👤 Dan J Rader

Lp(a) is a genetically determined lipoprotein targeted by emerging therapies. In a UK Biobank analysis (1,026 abdominal aortic aneurysm [AAA] cases, 469,989 controls), elevated Lp(a) was associated with increased risk of AAA, including at clinically relevant thresholds while controlling for traditional risk factors, including ApoB. Multivariable Mendelian randomization confirmed a causal relationship between lipoprotein(a) [Lp(a)] and AAA independent of apolipoprotein B. These findings support Lp(a) as a modifiable risk factor and potential therapeutic target for AAA, a condition with limited medical treatment options. AAA should be considered as an outcome in future clinical trials of Lp(a)-lowering therapies. Show less

Patients with severe hypertriglyceridemia (sHTG) have variable lipoprotein lipase (LPL) activity levels that may influence therapeutic response. This exploratory analysis investigated post-heparin triglyceride lipase and phospholipase activities in three cohorts of patients with sHTG who received evinacumab (angiopoietin-like 3 inhibitor) for 12 or 24 weeks during a phase 2 trial: cohort 1, familial chylomicronemia syndrome with bi-allelic loss-of-function (LOF) LPL pathway mutations; cohort 2, multifactorial chylomicronemia syndrome (MCS) with heterozygous LOF LPL pathway mutations; and cohort 3, MCS without LPL pathway mutations. Post-heparin plasma samples were obtained at baseline and at week 24 (end of the treatment period). Triglyceride lipase activities (LPL and hepatic lipase [HL]) were measured using both a colorimetric and a scintillation assays. Phospholipase activities (HL and endothelial lipase [EL]) were measured using a colorimetric assay. Baseline post-heparin LPL triglyceride lipase activity was lowest in cohort 1; treatment with evinacumab for 12 or 24 weeks did not alter activity at week 24 versus baseline across cohorts using the colorimetric assay. Non-HL triglyceride lipase activity (mostly LPL) assessed using the scintillation assay showed a significant increase in cohort 1 at 24 weeks versus baseline (P = 0.04). Neither HL nor EL phospholipase activities differed among cohorts or changed with evinacumab treatment. High intra- and inter-patient variability in lipase activity was observed with all methods. Post-heparin LPL triglyceride lipase activity was lower in patients with sHTG with bi-allelic LPL pathway mutations and increased in that group with evinacumab. The high variability in lipase activities observed via differing methods supports the need for more robust assays. Show less

Genome-wide association studies (GWAS) have identified nearly 100 loci associated with metabolic dysfunction-associated steatotic liver disease (MASLD), but the molecular functions of these variant alleles remain elusive, particularly when they occur in non-coding regions. Here we profiled the chromatin accessibility landscape of liver nuclei from MASLD individuals, and demonstrated these accessible genomic sites were bound by cell type-specific transcription factors (TFs) and enriched for MASLD risk variants, highlighting lineage- and disease state-specific regulation. Using a massively parallel reporter assay (MPRA), we identified hundreds of differential activity variants (DAVs) that operate in a cell type-specific manner or in a stimulus-dependent context by disrupting liver pathogenesis-associated transcriptional regulatory network. Integrative analyses combining liver eQTLs, chromatin looping, and single-cell CRISPRi screening linked these DAVs to functional target genes. Notably, we demonstrated that DAVs located near Show less

Cirrhosis and hepatocellular carcinoma (HCC) are long-term complications of chronic liver disease (CLD). In this large multi-ancestry genome-wide association study of all-cause cirrhosis (35,481 cases, 2.36M controls) and HCC (6,680 cases, 1.76M controls), we identified 27 loci associated with cirrhosis (10 novel) and 11 with HCC (three novel). Three novel cirrhosis loci were replicated in independent cohorts (e.g. Show less

