👤 Chintha M Kathiresan

Activation of cell cycle regulatory pathways has been detected during pathological cardiomyocyte growth. However, it has remained unclear whether DNA synthesis pathways play a direct role in cardiomyocyte hypertrophy. We previously discovered in a mouse model of hypertrophic cardiomyopathy that there was increased DNA synthesis, which led to cardiomyocyte endoreplication and replication stress-induced DNA damage. We hypothesized that targeting cardiomyocyte endoreplication pathways could reduce pathological myocardial hypertrophy. We utilized murine models of hypertrophic cardiomyopathy secondary to mutations in cardiac Mybpc3 (myosin-binding protein C3) We discovered that p21 protein peaked during the early stages of hypertrophic growth in both murine hypertrophic cardiomyopathy models and a pressure overload hypertrophy model. Using genetic manipulation of p21 expression, we discovered that cardiomyocyte endoreplication and hypertrophic growth were negatively correlated with p21 expression. Mechanistically, we discovered that p21 bound to PCNA (proliferating cell nuclear antigen), which led to a reduction of PCNA binding to POLD1 (DNA polymerase delta 1). Directly targeting PCNA or POLD1 prevented cardiomyocyte DNA synthesis and hypertrophic cardiomyocyte growth. Cardiomyocyte-selective overexpression of p21 using an adeno-associated virus vector reduced long-term pathological left ventricular hypertrophy and improved diastolic function in a preclinical murine model of hypertrophic cardiomyopathy (Myh6 Our results demonstrate that PCNA-POLD1-mediated cardiomyocyte endoreplication drives hypertrophic cardiomyocyte growth, and p21 serves as a negative regulator of this process. Targeting these pathways demonstrates therapeutic potential in preventing pathological myocardial hypertrophy. Show less

Genome-wide association studies (GWAS) have identified >250 loci for body mass index (BMI), implicating pathways related to neuronal biology. Most GWAS loci represent clusters of common, noncoding variants from which pinpointing causal genes remains challenging. Here we combined data from 718,734 individuals to discover rare and low-frequency (minor allele frequency (MAF) < 5%) coding variants associated with BMI. We identified 14 coding variants in 13 genes, of which 8 variants were in genes (ZBTB7B, ACHE, RAPGEF3, RAB21, ZFHX3, ENTPD6, ZFR2 and ZNF169) newly implicated in human obesity, 2 variants were in genes (MC4R and KSR2) previously observed to be mutated in extreme obesity and 2 variants were in GIPR. The effect sizes of rare variants are ~10 times larger than those of common variants, with the largest effect observed in carriers of an MC4R mutation introducing a stop codon (p.Tyr35Ter, MAF = 0.01%), who weighed ~7 kg more than non-carriers. Pathway analyses based on the variants associated with BMI confirm enrichment of neuronal genes and provide new evidence for adipocyte and energy expenditure biology, widening the potential of genetically supported therapeutic targets in obesity. Show less

The Million Veteran Program (MVP) was established in 2011 as a national research initiative to determine how genetic variation influences the health of US military veterans. Here we genotyped 312,571 MVP participants using a custom biobank array and linked the genetic data to laboratory and clinical phenotypes extracted from electronic health records covering a median of 10.0 years of follow-up. Among 297,626 veterans with at least one blood lipid measurement, including 57,332 black and 24,743 Hispanic participants, we tested up to around 32 million variants for association with lipid levels and identified 118 novel genome-wide significant loci after meta-analysis with data from the Global Lipids Genetics Consortium (total n > 600,000). Through a focus on mutations predicted to result in a loss of gene function and a phenome-wide association study, we propose novel indications for pharmaceutical inhibitors targeting PCSK9 (abdominal aortic aneurysm), ANGPTL4 (type 2 diabetes) and PDE3B (triglycerides and coronary disease). Show less

Angiopoietin-like 4 (ANGPTL4) is an endogenous inhibitor of lipoprotein lipase that modulates lipid levels, coronary atherosclerosis risk, and nutrient partitioning. We hypothesize that loss of ANGPTL4 function might improve glucose homeostasis and decrease risk of type 2 diabetes (T2D). We investigate protein-altering variants in ANGPTL4 among 58,124 participants in the DiscovEHR human genetics study, with follow-up studies in 82,766 T2D cases and 498,761 controls. Carriers of p.E40K, a variant that abolishes ANGPTL4 ability to inhibit lipoprotein lipase, have lower odds of T2D (odds ratio 0.89, 95% confidence interval 0.85-0.92, p = 6.3 × 10 Show less

