A Common Polymorphism in the FADS1 Locus Links miR1908 to Low-Density Lipoprotein Cholesterol Through BMP1.

OBJECTIVE: Leveraging microRNA-Seq data and the 1000 Genomes imputed genotypes, we identified rs174561 as a strong microRNA quantitative trait loci for circulating microRNA-1908-5p with higher miR-1908-5p and reduced LDL (lowdensity lipoprotein)-cholesterol, fasting glucose and A1c concentrations in carriers of the rs-174561-C allele. Here, we have investigated the molecular mechanism(s) linking miR-1908-5p to LDL-C concentrations. APPROACH AND RESULTS: Transfection experiments demonstrate that the presence of the C allele significantly increases miR- 1908-5p abundance relative to the T allele. LDLR mRNA and low-density lipoprotein receptor (LDLR) total protein were unchanged in response to differential miR-1908-5p expression. However, the ratio of the cleaved to full-length form of LDLR decreased with miR-1908-5p mimic and increased with miR-1908-5p inhibitor treatment. BMP1 (bone morphogenetic protein 1) is a protease responsible for LDLR cleavage, and we show that miR-1908-5p mimic reduces BMP1 mRNA. Using a reporter array, we identified the TGF-ß (transforming growth factor-beta) signaling pathway activity to be reduced by miR- 1908-5p mimic treatment, and this was associated with reduced TGFB1 expression. TGF-ß signaling increases BMP1, and we further demonstrate that the effect of miR-1908-5p on LDLR cleavage is abolished by exogenous TGF-ß treatment. CONCLUSIONS: These findings uncover a mechanism whereby miR-1908-5p reduces TGFB1 abundance resulting in lower expression of BMP1, ultimately leading to reduced LDLR cleavage. Cleavage of the mature LDLR is known to reduce cell surface affinity for LDL, thereby linking miR-1908-5p to lower circulating LDL-cholesterol levels.

Role of Tribbles Pseudokinase 1 (TRIB1) in human hepatocyte metabolism.

Genome-wide association studies for plasma triglycerides and hepatic steatosis identified a risk locus on chromosome 8q24 close to the TRIB1 gene, encoding Tribbles Pseudokinase 1 (TRIB1). In previous studies conducted in murine models, hepatic over-expression of Trib1 was shown to increase fatty acid oxidation and decrease triglyceride synthesis whereas Trib1 knockdown mice exhibited hypertriglyceridemia. Here we have examined the impact of TRIB1 suppression in human and mouse hepatocytes. Examination of a panel of lipid regulator transcripts revealed species-specific effects, prompting us to focus on human models for the remainder of the study. Acute knockdown of TRIB1 in human primary hepatocytes resulted in decreased expression of MTTP and APOB, required for very low density lipoprotein (VLDL) assembly although particle secretion was not significantly affected. A parallel analysis performed in HepG2 revealed reduced MTTP, but not APOB, protein as a result of TRIB1 suppression. Global gene expression changes of human primary hepatocytes upon TRIB1 suppression were analyzed by clustering algorithms and found to be consistent with dysregulation of several pathways fundamental to liver function, including altered CEBPA and B transcript levels and impaired glucose handling. Indeed, TRIB1 expression in HepG2 cells was found to be inversely proportional to glucose concentration. Lastly TRIB1 downregulation in primary hepatocytes was associated with suppression of the HNF4A axis. In HepG2 cells, TRIB1 suppression resulted in reduced HNF4A protein levels while HNF4A suppression increased TRIB1 expression. Taken together these studies reveal an important role for TRIB1 in human hepatocyte biology.

Functional Analysis of a Novel Genome-Wide Association Study Signal in SMAD3 That Confers Protection From Coronary Artery Disease.