The mammalian microtubule-associated serine/threonine (MAST) kinases are a highly conserved subfamily of AGC kinases that are implicated as therapeutic targets for cancer and diabetes. However, the activity, regulation, and substrates of MAST kinases are poorly understood. We examined the biochemical activity of Mast2, as a representative of the MAST family. The domain of unknown function (DUF1908) is necessary for Mast2 kinase activity in vitro, while the PDZ domain is dispensable. Mast2 kinase activity does not appear to be compatible with the AGC kinase model of T-loop phospho-activation. Instead, it contains a unique insertion that is likely stabilized by ion-pair interactions. The C terminus of the kinase domain contains motifs regulated by mechanistic target of rapamycin (mTOR) in other AGC kinases, and mutation of these conserved residues reduces Mast2 kinase activity. Consistent with mTOR regulation, Mast2 purified from insulin-stimulated cells has increased activity compared to serum-starved cells, and this increase in activity is dependent on mTOR. Finally, stable Show less

The genetic etiology of hypertrophic cardiomyopathy (HCM) and the critical role of sarcomeric variants in its pathogenesis are well recognized (1). Among these, loss-of-function variants in the myosin binding protein C gene ( This was a prospective, cross-sectional study of 100 adults (aged 18-65 years) with symptomatic Pre-existing anti-AAV9 NAb were undetectable in 50% of patients. Among those with detectable titers (range: 1:10-1:720), only 16% exceeded 1:40. TAb were undetectable in 53%; titers ranged from 1:10 to 1:65,600. A strong correlation was observed between NAb and TAb titers (r = 0.671, Pre-existing immunity to AAV9 was absent or low in most Show less

Heart failure is a complex trait, influenced by environmental and genetic factors, affecting over 30 million individuals worldwide. Here we report common-variant and rare-variant association studies of all-cause heart failure and examine how different classes of genetic variation impact its heritability. We identify 176 common-variant risk loci at genome-wide significance in 2,358,556 individuals and cluster these signals into five broad modules based on pleiotropic associations with anthropomorphic traits/obesity, blood pressure/renal function, atherosclerosis/lipids, immune activity and arrhythmias. In parallel, we uncover exome-wide significant associations for heart failure and rare predicted loss-of-function variants in TTN, MYBPC3, FLNC and BAG3 using exome sequencing of 376,334 individuals. We find that total burden heritability of rare coding variants is highly concentrated in a small set of Mendelian cardiomyopathy genes, while common-variant heritability is diffusely spread throughout the genome. Finally, we show that common-variant background modifies heart failure risk among carriers of rare pathogenic truncating variants in TTN. Together, these findings discern genetic links between dysregulated metabolism and heart failure and highlight a polygenic component to heart failure not captured by current clinical genetic testing. Show less

Atrial fibrillation (AF) is a prevalent and morbid abnormality of the heart rhythm with a strong genetic component. Here, we meta-analyzed genome and exome sequencing data from 36 studies that included 52,416 AF cases and 277,762 controls. In burden tests of rare coding variation, we identified novel associations between AF and the genes MYBPC3, LMNA, PKP2, FAM189A2 and KDM5B. We further identified associations between AF and rare structural variants owing to deletions in CTNNA3 and duplications of GATA4. We broadly replicated our findings in independent samples from MyCode, deCODE and UK Biobank. Finally, we found that CRISPR knockout of KDM5B in stem-cell-derived atrial cardiomyocytes led to a shortening of the action potential duration and widespread transcriptomic dysregulation of genes relevant to atrial homeostasis and conduction. Our results highlight the contribution of rare coding and structural variants to AF, including genetic links between AF and cardiomyopathies, and expand our understanding of the rare variant architecture for this common arrhythmia. Show less

To broaden our understanding of bradyarrhythmias and conduction disease, we performed common variant genome-wide association analyses in up to 1.3 million individuals and rare variant burden testing in 460,000 individuals for sinus node dysfunction (SND), distal conduction disease (DCD) and pacemaker (PM) implantation. We identified 13, 31 and 21 common variant loci for SND, DCD and PM, respectively. Four well-known loci (SCN5A/SCN10A, CCDC141, TBX20 and CAMK2D) were shared for SND and DCD, while others were more specific for SND or DCD. SND and DCD showed a moderate genetic correlation (r Show less