Angiopoietin-like 3 (ANGPTL3) has emerged as a key regulator of lipoprotein metabolism in humans. Homozygous loss of ANGPTL3 function causes familial combined hypolipidemia characterized by low plasma levels of triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C). While known effects of ANGPTL3 in inhibiting lipoprotein lipase and endothelial lipase contribute to the low TG and HDL-C, respectively, the basis of low LDL-C remains unclear. Our aim was to explore the role of ANGPTL3 in modulating plasma LDL-C. We performed RNAi-mediated gene silencing of ANGPTL3 in five mouse models and in human hepatoma cells. We validated results by deleting ANGPTL3 gene using the CRISPR/Cas9 genome editing system. RNAi-mediated Angptl3 silencing in mouse livers resulted in very low TG, HDL-C and LDL-C, a pattern similar to the human phenotype. The effect was observed in wild-type and obese mice, while in hCETP/apolipoprotein (Apo) B-100 double transgenic mice, the silencing decreased LDL-C and TG, but not HDL-C. In a humanized mouse model (Apobec1 Reduced secretion and increased uptake of ApoB-containing lipoproteins may contribute to the low LDL-C observed in mice and humans with genetic ANGPTL3 deficiency. Show less

We screened variants on an exome-focused genotyping array in >300,000 participants (replication in >280,000 participants) and identified 444 independent variants in 250 loci significantly associated with total cholesterol (TC), high-density-lipoprotein cholesterol (HDL-C), low-density-lipoprotein cholesterol (LDL-C), and/or triglycerides (TG). At two loci (JAK2 and A1CF), experimental analysis in mice showed lipid changes consistent with the human data. We also found that: (i) beta-thalassemia trait carriers displayed lower TC and were protected from coronary artery disease (CAD); (ii) excluding the CETP locus, there was not a predictable relationship between plasma HDL-C and risk for age-related macular degeneration; (iii) only some mechanisms of lowering LDL-C appeared to increase risk for type 2 diabetes (T2D); and (iv) TG-lowering alleles involved in hepatic production of TG-rich lipoproteins (TM6SF2 and PNPLA3) tracked with higher liver fat, higher risk for T2D, and lower risk for CAD, whereas TG-lowering alleles involved in peripheral lipolysis (LPL and ANGPTL4) had no effect on liver fat but decreased risks for both T2D and CAD. Show less

Recent large-scale genetic sequencing efforts have identified rare coding variants in genes in the triglyceride-rich lipoprotein (TRL) clearance pathway that are protective against coronary heart disease (CHD), independently of LDL cholesterol (LDL-C) levels. Insight into the mechanisms of protection of these variants may facilitate the development of new therapies for lowering TRL levels. The gene APOC3 encodes apoC-III, a critical inhibitor of triglyceride (TG) lipolysis and remnant TRL clearance. Here we report a detailed interrogation of the mechanism of TRL lowering by the APOC3 Ala43Thr (A43T) variant, the only missense (rather than protein-truncating) variant in APOC3 reported to be TG lowering and protective against CHD. We found that both human APOC3 A43T heterozygotes and mice expressing human APOC3 A43T display markedly reduced circulating apoC-III levels. In mice, this reduction is due to impaired binding of A43T apoC-III to lipoproteins and accelerated renal catabolism of free apoC-III. Moreover, the reduced content of apoC-III in TRLs resulted in accelerated clearance of circulating TRLs. On the basis of this protective mechanism, we developed a monoclonal antibody targeting lipoprotein-bound human apoC-III that promotes circulating apoC-III clearance in mice expressing human APOC3 and enhances TRL catabolism in vivo. These data reveal the molecular mechanism by which a missense variant in APOC3 causes reduced circulating TG levels and, hence, protects from CHD. This protective mechanism has the potential to be exploited as a new therapeutic approach to reduce apoC-III levels and circulating TRL burden. Show less

