Sumeet A. Khetarpal

Rare variant in scavenger receptor BI raises HDL cholesterol and increases risk of coronary heart disease

A scavenger that protects the heart Coronary heart disease is a tale of two forms of plasma cholesterol. In contrast to the well-established effects of “bad” cholesterol (LDL-C), the role of “good” cholesterol (HDL-C) is mysterious. Elevated HDL-C correlates with a lower risk of heart disease, yet drugs that raise HDL-C levels do not reduce risk. Zanoni et al. found that some people with exceptionally high levels of HDL-C carry a rare sequence variant in the gene encoding the major HDL-C receptor, scavenger receptor BI. This variant destroys the receptors ability to take up HDL-C. Interestingly, people with this variant have a higher risk of heart disease despite having high levels of HDL-C. Science, this issue p. 1166 A human genetics study sheds light on how HDL (good) cholesterol protects against cardiovascular disease. Scavenger receptor BI (SR-BI) is the major receptor for high-density lipoprotein (HDL) cholesterol (HDL-C). In humans, high amounts of HDL-C in plasma are associated with a lower risk of coronary heart disease (CHD). Mice that have depleted Scarb1 (SR-BI knockout mice) have markedly elevated HDL-C levels but, paradoxically, increased atherosclerosis. The impact of SR-BI on HDL metabolism and CHD risk in humans remains unclear. Through targeted sequencing of coding regions of lipid-modifying genes in 328 individuals with extremely high plasma HDL-C levels, we identified a homozygote for a loss-of-function variant, in which leucine replaces proline 376 (P376L), in SCARB1, the gene encoding SR-BI. The P376L variant impairs posttranslational processing of SR-BI and abrogates selective HDL cholesterol uptake in transfected cells, in hepatocyte-like cells derived from induced pluripotent stem cells from the homozygous subject, and in mice. Large population-based studies revealed that subjects who are heterozygous carriers of the P376L variant have significantly increased levels of plasma HDL-C. P376L carriers have a profound HDL-related phenotype and an increased risk of CHD (odds ratio = 1.79, which is statistically significant).

Loss-of-function variants in endothelial lipase are a cause of elevated HDL cholesterol in humans

Elevated plasma concentrations of HDL cholesterol (HDL-C) are associated with protection from atherosclerotic cardiovascular disease. Animal models indicate that decreased expression of endothelial lipase (LIPG) is inversely associated with HDL-C levels, and genome-wide association studies have identified LIPG variants as being associated with HDL-C levels in humans. We hypothesized that loss-of-function mutations in LIPG may result in elevated HDL-C and therefore performed deep resequencing of LIPG exons in cases with elevated HDL-C levels and controls with decreased HDL-C levels. We identified a significant excess of nonsynonymous LIPG variants unique to cases with elevated HDL-C. In vitro lipase activity assays demonstrated that these variants significantly decreased endothelial lipase activity. In addition, a meta-analysis across 5 cohorts demonstrated that the low-frequency Asn396Ser variant is significantly associated with increased HDL-C, while the common Thr111Ile variant is not. Functional analysis confirmed that the Asn396Ser variant has significantly decreased lipase activity both in vitro and in vivo, while the Thr111Ile variant has normal lipase activity. Our results establish that loss-of-function mutations in LIPG lead to increased HDL-C levels and support the idea that inhibition of endothelial lipase may be an effective mechanism to raise HDL-C.

Human knockouts and phenotypic analysis in a cohort with a high rate of consanguinity

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.

A systematic study of modulation of ADAM-mediated ectodomain shedding by site-specific O-glycosylation.