Heart failure (HF) is a complex trait, influenced by environmental and genetic factors, which affects over 30 million individuals worldwide. Historically, the genetics of HF have been studied in Mendelian forms of disease, where rare genetic variants have been linked to familial cardiomyopathies. More recently, genome-wide association studies (GWAS) have successfully identified common genetic variants associated with risk of HF. However, the relative importance of genetic variants across the allele-frequency spectrum remains incompletely characterized. Here, we report the results of common- and rare-variant association studies of all-cause heart failure, applying recently developed methods to quantify the heritability of HF attributable to different classes of genetic variation. We combine GWAS data across multiple populations including 207,346 individuals with HF and 2,151,210 without, identifying 176 risk loci at genome-wide significance (P-value < 5×10 Show less

Common variants of the max-like protein X (MLX)-interacting protein-like (MLXIPL) gene, encoding the transcription factor carbohydrate-responsive element-binding protein, have been shown to be associated with plasma triglyceride levels. However, the role of these variants in steatotic liver disease (SLD) is unclear. We used a genome-first approach to analyze a variety of metabolic phenotypes and clinical outcomes associated with a common missense variant in MLXIPL, Gln241His, in 2 large biobanks: the UK Biobank and the Penn Medicine Biobank. Carriers of MLXIPL Gln241His were associated with significantly lower serum levels of triglycerides, apolipoprotein-B, gamma-glutamyl transferase, and alkaline phosphatase. Additionally, MLXIPL Gln241His carriers were associated with significantly higher serum levels of HDL cholesterol and alanine aminotransferase. Carriers homozygous for MLXIPL Gln241His showed a higher risk of SLD in 2 unrelated cohorts. Carriers of MLXIPL Gln241His were especially more likely to be diagnosed with SLD if they were female, obese, and/or also carried the PNPLA3 I148M variant. Furthermore, the heterozygous carriage of MLXIPL Gln241His was associated with significantly higher all-cause, liver-related, and cardiovascular mortality rates. Nuclear magnetic resonance metabolomics data indicated that carriage of MLXIPL Gln241His was significantly associated with lower serum levels of VLDL and increased serum levels of HDL cholesterol. Analyses of the MLXIPL Gln241His polymorphism showed a significant association with a higher risk of SLD diagnosis and elevated serum alanine aminotransferase as well as significantly lower serum triglycerides and apolipoprotein-B levels. MLXIPL might, therefore, be a potential pharmacological target for the treatment of SLD and hyperlipidemia, notably for patients at risk. More mechanistic studies are needed to better understand the role of MLXIPL Gln241His on lipid metabolism and steatosis development. Show less

Plasma triglycerides (TGs) are causally associated with coronary artery disease and acute pancreatitis. Apolipoprotein A-V (apoA-V, gene We used hydrogen-deuterium exchange mass spectrometry to determine the secondary structure of human apoA-V in lipid-free and lipid-associated conditions and identified a C-terminal hydrophobic face. Then, we used genomic data in the Penn Medicine Biobank to identify a rare variant, Q252X, predicted to specifically eliminate this region. We interrogated the function of apoA-V Q252X using recombinant protein Human apoA-V Q252X carriers exhibited elevated plasma TG levels consistent with loss of function. Deletion of apoA-V's C-terminus leads to reduced apoA-V bioavailability Show less