A major goal of biomedicine is to understand the function of every gene in the human genome. Loss-of-function mutations can disrupt both copies of a given gene in humans and phenotypic analysis of such 'human knockouts' can provide insight into gene function. Consanguineous unions are more likely to result in offspring carrying homozygous loss-of-function mutations. In Pakistan, consanguinity rates are notably high. Here we sequence the protein-coding regions of 10,503 adult participants in the Pakistan Risk of Myocardial Infarction Study (PROMIS), designed to understand the determinants of cardiometabolic diseases in individuals from South Asia. We identified individuals carrying homozygous predicted loss-of-function (pLoF) mutations, and performed phenotypic analysis involving more than 200 biochemical and disease traits. We enumerated 49,138 rare (<1% minor allele frequency) pLoF mutations. These pLoF mutations are estimated to knock out 1,317 genes, each in at least one participant. Homozygosity for pLoF mutations at PLA2G7 was associated with absent enzymatic activity of soluble lipoprotein-associated phospholipase A2; at CYP2F1, with higher plasma interleukin-8 concentrations; at TREH, with lower concentrations of apoB-containing lipoprotein subfractions; at either A3GALT2 or NRG4, with markedly reduced plasma insulin C-peptide concentrations; and at SLC9A3R1, with mediators of calcium and phosphate signalling. Heterozygous deficiency of APOC3 has been shown to protect against coronary heart disease; we identified APOC3 homozygous pLoF carriers in our cohort. We recruited these human knockouts and challenged them with an oral fat load. Compared with family members lacking the mutation, individuals with APOC3 knocked out displayed marked blunting of the usual post-prandial rise in plasma triglycerides. Overall, these observations provide a roadmap for a 'human knockout project', a systematic effort to understand the phenotypic consequences of complete disruption of genes in humans. Show less

Therapies that inhibit CETP (cholesteryl ester transfer protein) have failed to demonstrate a reduction in risk for coronary heart disease (CHD). Human DNA sequence variants that truncate the To test whether protein-truncating variants (PTVs) at the We sequenced the exons of the Compared with noncarriers, carriers of PTV at Show less

Genome-wide association studies have so far identified 56 loci associated with risk of coronary artery disease (CAD). Many CAD loci show pleiotropy; that is, they are also associated with other diseases or traits. This study sought to systematically test if genetic variants identified for non-CAD diseases/traits also associate with CAD and to undertake a comprehensive analysis of the extent of pleiotropy of all CAD loci. In discovery analyses involving 42,335 CAD cases and 78,240 control subjects we tested the association of 29,383 common (minor allele frequency >5%) single nucleotide polymorphisms available on the exome array, which included a substantial proportion of known or suspected single nucleotide polymorphisms associated with common diseases or traits as of 2011. Suggestive association signals were replicated in an additional 30,533 cases and 42,530 control subjects. To evaluate pleiotropy, we tested CAD loci for association with cardiovascular risk factors (lipid traits, blood pressure phenotypes, body mass index, diabetes, and smoking behavior), as well as with other diseases/traits through interrogation of currently available genome-wide association study catalogs. We identified 6 new loci associated with CAD at genome-wide significance: on 2q37 (KCNJ13-GIGYF2), 6p21 (C2), 11p15 (MRVI1-CTR9), 12q13 (LRP1), 12q24 (SCARB1), and 16q13 (CETP). Risk allele frequencies ranged from 0.15 to 0.86, and odds ratio per copy of the risk allele ranged from 1.04 to 1.09. Of 62 new and known CAD loci, 24 (38.7%) showed statistical association with a traditional cardiovascular risk factor, with some showing multiple associations, and 29 (47%) showed associations at p < 1 × 10 We identified 6 loci associated with CAD at genome-wide significance. Several CAD loci show substantial pleiotropy, which may help us understand the mechanisms by which these loci affect CAD risk. Show less

Genome-wide association studies have struggled to identify functional genes and variants underlying complex phenotypes. We recruited a multi-ethnic cohort of healthy volunteers (n = 91) and used their tissue to generate induced pluripotent stem cells (iPSCs) and hepatocyte-like cells (HLCs) for genome-wide mapping of expression quantitative trait loci (eQTLs) and allele-specific expression (ASE). We identified many eQTL genes (eGenes) not observed in the comparably sized Genotype-Tissue Expression project's human liver cohort (n = 96). Focusing on blood lipid-associated loci, we performed massively parallel reporter assays to screen candidate functional variants and used genome-edited stem cells, CRISPR interference, and mouse modeling to establish rs2277862-CPNE1, rs10889356-DOCK7, rs10889356-ANGPTL3, and rs10872142-FRK as functional SNP-gene sets. We demonstrated HLC eGenes CPNE1, VKORC1, UBE2L3, and ANGPTL3 and HLC ASE gene ACAA2 to be lipid-functional genes in mouse models. These findings endorse an iPSC-based experimental framework to discover functional variants and genes contributing to complex human traits. Show less