OBJECTIVE: A recent genome-wide association study meta-analysis identified an intronic single nucleotide polymorphism in SMAD3, rs56062135C>T, the minor allele (T) which associates with protection from coronary artery disease. Relevant to atherosclerosis, SMAD3 is a key contributor to transforming growth factor-ß pathway signaling. Here, we seek to identify ≥1 causal coronary artery disease-associated single nucleotide polymorphisms at the SMAD3 locus and characterize mechanisms whereby the risk allele(s) contribute to coronary artery disease risk. APPROACH AND RESULTS: By genetic and epigenetic fine mapping, we identified a candidate causal single nucleotide polymorphism rs17293632C>T (D', 0.97; r(2), 0.94 with rs56062135) in intron 1 of SMAD3 with predicted functional effects. We show that the sequence encompassing rs17293632 acts as a strong enhancer in human arterial smooth muscle cells. The common allele (C) preserves an activator protein (AP)-1 site and enhancer function, whereas the protective (T) allele disrupts the AP-1 site and significantly reduces enhancer activity (P<0.001). Pharmacological inhibition of AP-1 activity upstream demonstrates that this allele-specific enhancer effect is AP-1 dependent (P<0.001). Chromatin immunoprecipitation experiments reveal binding of several AP-1 component proteins with preferential binding to the (C) allele. We show that rs17293632 is an expression quantitative trait locus for SMAD3 in blood and atherosclerotic plaque with reduced expression of SMAD3 in carriers of the protective allele. Finally, siRNA knockdown of SMAD3 in human arterial smooth muscle cells increases cell viability, consistent with an antiproliferative role. CONCLUSIONS: The coronary artery disease-associated rs17293632C>T single nucleotide polymorphism represents a novel functional cis-acting element at the SMAD3 locus. The protective (T) allele of rs17293632 disrupts a consensus AP-1 binding site in a SMAD3 intron 1 enhancer, reduces enhancer activity and SMAD3 expression, altering human arterial smooth muscle cell proliferation.

Adiposity significantly modifies genetic risk for dyslipidemia.

Recent genome-wide association studies have identified multiple loci robustly associated with plasma lipids, which also contribute to extreme lipid phenotypes. However, these common genetic variants explain <12% of variation in lipid traits. Adiposity is also an important determinant of plasma lipoproteins, particularly plasma TGs and HDL cholesterol (HDLc) concentrations. Thus, interactions between genes and clinical phenotypes may contribute to this unexplained heritability. We have applied a weighted genetic risk score (GRS) for both plasma TGs and HDLc in two large cohorts at the extremes of BMI. Both BMI and GRS were strongly associated with these lipid traits. A significant interaction between obese/lean status and GRS was noted for each of TG (P(Interaction) = 2.87 × 10(-4)) and HDLc (P(Interaction) = 1.05 × 10(-3)). These interactions were largely driven by SNPs tagging APOA5, glucokinase receptor (GCKR), and LPL for TG, and cholesteryl ester transfer protein (CETP), GalNAc-transferase (GALNT2), endothelial lipase (LIPG), and phospholipid transfer protein (PLTP) for HDLc. In contrast, the GRSLDL cholesterol × adiposity interaction was not significant. Sexual dimorphism was evident for the GRSHDL on HDLc in obese (P(Interaction) = 0.016) but not lean subjects. SNP by BMI interactions may provide biological insight into specific genetic associations and missing heritability.

Regulation of TRIB1 abundance in hepatocyte models in response to proteasome inhibition.