'Genome-first' approaches to analyzing rare variants can reveal new insights into human biology and disease. Because pathogenic variants are often rare, new discovery requires aggregating rare coding variants into 'gene burdens' for sufficient power. However, a major challenge is deciding which variants to include in gene burden tests. Pathogenic variants in MYBPC3 and MYH7 are well-known causes of hypertrophic cardiomyopathy (HCM), and focusing on these 'positive control' genes in a genome-first approach could help inform variant selection methods and gene burdening strategies for other genes and diseases. Integrating exome sequences with electronic health records among 41 759 participants in the Penn Medicine BioBank, we evaluated the performance of aggregating predicted loss-of-function (pLOF) and/or predicted deleterious missense (pDM) variants in MYBPC3 and MYH7 for gene burden phenome-wide association studies (PheWAS). The approach to grouping rare variants for these two genes produced very different results: pLOFs but not pDM variants in MYBPC3 were strongly associated with HCM, whereas the opposite was true for MYH7. Detailed review of clinical charts revealed that only 38.5% of patients with HCM diagnoses carrying an HCM-associated variant in MYBPC3 or MYH7 had a clinical genetic test result. Additionally, 26.7% of MYBPC3 pLOF carriers without HCM diagnoses had clear evidence of left atrial enlargement and/or septal/LV hypertrophy on echocardiography. Our study shows the importance of evaluating both pLOF and pDM variants for gene burden testing in future studies to uncover novel gene-disease relationships and identify new pathogenic loss-of-function variants across the human genome through genome-first analyses of healthcare-based populations. Show less

Large-scale human exome sequencing can identify rare protein-coding variants with a large impact on complex traits such as body adiposity. We sequenced the exomes of 645,626 individuals from the United Kingdom, the United States, and Mexico and estimated associations of rare coding variants with body mass index (BMI). We identified 16 genes with an exome-wide significant association with BMI, including those encoding five brain-expressed G protein-coupled receptors ( Show less

Among many causes of hypertriglyceridemia (HTG), familial chylomicronemia syndrome (FCS) is a rare monogenic disorder that manifests as severe HTG and acute pancreatitis. Among the known causal genes for FCS, mutations in We present the challenging care of a 43-year-old man with FCS with apoC-II deficiency and the results of two types of TPE and of investigational TG-lowering biologic therapies. The patient's lipid profile was consistent with FCS. A novel homozygous variant was identified in Our case demonstrates the importance of delineating and defining the underlying etiology of a rare disorder to optimize therapy and to minimize unfavorable outcomes. Show less

Aortic stenosis (AS) has no approved medical treatment. Identifying etiological pathways for AS could identify pharmacological targets. To identify novel genetic loci and pathways associated with AS. This genome-wide association study used a case-control design to evaluate 44 703 participants (3469 cases of AS) of self-reported European ancestry from the Genetic Epidemiology Research on Adult Health and Aging (GERA) cohort (from January 1, 1996, to December 31, 2015). Replication was performed in 7 other cohorts totaling 256 926 participants (5926 cases of AS), with additional analyses performed in 6942 participants from the Cohorts for Heart and Aging Research in Genomic Epidemiology (CHARGE) Consortium. Follow-up biomarker analyses with aortic valve calcium (AVC) were also performed. Data were analyzed from May 1, 2017, to December 5, 2019. Genetic variants (615 643 variants) and polyunsaturated fatty acids (ω-6 and ω-3) measured in blood samples. Aortic stenosis and aortic valve replacement defined by electronic health records, surgical records, or echocardiography and the presence of AVC measured by computed tomography. The mean (SD) age of the 44 703 GERA participants was 69.7 (8.4) years, and 22 019 (49.3%) were men. The rs174547 variant at the FADS1/2 locus was associated with AS (odds ratio [OR] per C allele, 0.88; 95% CI, 0.83-0.93; P = 3.0 × 10-6), with genome-wide significance after meta-analysis with 7 replication cohorts totaling 312 118 individuals (9395 cases of AS) (OR, 0.91; 95% CI, 0.88-0.94; P = 2.5 × 10-8). A consistent association with AVC was also observed (OR, 0.91; 95% CI, 0.83-0.99; P = .03). A higher ratio of arachidonic acid to linoleic acid was associated with AVC (OR per SD of the natural logarithm, 1.19; 95% CI, 1.09-1.30; P = 6.6 × 10-5). In mendelian randomization, increased FADS1 liver expression and arachidonic acid were associated with AS (OR per unit of normalized expression, 1.31 [95% CI, 1.17-1.48; P = 7.4 × 10-6]; OR per 5-percentage point increase in arachidonic acid for AVC, 1.23 [95% CI, 1.01-1.49; P = .04]; OR per 5-percentage point increase in arachidonic acid for AS, 1.08 [95% CI, 1.04-1.13; P = 4.1 × 10-4]). Variation at the FADS1/2 locus was associated with AS and AVC. Findings from biomarker measurements and mendelian randomization appear to link ω-6 fatty acid biosynthesis to AS, which may represent a therapeutic target. 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