Coronary artery disease (CAD) is a leading cause of morbidity and mortality worldwide. Although 58 genomic regions have been associated with CAD thus far, most of the heritability is unexplained, indicating that additional susceptibility loci await identification. An efficient discovery strategy may be larger-scale evaluation of promising associations suggested by genome-wide association studies (GWAS). Hence, we genotyped 56,309 participants using a targeted gene array derived from earlier GWAS results and performed meta-analysis of results with 194,427 participants previously genotyped, totaling 88,192 CAD cases and 162,544 controls. We identified 25 new SNP-CAD associations (P < 5 × 10 Show less

Genome-wide association studies (GWAS) for plasma lipid levels have mapped numerous genomic loci, with each region often containing many protein-coding genes. Targeted re-sequencing of exons is a strategy to pinpoint causal variants and genes. We performed solution-based hybrid selection of 9008 exons at 939 genes within 95 GWAS loci for plasma lipid levels and sequenced using next-generation sequencing technology individuals with extremely high as well as low to normal levels of low-density lipoprotein cholesterol (LDL-C, n = 311; mean low = 71 mg/dl versus high = 241 mg/dl), triglycerides (TG, n = 308; mean low = 75 mg/dl versus high = 1938 mg/dl), and high-density lipoprotein cholesterol (HDL-C, n = 684; mean low = 32 mg/dl versus high = 102 mg/dl). We identified 15,002 missense, nonsense, or splice site variants with a frequency <5%. We tested whether coding sequence variants, individually or aggregated within a gene, were associated with plasma lipid levels. To replicate findings, we performed sequencing in independent participants (n = 6424). Across discovery and replication sequencing, we found 6 variants with significant associations with plasma lipids. Of these, one was a novel association: p.Ser147Asn variant in APOA4 (14.3% frequency, TG OR = 0.49, P = 7.1 × 10(-4)) with TG. In gene-level association analyses where rare variants within each gene are collapsed, APOC3 (P = 2.1 × 10(-5)) and LDLR (P = 5.0 × 10(-12)) were associated with plasma lipids. After sequencing genes from 95 GWAS loci in participants with extremely high plasma lipid levels, we identified one new coding variant associated with TG. These results provide insight regarding design of similar sequencing studies with respect to sample size, follow-up, and analysis methodology. Show less

Observational epidemiological studies have associated plasma lipid concentrations with risk for coronary heart disease (CHD), but these studies cannot distinguish cause from mere correlation. Human genetic studies, when considered with the results of randomized controlled trials of medications, can potentially shed light on whether lipid biomarkers are causal for diseases. Genetic analyses and randomized trials suggest that low-density lipoprotein is causal for CHD, whereas high-density lipoprotein is not. Surprisingly, human genetic evidence suggests that lipoprotein(a) and triglyceride-rich lipoproteins causally contribute to CHD. Gene variants leading to higher levels of plasma apolipoprotein B-containing lipoproteins [low-density lipoprotein, triglyceride-rich lipoproteins, or lipoprotein(a)] consistently increase risk for CHD. For triglyceride-rich lipoproteins, the most compelling evidence revolves around lipoprotein lipase and its endogenous facilitator (APOA5 [apolipoprotein A-V]) and inhibitory proteins (APOC3 [apolipoprotein C-III], ANGPTL4 [angiopoietin like 4]). Combined, these genetic results anticipate that, beyond low-density lipoprotein, pharmacological lowering of triglyceride-rich lipoproteins or lipoprotein(a) will reduce risk for CHD, but this remains to be proven through randomized controlled trials. Show less