Significance Ectodomain shedding is a central event in a range of biological processes and pathways, however the underlying mechanisms are still not fully understood. Here we present evidence that site-specific O-glycosylation regulated by individual GalNAc-Transferase isoforms, serve to coregulate ectodomain shedding, predominantly through blocking of cleavage. Our general finding is exemplified by the specific role of a single GalNAc-T isoform (GalNAc-T2) in coregulating TNF-alpha release in vitro, ex vivo in isogenic cell models, and in vivo in mouse Galnt2 knockouts. Adding the large family of GalNAc-T isoforms to regulation of ectodomain shedding substantially increase the ability to fine-tune this important process on a substrate level. Regulated shedding of the ectodomain of cell membrane proteins by proteases is a common process that releases the extracellular domain from the cell and activates cell signaling. Ectodomain shedding occurs in the immediate extracellular juxtamembrane region, which is also where O-glycosylation is often found and examples of crosstalk between shedding and O-glycosylation have been reported. Here, we systematically investigated the potential of site-specific O-glycosylation mediated by distinct polypeptide GalNAc-transferase (GalNAc-T) isoforms to coregulate ectodomain shedding mediated by the A Disintegrin And Metalloproteinase (ADAM) subfamily of proteases and in particular ADAM17. We analyzed 25 membrane proteins that are known to undergo ADAM17 shedding and where the processing sites included Ser/Thr residues within ± 4 residues that could represent O-glycosites. We used in vitro GalNAc-T enzyme and ADAM cleavage assays to demonstrate that shedding of at least 12 of these proteins are potentially coregulated by O-glycosylation. Using TNF-α as an example, we confirmed that shedding mediated by ADAM17 is coregulated by O-glycosylation controlled by the GalNAc-T2 isoform both ex vivo in isogenic cell models and in vivo in mouse Galnt2 knockouts. The study provides compelling evidence for a wider role of site-specific O-glycosylation in ectodomain shedding.

Triglyceride-Rich Lipoproteins and Coronary Artery Disease Risk New Insights From Human Genetics

Despite ample success in reducing coronary artery disease (CAD) risk through reduction of low-density lipoprotein cholesterol (LDL-C), there remains substantial residual risk.1–4 Recent prospective studies have demonstrated that elevated triglycerides (TGs) are independent predictors of CAD risk.5–9 Furthermore, TGs are strongly associated with incident CAD events in patients with low LDL-C levels treated with statin.10 Thus, triglyceride-rich lipoproteins (TRLs) offer a potentially orthogonal risk factor to LDL-C for lowering CAD risk, but only if TRLs are causally associated with atherosclerotic disease.11 Human genetics has the potential to reveal the causal relationships of biomarkers found to be associated with disease outcomes.12–15 For example, genetic variants associated with plasma LDL-C levels are consistently associated with CAD risk in the right direction,15–18 consistent with a causal relationship. Importantly, similar studies have causally implicated the key TG-regulating enzyme lipoprotein lipase (LPL) in CAD risk. A common gain-of-function LPL variant, S447X, confers an antiatherogenic lipid profile characterized by low levels of TGs, and in several studies, it has been associated with lower incidence of vascular disease or myocardial infarction (MI).19–25 Conversely, several loss-of-function (LOF) LPL variants associated with elevated TG levels have been reported to be associated with increased CAD risk.21,26 Furthermore, multiple genome-wide association studies in the last 5 years have identified common noncoding variants at the LPL gene locus associated with both TG and CAD risk in the same direction.27–29 Beyond LPL itself, common variants that influence TG levels are significantly associated with CAD risk even after adjusting for their effects on other lipid traits.30 Do et al30 surveyed 185 single-nucleotide polymorphisms (SNPs) that were genome-wide significantly associated with ≥1 plasma lipid trait and identified a subset of …

Plasma Apolipoprotein C-III Levels, Triglycerides, and Coronary Artery Calcification in Type 2 Diabetics