The angiopoietin-like proteins are a family of proteins that share structural similarities to the angiopoietins. These proteins have been described to play a key role in the regulation of a myriad of physiologic processes, including serving as essential modulators of lipid and glucose metabolism. Angiopoietin-like protein 4 (ANGPTL4) is a key regulator of lipoprotein lipase, and its modulation has been shown to significantly impact the body's processing and distribution of triglycerides and cholesterol. Although more research remains to elucidate the full range of mechanisms through which ANGPTL4 affects triglyceride and cholesterol homeostasis, current research has associated decreased ANGPTL4 function with a beneficial effect on lipid parameters and overall cardiovascular disease risk, opening the possibility of ANGPTL4 as a new therapeutic target for the treatment of cardiovascular disease. Show less

Genome-wide association studies have uncovered common variants at many loci influencing human complex traits, such as high-density lipoprotein cholesterol (HDL-C). However, the contribution of the identified genes is difficult to ascertain from current efforts interrogating common variants with small effects. Thus, there is a pressing need for scalable, cost-effective strategies for uncovering causal variants, many of which may be rare and noncoding. Here, we used a molecular inversion probe target capture approach to resequence both coding and regulatory regions at 7 HDL-C-associated loci in 797 individuals with extremely high HDL-C versus 735 low-to-normal HDL-C controls. Our targets included protein-coding regions of Exome-wide genotyping in 1114 of the 1532 participants yielded a >90% genotyping concordance rate with molecular inversion probe-identified variants in ≈90% of participants. This approach rediscovered nearly all established genome-wide association studies associations in Our targeted resequencing of individuals at the HDL-C phenotypic extremes offers a novel, efficient, and cost-effective approach for identifying rare coding and noncoding variation differences in extreme phenotypes and supports the rationale for applying this methodology to uncover rare variation-particularly noncoding variation-underlying myriad complex traits. 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

The development of CETP (cholesteryl ester transfer protein) inhibitors has had a long and difficult course with 3 compounds failing in phase III clinical trials. Finally, the REVEAL (Randomized Evaluation of the Effects of Anacetrapib through Lipid modification) trial has shown that the CETP inhibitor anacetrapib decreased coronary heart disease when added to statin therapy. Although the result is different to earlier studies, this is likely related to the size and duration of the trial. The benefit of anacetrapib seems to be largely explained by lowering of non-HDL-C (high-density lipoprotein cholesterol), rather than increases in HDL-C. Although the magnitude of benefit for coronary heart disease appeared to be moderate, in part this may have reflected aspects of the trial design. Anacetrapib treatment was associated with a small increase in blood pressure, but was devoid of major side effects and was also associated with a small reduction in diabetes mellitus. Treatment with CETP inhibitors, either alone or in combination with statins, could provide another option for patients with coronary disease who require further reduction in LDL (low-density lipoprotein) and non-HDL-C. 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