Myocardial infarction (MI), a leading cause of death around the world, displays a complex pattern of inheritance. When MI occurs early in life, genetic inheritance is a major component to risk. Previously, rare mutations in low-density lipoprotein (LDL) genes have been shown to contribute to MI risk in individual families, whereas common variants at more than 45 loci have been associated with MI risk in the population. Here we evaluate how rare mutations contribute to early-onset MI risk in the population. We sequenced the protein-coding regions of 9,793 genomes from patients with MI at an early age (≤50 years in males and ≤60 years in females) along with MI-free controls. We identified two genes in which rare coding-sequence mutations were more frequent in MI cases versus controls at exome-wide significance. At low-density lipoprotein receptor (LDLR), carriers of rare non-synonymous mutations were at 4.2-fold increased risk for MI; carriers of null alleles at LDLR were at even higher risk (13-fold difference). Approximately 2% of early MI cases harbour a rare, damaging mutation in LDLR; this estimate is similar to one made more than 40 years ago using an analysis of total cholesterol. Among controls, about 1 in 217 carried an LDLR coding-sequence mutation and had plasma LDL cholesterol > 190 mg dl(-1). At apolipoprotein A-V (APOA5), carriers of rare non-synonymous mutations were at 2.2-fold increased risk for MI. When compared with non-carriers, LDLR mutation carriers had higher plasma LDL cholesterol, whereas APOA5 mutation carriers had higher plasma triglycerides. Recent evidence has connected MI risk with coding-sequence mutations at two genes functionally related to APOA5, namely lipoprotein lipase and apolipoprotein C-III (refs 18, 19). Combined, these observations suggest that, as well as LDL cholesterol, disordered metabolism of triglyceride-rich lipoproteins contributes to MI risk. Show less

Plasma triglyceride levels are heritable and are correlated with the risk of coronary heart disease. Sequencing of the protein-coding regions of the human genome (the exome) has the potential to identify rare mutations that have a large effect on phenotype. We sequenced the protein-coding regions of 18,666 genes in each of 3734 participants of European or African ancestry in the Exome Sequencing Project. We conducted tests to determine whether rare mutations in coding sequence, individually or in aggregate within a gene, were associated with plasma triglyceride levels. For mutations associated with triglyceride levels, we subsequently evaluated their association with the risk of coronary heart disease in 110,970 persons. An aggregate of rare mutations in the gene encoding apolipoprotein C3 (APOC3) was associated with lower plasma triglyceride levels. Among the four mutations that drove this result, three were loss-of-function mutations: a nonsense mutation (R19X) and two splice-site mutations (IVS2+1G→A and IVS3+1G→T). The fourth was a missense mutation (A43T). Approximately 1 in 150 persons in the study was a heterozygous carrier of at least one of these four mutations. Triglyceride levels in the carriers were 39% lower than levels in noncarriers (P<1×10(-20)), and circulating levels of APOC3 in carriers were 46% lower than levels in noncarriers (P=8×10(-10)). The risk of coronary heart disease among 498 carriers of any rare APOC3 mutation was 40% lower than the risk among 110,472 noncarriers (odds ratio, 0.60; 95% confidence interval, 0.47 to 0.75; P=4×10(-6)). Rare mutations that disrupt APOC3 function were associated with lower levels of plasma triglycerides and APOC3. Carriers of these mutations were found to have a reduced risk of coronary heart disease. (Funded by the National Heart, Lung, and Blood Institute and others.). Show less

The strong observational association between total homocysteine (tHcy) concentrations and risk of coronary artery disease (CAD) and the null associations in the homocysteine-lowering trials have prompted the need to identify genetic variants associated with homocysteine concentrations and risk of CAD. We tested whether common genetic polymorphisms associated with variation in tHcy are also associated with CAD. We conducted a meta-analysis of genome-wide association studies (GWAS) on tHcy concentrations in 44,147 individuals of European descent. Polymorphisms associated with tHcy (P < 10(⁻⁸) were tested for association with CAD in 31,400 cases and 92,927 controls. Common variants at 13 loci, explaining 5.9% of the variation in tHcy, were associated with tHcy concentrations, including 6 novel loci in or near MMACHC (2.1 × 10⁻⁹), SLC17A3 (1.0 × 10⁻⁸), GTPB10 (1.7 × 10⁻⁸), CUBN (7.5 × 10⁻¹⁰), HNF1A (1.2 × 10⁻¹²)), and FUT2 (6.6 × 10⁻⁹), and variants previously reported at or near the MTHFR, MTR, CPS1, MUT, NOX4, DPEP1, and CBS genes. Individuals within the highest 10% of the genotype risk score (GRS) had 3-μmol/L higher mean tHcy concentrations than did those within the lowest 10% of the GRS (P = 1 × 10⁻³⁶). The GRS was not associated with risk of CAD (OR: 1.01; 95% CI: 0.98, 1.04; P = 0.49). We identified several novel loci that influence plasma tHcy concentrations. Overall, common genetic variants that influence plasma tHcy concentrations are not associated with risk of CAD in white populations, which further refutes the causal relevance of moderately elevated tHcy concentrations and tHcy-related pathways for CAD. Show less