Objective—Triglyceride-rich lipoproteins have emerged as causal risk factors for developing coronary heart disease independent of low-density lipoprotein cholesterol levels. Apolipoprotein C-III (ApoC-III) modulates triglyceride-rich lipoprotein metabolism through inhibition of lipoprotein lipase and hepatic uptake of triglyceride-rich lipoproteins. Mutations causing loss-of-function of ApoC-III lower triglycerides and reduce coronary heart disease risk, suggestive of a causal role for ApoC-III. Little data exist about the relationship of ApoC-III, triglycerides, and atherosclerosis in patients with type 2 diabetes mellitus (T2DM). Here, we examined the relationships between plasma ApoC-III, triglycerides, and coronary artery calcification in patients with T2DM. Approach and Results—Plasma ApoC-III levels were measured in a cross-sectional study of 1422 subjects with T2DM but without clinically manifest coronary heart disease. ApoC-III levels were positively associated with total cholesterol (Spearman r=0.36), triglycerides (r=0.59), low-density lipoprotein cholesterol (r=0.16), fasting glucose (r=0.16), and glycosylated hemoglobin (r=0.12; P<0.0001 for all). In age, sex, and race-adjusted analysis, ApoC-III levels were positively associated with coronary artery calcification (Tobit regression ratio, 1.78; 95% confidence interval, 1.27–2.50 per SD increase in ApoC-III; P<0.001). As expected for an intermediate mediator, these findings were attenuated when adjusted for both triglycerides (Tobit regression ratio, 1.43; 95% confidence interval, 0.94–2.18; P=0.086) and separately for very low–density lipoprotein cholesterol (Tobit regression ratio, 1.14; 95% confidence interval, 0.75–1.71; P=0.53). Conclusions—In persons with T2DM, increased plasma ApoC-III is associated with higher triglycerides, less favorable cardiometabolic phenotypes, and higher coronary artery calcification, a measure of subclinical atherosclerosis. Therapeutic inhibition of ApoC-III may thus be a novel strategy for reducing plasma triglyceride-rich lipoproteins and cardiovascular risk in T2DM.

Mining the LIPG Allelic Spectrum Reveals the Contribution of Rare and Common Regulatory Variants to HDL Cholesterol

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.

HDL Cholesterol Metabolism and the Risk of CHD: New Insights from Human Genetics

Purpose of ReviewElevated 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.Recent FindingsMethodologic 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.SummaryRecent 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.

The effects of apolipoprotein F deficiency on high density lipoprotein cholesterol metabolism in mice.

Apolipoprotein F (apoF) is 29 kilodalton secreted sialoglycoprotein that resides on the HDL and LDL fractions of human plasma. Human ApoF is also known as Lipid Transfer Inhibitor protein (LTIP) based on its ability to inhibit cholesteryl ester transfer protein (CETP)-mediated transfer events between lipoproteins. In contrast to other apolipoproteins, ApoF is predicted to lack strong amphipathic alpha helices and its true physiological function remains unknown. We previously showed that overexpression of Apolipoprotein F in mice reduced HDL cholesterol levels by 20–25% by accelerating clearance from the circulation. In order to investigate the effect of physiological levels of ApoF expression on HDL cholesterol metabolism, we generated ApoF deficient mice. Unexpectedly, deletion of ApoF had no substantial impact on plasma lipid concentrations, HDL size, lipid or protein composition. Sex-specific differences were observed in hepatic cholesterol content as well as serum cholesterol efflux capacity. Female ApoF KO mice had increased liver cholesteryl ester content relative to wild type controls on a chow diet (KO: 3.4+/−0.9 mg/dl vs. WT: 1.2+/−0.3 mg/dl, p<0.05). No differences were observed in ABCG1-mediated cholesterol efflux capacity in either sex. Interestingly, ApoB-depleted serum from male KO mice was less effective at promoting ABCA1-mediated cholesterol efflux from J774 macrophages relative to WT controls.

Targeting ApoC-III to Reduce Coronary Disease Risk

Triglyceride-rich lipoproteins (TRLs) are causal contributors to the risk of developing coronary artery disease (CAD). Apolipoprotein C-III (apoC-III) is a component of TRLs that elevates plasma triglycerides (TGs) through delaying the lipolysis of TGs and the catabolism of TRL remnants. Recent human genetics approaches have shown that heterozygous loss-of-function mutations in APOC3, the gene encoding apoC-III, lower plasma TGs and protect from CAD. This observation has spawned new interest in therapeutic efforts to target apoC-III. Here, we briefly review both currently available as well as developing therapies for reducing apoC-III levels and function to lower TGs and cardiovascular risk. These therapies include existing options including statins, fibrates, thiazolidinediones, omega-3-fatty acids, and niacin, as well as an antisense oligonucleotide targeting APOC3 currently in clinical development. We review the mechanisms of action by which these drugs reduce apoC-III and the current understanding of how reduction in apoC-III may impact CAD risk.