and genetic loci from genome-wide association studies (GWAS) for association with low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and triglycerides (TG) in 25,000 European Americans and 9,000 African Americans in the National Heart, Lung, and Blood Institute (NHLBI) Candidate Gene Association Resource (CARe). We replicated associations for a number of genes in one or both ethnicities and identified a novel lipid-associated variant in a locus harboring ICAM1. We compared the architecture of genetic loci associated with lipids in both African Americans and European Americans and found that the same genes were relevant across ethnic groups but the specific associated variants at each gene often differed.
We identify or provide further evidence for a number of genetic determinants of plasma lipid levels through population association studies. In many loci the determinants appear to differ substantially between African Americans and European Americans.
genome-wide association study; genetic epidemiology; genetics; subclinical atherosclerosis; carotid intima media thickness; cardiovascular disease; cohort study; meta-analysis; risk
We tested whether genetic factors distinctly contribute to either development of coronary atherosclerosis or, specifically, to myocardial infarction in existing coronary atherosclerosis.
We did two genome-wide association studies (GWAS) with coronary angiographic phenotyping in participants of European ancestry. To identify loci that predispose to angiographic coronary artery disease (CAD), we compared individuals who had this disorder (n=12 393) with those who did not (controls, n=7383). To identify loci that predispose to myocardial infarction, we compared patients who had angiographic CAD and myocardial infarction (n=5783) with those who had angiographic CAD but no myocardial infarction (n=3644).
In the comparison of patients with angiographic CAD versus controls, we identified a novel locus, ADAMTS7 (p=4·98×10−13). In the comparison of patients with angiographic CAD who had myocardial infarction versus those with angiographic CAD but no myocardial infarction, we identified a novel association at the ABO locus (p=7·62×10−9). The ABO association was attributable to the glycotransferase-deficient enzyme that encodes the ABO blood group O phenotype previously proposed to protect against myocardial infarction.
Our findings indicate that specific genetic predispositions promote the development of coronary atherosclerosis whereas others lead to myocardial infarction in the presence of coronary atherosclerosis. The relation to specific CAD phenotypes might modify how novel loci are applied in personalised risk assessment and used in the development of novel therapies for CAD.
The PennCath and MedStar studies were supported by the Cardiovascular Institute of the University of Pennsylvania, by the MedStar Health Research Institute at Washington Hospital Center and by a research grant from GlaxoSmithKline. The funding and support for the other cohorts contributing to the paper are described in the webappendix.
Background. Inflammation and chronic kidney disease (CKD) are both associated with cardiovascular disease (CVD). Whether inflammatory biomarkers are associated with kidney function and albuminuria after accounting for traditional CVD risk factors is not completely understood.
Methods. The sample comprised Framingham Offspring cohort participants (n = 3294, mean age 61, 53% women) who attended the seventh examination cycle (1998–2001). Inflammatory biomarkers [C-reactive protein (CRP), tumour necrosis factor (TNF)-alpha, interleukin-6, TNF receptor 2 (TNFR2), intercellular adhesion molecule-1 (ICAM-1), monocyte chemoattractant protein-1 (MCP-1), P-selectin, CD-40 ligand, osteoprotegerin, urinary isoprostanes, myeloperoxidase and fibrinogen] were measured on fasting blood samples. Serum creatinine-based estimated glomerular filtration rate (eGFR) and serum cystatin C concentration were used to assess kidney function. Urinary albumin-to-creatinine ratio (UACR) was used to assess albuminuria. Linear or logistic regression was used to test associations between biomarkers and kidney measures.
Results. Chronic kidney disease (CKD), defined as eGFR < 59/64 mL/min/1.73 m2 in women/men, was present in 8.8% (n = 291) of participants. TNF-alpha, interleukin-6, TNFR2, MCP-1, osteoprotegerin, myeloperoxidase and fibrinogen were higher among individuals with CKD; all biomarkers except for urinary isoprostanes were elevated in higher cystatin C quartiles; and TNF-alpha, interleukin-6, TNFR2, ICAM-1 and osteoprotegerin were elevated in higher UACR quartiles—all assessed after multivariable adjustment. Almost 6% and 17% of variability in TNFR2 were explained by CKD status and higher cystatin C quartiles, respectively.