Epidemiological studies support the role for a strong genetic component in the occurrence of early-onset myocardial infarction (MI), although the specific genetic variants responsible for familial clustering remain largely unknown. The Italian study of early-onset MI is a nationwide case-control study involving 1864 case patients <45 years old who were hospitalized for a first MI, and age/sex/place of origin-matched controls (n = 1864). We investigated the association between early-onset MI, lipid levels and 20 single nucleotide polymorphisms (SNPs) in the candidate genes ADIPOQ, APOA5, ALOX5AP, CYBA, IL6, LPL, PECAM1, PLA2G2A and PLA2G7, chosen because of previously reported associations with Coronary Heart Disease (CHD) or with CHD risk factors. Of all the SNPs investigated, APOA5-1131T>C [(rs662799), minor allele frequency 0.084 (95% confidence interval (CI) 0.07-0.09)] alone showed a statistically significant association with risk of early-onset MI (p = 6.7 × 10(-5)), after Bonferroni correction, with a per C allele odds ratio of 1.44 (95% CI 1.23-1.69). In controls, APOA5-1131T>C was significantly associated with raised plasma triglyceride levels (p = 0.001), compared with non-carriers, the per C allele increase being 11.4% (95% CI 4-19%), equivalent to 0.15 mmol/L (95% CI 0.11-0.20 mmol/L). In cases, the association with early MI risk remained statistically significant after adjustment for triglycerides (p = 0.006). The APOA5-1131C allele, associated with higher fasting triglyceride levels, strongly affects the risk for early-onset MI, even after adjusting for triglycerides. This raises the possibility that APOA5-1131T>C may affect the risk of early MI over and above effects mediated by triglycerides. Show less

Plasma triglyceride (TG) concentration is reemerging as an important cardiovascular disease risk factor. More complete understanding of the genes and variants that modulate plasma TG should enable development of markers for risk prediction, diagnosis, prognosis, and response to therapies and might help specify new directions for therapeutic interventions. Recent genome-wide association studies (GWAS) have identified both known and novel loci associated with plasma TG concentration. However, genetic variation at these loci explains only ∼10% of overall TG variation within the population. As the GWAS approach may be reaching its limit for discovering genetic determinants of TG, alternative genetic strategies, such as rare variant sequencing studies and evaluation of animal models, may provide complementary information to flesh out knowledge of clinically and biologically important pathways in TG metabolism. Herein, we review genes recently implicated in TG metabolism and describe how some of these genes likely modulate plasma TG concentration. We also discuss lessons regarding plasma TG metabolism learned from various genomic and genetic experimental approaches. Treatment of patients with moderate to severe hypertriglyceridemia with existing therapies is often challenging; thus, gene products and pathways found in recent genetic research studies provide hope for development of more effective clinical strategies. Show less

Genome-wide association (GWA) studies represent a powerful strategy for identifying susceptibility genes for complex diseases in human populations but results must be confirmed and replicated. Because of the close homology between mouse and human genomes, the mouse can be used to add evidence to genes suggested by human studies. We used the mouse quantitative trait loci (QTL) map to interpret results from a GWA study for genes associated with plasma HDL cholesterol levels. We first positioned single nucleotide polymorphisms (SNPs) from a human GWA study on the genomic map for mouse HDL QTL. We then used mouse bioinformatics, sequencing, and expression studies to add evidence for one well-known HDL gene (Abca1) and three newly identified genes (Galnt2, Wwox, and Cdh13), thus supporting the results of the human study. For GWA peaks that occur in human haplotype blocks with multiple genes, we examined the homologous regions in the mouse to prioritize the genes using expression, sequencing, and bioinformatics from the mouse model, showing that some genes were unlikely candidates and adding evidence for candidate genes Mvk and Mmab in one haplotype block and Fads1 and Fads2 in the second haplotype block. Our study highlights the value of mouse genetics for evaluating genes found in human GWA studies. Show less