Tribbles related homolog 1 (TRIB1) contributes to lipid and glucose homeostasis by facilitating the degradation of cognate cargos by the proteasome. In view of the key metabolic role of TRIB1 and the impact of proteasome inhibition on hepatic function, we continue our exploration of TRIB1 regulation in two commonly used human hepatocyte models, transformed cell lines HuH-7 and HepG2. In both models, proteasome inhibitors potently upregulated both endogenous and recombinant TRIB1 mRNA and protein levels. Increased transcript abundance was unaffected by MAPK inhibitors while ER stress was a weaker inducer. Suppressing proteasome function via PSMB3 silencing was sufficient to increase TRIB1 mRNA expression. ATF3 was required to sustain basal TRIB1 expression and support maximal induction. Despite increasing TRIB1 protein abundance and stabilizing bulk ubiquitylation, proteasome inhibition delayed but did not prevent TRIB1 loss upon translation block. Immunoprecipitation experiments indicated that TRIB1 was not ubiquitylated in response to proteasome inhibition. A control bona fide proteasome substrate revealed that high doses of proteasome inhibitors resulted in incomplete proteasome inhibition. Cytoplasm retained TRIB1 was unstable, suggesting that TRIB1 lability is regulated prior to its nuclear import. N-terminal deletion and substitutions were insufficient to stabilize TRIB1. These findings identify transcriptional regulation as a prominent mechanism increasing TRIB1 abundance in transformed hepatocyte cell lines in response to proteasome inhibition and provide evidence of an inhibitor resistant proteasome activity responsible for TRIB1 degradation.

Common Polymorphism That Protects From Cardiovascular Disease Increases Fibronectin Processing and Secretion.

BACKGROUND: Fibronectin (FN1) is an essential regulator of homodynamic processes and tissue remodeling that have been proposed to contribute to atherosclerosis. Moreover, recent large-scale genome-wide association studies (GWAS) have linked common genetic variants within the FN1 gene to coronary artery disease risk. METHODS: Public databases were analyzed by 2-Sample Mendelian Randomization. Expression constructs encoding short FN1 reporter constructs and full-length plasma FN1 variants were introduced in various cell models. Secreted and cellular levels were then analyzed and quantified by SDS-PAGE and fluorescence microscopy. Mass spectrometry and glycosylation analyses were performed to probe possible posttranscriptional differences. RESULTS: Bioinformatic analyses revealed that common coronary artery disease risk single nucleotide polymorphisms in the FN1 locus associate with circulating levels of FN1 and that higher FN1 (fibronectin 1) protein levels in plasma are linked to lower coronary artery disease risk. The coronary artery disease-associated FN1 locus encompasses a common polymorphism that translates a L15Q variant situated within the FN1 signal peptide. Introduction of FN1 reporter constructs, differing at position 15, revealed differences in secretion, with the FN1 Q15 variant being less well secreted. Moreover, the L15Q polymorphism was found to alter glycosylation in some cell models but not in human plasma. CONCLUSIONS: In addition to providing novel functional evidence implicating FN1 in cardioprotection, these findings demonstrate that a common variant within a secretion signal peptide regulates protein function.

A novel anti-inflammatory role links the CARS2 locus to protection from coronary artery disease.

BACKGROUND AND AIMS: Genome-wide association studies (GWAS) identified a coronary artery disease (CAD) risk locus on 13.q34 tagged by rs61969072 (T/G). This variant lies in an intergenic region, proximal to ING1, CARKD and CARS2 but its causal relationship to CAD is unknown. METHODS AND RESULTS: We first demonstrated that rs61969072 and tightly linked single nucleotide polymorphisms (SNPs) associate with CARS2 but not ING1 or CARKD expression in carotid endarterectomy samples, with reduced CARS2 abundance in carriers of the CAD risk allele (G). THP-1 monocytes were differentiated and polarized to proinflammatory (M1) and anti-inflammatory (M2) macrophages. CARS2 gene expression decreased in M1 and increased in M2 macrophages, consistent with a role for CARS2 in inflammation. Gene expression profiling revealed an increase in pro-inflammatory markers in response to CARS2 siRNA knockdown in THP-1 derived macrophages, accompanied by an increased abundance of inflammatory cytokines in the cell supernatant. Functional enrichment analysis of impacted transcripts identified the anti-inflammatory IL10 signalling pathway. Western blot analysis of CARS2 silenced macrophages revealed reduced STAT3 phosphorylation in response to IL-10 and increased expression of LPS-induced genes that are repressed by IL-10, indicating a role for CARS2 in anti-inflammatory signalling. Finally, to simulate vessel wall conditions, macrophages, and smooth muscle cells (SMC) were maintained in co-culture. Significantly, CARS2 silencing in macrophages altered the SMC phenotype, decreasing expression of contractile genes and increasing expression of inflammatory genes. CONCLUSIONS: These data highlight a novel anti-inflammatory novel role for CARS2 in human macrophages and SMCs that may underlie the protective effect of a common GWAS-identified variant.

miR1908-5p regulates energy homeostasis in hepatocyte models.