Elevated high-density lipoprotein cholesterol levels in the blood (HDL-C) represent one of the strongest epidemiological surrogates for protection against coronary heart disease (CHD), but recent human genetic and pharmacological intervention studies have raised controversy about the causality of this relationship. Here, we review recent discoveries from human genome studies using new analytic tools as well as relevant animal studies that have both addressed, and in some cases, fueled this controversy. Methodologic developments in genotyping and sequencing, such as genome-wide association studies (GWAS), exome sequencing, and exome array genotyping, have been applied to the study of HDL-C and risk of CHD in large, multi-ethnic populations. Some of these efforts focused on population-wide variation in common variants have uncovered new polymorphisms at novel loci associated with HDL-C and, in some cases, CHD risk. Other efforts have discovered loss-of-function variants for the first time in genes previously implicated in HDL metabolism through common variant studies or animal models. These studies have allowed the genetic relationship between these pathways, HDL-C and CHD to be explored in humans for the first time through analysis tools such as Mendelian randomization. We explore these discoveries for selected key HDL-C genes CETP, LCAT, LIPG, SCARB1, and novel loci implicated from GWAS including GALNT2, KLF14, and TTC39B. Recent human genetics findings have identified new nodes regulating HDL metabolism while reshaping our current understanding of known candidate genes to HDL and CHD risk through the study of critical variants across model systems. Despite their effect on HDL-C, variants in many of the reviewed genes were found to lack any association with CHD. These data collectively indicate that HDL-C concentration, which represents a static picture of a very dynamic and heterogeneous metabolic milieu, is unlikely to be itself causally protective against CHD. In this context, human genetics represent an extremely valuable tool to further explore the biological mechanisms regulating HDL metabolism and investigate what role, if any, HDL plays in the pathogenesis of CHD. Show less

Lp(a) [lipoprotein (a)] is composed of apoB (apolipoprotein B) and apo(a) [apolipoprotein (a)] and is an independent risk factor for cardiovascular disease and aortic stenosis. In clinical trials, anacetrapib, a CETP (cholesteryl ester transfer protein) inhibitor, causes significant reductions in plasma Lp(a) levels. We conducted an exploratory study to examine the mechanism for Lp(a) lowering by anacetrapib. We enrolled 39 participants in a fixed-sequence, double-blind study of the effects of anacetrapib on the metabolism of apoB and high-density lipoproteins. Twenty-nine patients were randomized to atorvastatin 20 mg/d, plus placebo for 4 weeks, and then atorvastatin plus anacetrapib (100 mg/d) for 8 weeks. The other 10 subjects were randomized to double placebo for 4 weeks followed by placebo plus anacetrapib for 8 weeks. We examined the mechanisms of Lp(a) lowering in a subset of 12 subjects having both Lp(a) levels >20 nmol/L and more than a 15% reduction in Lp(a) by the end of anacetrapib treatment. We performed stable isotope kinetic studies using Anacetrapib reduces Lp(a) levels by decreasing its production. URL: http://www.clinicaltrials.gov. Unique identifier: NCT00990808. 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

Cholesteryl ester transfer protein (CETP) mediates the transfer of HDL cholesteryl esters for triglyceride (TG) in VLDL/LDL. CETP inhibition, with anacetrapib, increases HDL-cholesterol, reduces LDL-cholesterol, and lowers TG levels. This study describes the mechanisms responsible for TG lowering by examining the kinetics of VLDL-TG, apoC-II, apoC-III, and apoE. Mildly hypercholesterolemic subjects were randomized to either placebo (N = 10) or atorvastatin 20 mg/qd (N = 29) for 4 weeks (period 1) followed by 8 weeks of anacetrapib, 100 mg/qd (period 2). Following each period, subjects underwent stable isotope metabolic studies to determine the fractional catabolic rates (FCRs) and production rates (PRs) of VLDL-TG and plasma apoC-II, apoC-III, and apoE. Anacetrapib reduced the VLDL-TG pool on a statin background due to an increased VLDL-TG FCR (29%; Show less