Coronary heart disease (CHD) is the leading cause of mortality in African Americans. To identify common genetic polymorphisms associated with CHD and its risk factors (LDL- and HDL-cholesterol (LDL-C and HDL-C), hypertension, smoking, and type-2 diabetes) in individuals of African ancestry, we performed a genome-wide association study (GWAS) in 8,090 African Americans from five population-based cohorts. We replicated 17 loci previously associated with CHD or its risk factors in Caucasians. For five of these regions (CHD: CDKN2A/CDKN2B; HDL-C: FADS1-3, PLTP, LPL, and ABCA1), we could leverage the distinct linkage disequilibrium (LD) patterns in African Americans to identify DNA polymorphisms more strongly associated with the phenotypes than the previously reported index SNPs found in Caucasian populations. We also developed a new approach for association testing in admixed populations that uses allelic and local ancestry variation. Using this method, we discovered several loci that would have been missed using the basic allelic and global ancestry information only. Our conclusions suggest that no major loci uniquely explain the high prevalence of CHD in African Americans. Our project has developed resources and methods that address both admixture- and SNP-association to maximize power for genetic discovery in even larger African-American consortia. Show less

Obesity is globally prevalent and highly heritable, but its underlying genetic factors remain largely elusive. To identify genetic loci for obesity susceptibility, we examined associations between body mass index and ∼ 2.8 million SNPs in up to 123,865 individuals with targeted follow up of 42 SNPs in up to 125,931 additional individuals. We confirmed 14 known obesity susceptibility loci and identified 18 new loci associated with body mass index (P < 5 × 10⁻⁸), one of which includes a copy number variant near GPRC5B. Some loci (at MC4R, POMC, SH2B1 and BDNF) map near key hypothalamic regulators of energy balance, and one of these loci is near GIPR, an incretin receptor. Furthermore, genes in other newly associated loci may provide new insights into human body weight regulation. Show less

Genome-wide association studies (GWAS) have identified multiple loci associated with plasma lipid concentrations. Common variants at these loci together explain <10% of variation in each lipid trait. Rare variants with large individual effects may also contribute to the heritability of lipid traits; however, the extent to which rare variants affect lipid phenotypes remains to be determined. Here we show an accumulation of rare variants, or a mutation skew, in GWAS-identified genes in individuals with hypertriglyceridemia (HTG). Through GWAS, we identified common variants in APOA5, GCKR, LPL and APOB associated with HTG. Resequencing of these genes revealed a significant burden of 154 rare missense or nonsense variants in 438 individuals with HTG, compared to 53 variants in 327 controls (P = 6.2 x 10(-8)), corresponding to a carrier frequency of 28.1% of affected individuals and 15.3% of controls (P = 2.6 x 10(-5)). Considering rare variants in these genes incrementally increased the proportion of genetic variation contributing to HTG. Show less

The National Heart, Lung, and Blood Institute's Candidate Gene Association Resource (CARe), a planned cross-cohort analysis of genetic variation in cardiovascular, pulmonary, hematologic, and sleep-related traits, comprises >40,000 participants representing 4 ethnic groups in 9 community-based cohorts. The goals of CARe include the discovery of new variants associated with traits using a candidate gene approach and the discovery of new variants using the genome-wide association mapping approach specifically in African Americans. CARe has assembled DNA samples for >40,000 individuals self-identified as European American, African American, Hispanic, or Chinese American, with accompanying data on hundreds of phenotypes that have been standardized and deposited in the CARe Phenotype Database. All participants were genotyped for 7 single-nucleotide polymorphisms (SNPs) selected based on prior association evidence. We performed association analyses relating each of these SNPs to lipid traits, stratified by sex and ethnicity, and adjusted for age and age squared. In at least 2 of the ethnic groups, SNPs near CETP, LIPC, and LPL strongly replicated for association with high-density lipoprotein cholesterol concentrations, PCSK9 with low-density lipoprotein cholesterol levels, and LPL and APOA5 with serum triglycerides. Notably, some SNPs showed varying effect sizes and significance of association in different ethnic groups. The CARe Pilot Study validates the operational framework for phenotype collection, SNP genotyping, and analytic pipeline of the CARe project and validates the planned candidate gene study of approximately 2000 biological candidate loci in all participants and genome-wide association study in approximately 8000 African American participants. CARe will serve as a valuable resource for the scientific community. Show less