Conclusions. Biomarkers of inflammation are associated with kidney function and albuminuria. In particular, substantial variability in soluble TNFR2 is explained by CKD and cystatin C.
albuminuria; chronic kidney disease; C-reactive protein; inflammation
Genome-wide association studies (GWAS) have successfully identified loci associated with quantitative traits, such as blood lipids. Deep resequencing studies are being utilized to catalogue the allelic spectrum at GWAS loci. The goal of these studies is to identify causative variants and missing heritability, including heritability due to low frequency and rare alleles with large phenotypic impact. Whereas rare variant efforts have primarily focused on nonsynonymous coding variants, we hypothesized that noncoding variants in these loci are also functionally important. Using the HDL-C gene LIPG as an example, we explored the effect of regulatory variants identified through resequencing of subjects at HDL-C extremes on gene expression, protein levels, and phenotype. Resequencing a portion of the LIPG promoter and 5′ UTR in human subjects with extreme HDL-C, we identified several rare variants in individuals from both extremes. Luciferase reporter assays were used to measure the effect of these rare variants on LIPG expression. Variants conferring opposing effects on gene expression were enriched in opposite extremes of the phenotypic distribution. Minor alleles of a common regulatory haplotype and noncoding GWAS SNPs were associated with reduced plasma levels of the LIPG gene product endothelial lipase (EL), consistent with its role in HDL-C catabolism. Additionally, we found that a common nonfunctional coding variant associated with HDL-C (rs2000813) is in linkage disequilibrium with a 5′ UTR variant (rs34474737) that decreases LIPG promoter activity. We attribute the gene regulatory role of rs34474737 to the observed association of the coding variant with plasma EL levels and HDL-C. Taken together, the findings show that both rare and common noncoding regulatory variants are important contributors to the allelic spectrum in complex trait loci.
Genetic association studies have identified genomic regions that affect quantifiable traits such as lipid levels. When a gene and a trait are found to be associated with one another, the gene is often further studied to determine its role in affecting the trait. One approach is to sequence the gene in individuals at the extremes of the trait's distribution with the hope of finding rare mutations that directly contribute to the trait. Until now studies using this approach have focused on genetic variation in the protein coding sequence of these genes and have been largely successful in identifying functionally important mutations. However, other studies have found an abundance of noncoding variation in the genome that may also contribute to the heritability of these traits. Here we seek to determine the contribution of such noncoding mutations to high density lipoprotein cholesterol (HDL-C) levels in humans using the HDL-C candidate gene LIPG as an example. Through a sequencing study in individuals with high and low HDL-C levels, we demonstrate that both rare and common noncoding mutations are influential contributors to the allelic spectrum of such traits and should be further characterized after initial association with the trait.
We sought to replicate the association between the kinesin-like protein 6 (KIF6) Trp719Arg polymorphism (rs20455) and clinical coronary artery disease (CAD).
Recent prospective studies suggest that carriers of the 719Arg allele in KIF6 are at increased risk of clinical CAD compared with non-carriers.
The KIF6 Trp719Arg polymorphism (rs20455) was genotyped in nineteen case-control studies of non-fatal CAD either as part of a genome-wide association study or in a formal attempt to replicate the initial positive reports.
Over 17 000 cases and 39 000 controls of European descent as well as a modest number of South Asians, African Americans, Hispanics, East Asians, and admixed cases and controls were successfully genotyped. None of the nineteen studies demonstrated an increased risk of CAD in carriers of the 719Arg allele compared with non-carriers. Regression analyses and fixed effect meta-analyses ruled out with high degree of confidence an increase of ≥2% in the risk of CAD among European 719Arg carriers. We also observed no increase in the risk of CAD among 719Arg carriers in the subset of Europeans with early onset disease (<50 years of age for males and <60 years for females) compared with similarly aged controls as well as all non-European subgroups.
The KIF6 Trp719Arg polymorphism was not associated with the risk of clinical CAD in this large replication study.
kinesin-like protein 6; KIF6; coronary artery disease; myocardial infarction; polymorphism