Genome-wide association studies in cohorts of European descent have identified novel genomic regions as associated with lipids, but their relevance in African Americans remains unclear. We genotyped 8 index single nucleotide polymorphisms (SNPs) and 488 tagging SNPs across 8 novel lipid loci in the Jackson Heart Study, a community-based cohort of 4605 African Americans. For each trait, we calculated residuals adjusted for age, sex, and global ancestry and performed multivariable linear regression to detect genotype-phenotype association with adjustment for local ancestry. To explore admixture effects, we conducted stratified analyses in individuals with a high probability of 2 African ancestral alleles or at least 1 European allele at each locus. We confirmed 2 index SNPs as associated with lipid traits in African Americans, with suggestive association for 3 more. However, the effect sizes for 4 of the 5 associated SNPs were larger in the European local ancestry subgroup compared with the African local ancestry subgroup, suggesting that the replication is driven by European ancestry segments. Through fine-mapping, we discovered 3 new SNPs with significant associations, 2 with consistent effect on triglyceride levels across ancestral groups: rs636523 near DOCK7/ANGPTL3 and rs780093 in GCKR. African linkage disequilibrium patterns did not assist in narrowing association signals. We confirm that 5 genetic regions associated with lipid traits in European-derived populations are relevant in African Americans. To further evaluate these loci, fine-mapping in larger African American cohorts and/or resequencing will be required. Show less

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; 8 of these loci were novel. Whether these same SNPs associate with lipids in a broader range of ethnicities is unknown. 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 United States 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 3 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 5 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 3 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 5 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. Show less

Hypertriglyceridemia is a risk factor for cardiovascular disease. Variation in the apolipoprotein A5 (APOA5) and glucokinase regulatory protein (GCKR) genes has been associated with fasting plasma triacylglycerol. We investigated the combined effects of the GCKR rs780094C-->T, APOA5 -1131T-->C, and APOA5 56C-->G single nucleotide polymorphisms (SNPs) on fasting triacylglycerol in several independent populations and the response to a high-fat meal and fenofibrate interventions. We used a cross-sectional design to investigate the association with fasting triacylglycerol in 8 populations from America, Asia, and Europe (n = 7,730 men and women) and 2 intervention studies in US whites (n = 1,061) to examine postprandial triacylglycerol after a high-fat meal and the response to fenofibrate. We defined 3 combined genotype groups: 1) protective (homozygous for the wild-type allele for all 3 SNPs); 2) intermediate (any mixed genotype not included in groups 1 and 3); and 3) risk (carriers of the variant alleles at both genes). Subjects within the risk group had significantly higher fasting triacylglycerol and a higher prevalence of hypertriglyceridemia than did subjects in the protective group across all populations. Moreover, subjects in the risk group had a greater postprandial triacylglycerol response to a high-fat meal and greater fenofibrate-induced reduction of fasting triacylglycerol than did the other groups, especially among persons with hypertriglyceridemia. Subjects with the intermediate genotype had intermediate values (P for trend <0.001). SNPs in GCKR and APOA5 have an additive effect on both fasting and postprandial triacylglycerol and contribute to the interindividual variability in response to fenofibrate treatment. Show less

Blood low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol and triglyceride levels are risk factors for cardiovascular disease. To dissect the polygenic basis of these traits, we conducted genome-wide association screens in 19,840 individuals and replication in up to 20,623 individuals. We identified 30 distinct loci associated with lipoprotein concentrations (each with P < 5 x 10(-8)), including 11 loci that reached genome-wide significance for the first time. The 11 newly defined loci include common variants associated with LDL cholesterol near ABCG8, MAFB, HNF1A and TIMD4; with HDL cholesterol near ANGPTL4, FADS1-FADS2-FADS3, HNF4A, LCAT, PLTP and TTC39B; and with triglycerides near AMAC1L2, FADS1-FADS2-FADS3 and PLTP. The proportion of individuals exceeding clinical cut points for high LDL cholesterol, low HDL cholesterol and high triglycerides varied according to an allelic dosage score (P < 10(-15) for each trend). These results suggest that the cumulative effect of multiple common variants contributes to polygenic dyslipidemia. Show less