We previously identified genomic variants that are quantitative trait loci for circulating miR-1908-5p and then showed this microRNA to causally associate with plasma levels of LDL-C, fasting blood glucose and HbA1c. The link to LDL-C was subsequently validated and clarified by the identification of a miR1908-5p-TGFB-LDLR regulatory axis. Here, we continue our investigations on miR1908-5p function by leveraging human primary hepatocytes and HuH-7 hepatoma models. Expression of miR1908-5p was shown to be sensitive to glucose and agents affecting glucose metabolism. Transcriptome-wide changes in primary hepatocytes and HuH-7 cells treated with a miR1908-5p mimic were investigated by enrichment approaches to identify targeted transcripts and cognate pathways. Significant pathways included autophagy and increased mitochondrial function. Reduced activation and/or levels of several key energy and metabolic regulators (AKT, mTOR, ME1, G6PD, AMPK and LKB) were subsequently confirmed in mimic treated HuH-7 cells. These effects were associated with reduced NADPH to NADP+ ratio in HuH-7 cells. LKB1 was validated as a direct target of miR1908-5p, the reintroduction of which was however insufficient to compensate for the impact of the miR1908-5p mimic on AMPK and ACC1. These findings implicate miR1908-5p in metabolic and energy regulation in hepatocyte models via multiple, independent, pathways.

Functional analysis of the chromosome 9p21.3 coronary artery disease risk locus.

OBJECTIVE: We have investigated the functional significance of conserved sequences within the 9p21.3 risk locus for coronary artery disease (CAD) and determined the relationship of 9p21.3 to expression of ANRIL and to whole genome gene expression. METHODS AND RESULTS: We demonstrate that a conserved sequence within the 9p21.3 locus has enhancer activity and that the risk variant significantly increases reporter gene expression in primary aortic smooth muscle cells. Whole blood RNA expression of the short variants of ANRIL was increased by 2.2-fold whereas expression of the long ANRIL variant was decreased by 1.2-fold in healthy subjects homozygous for the risk allele. Expression levels of the long and short ANRIL variants were positively correlated with that of the cyclin-dependent kinase inhibitor, CDKN2B (p15) and TDGF1 (Cripto), respectively. Relevant to atherosclerosis, genome-wide expression profiling demonstrated upregulation of gene sets modulating cellular proliferation in carriers of the risk allele. CONCLUSIONS: These findings are consistent with the hypothesis that the 9p21.3 risk allele contains a functional enhancer, the activity of which is altered in carriers of the risk allele. 9p21.3 may promote atherosclerosis by regulating expression of ANRIL, which in turn is associated with altered expression of genes controlling cellular proliferation pathways.

Molecular mechanism linking a novel PCSK9 copy number variant to severe hypercholesterolemia.

BACKGROUND AND AIMS: A 42 year-old male with premature atherosclerosis, severe dyslipidemia and resistance to treatment with high dose statin and a recommended dose of a PCSK9 inhibitor, was found to have a duplication of the PCSK9 gene. However, the clinical phenotype, which included a more than 15-fold elevation in circulating PCSK9, was unexpected given that he had one additional gene copy. METHODS: Here we have carried out whole genome sequencing and transcriptional reporter assays to investigate the molecular mechanism leading to this unusual FH phenotype. RESULTS: The PCSK9 duplication was found to contain the full coding sequence but with an 829 bp shorter 3'-untranslated region (UTR) sequence. All possible rearrangements include a head-to-head fusion between a completely duplicated PCSK9 and a chromosomal region, normally situated ~80 kb away, that includes HNF4 and USF1 binding sites that could promote transcription of the PCSK9 gene. Transcriptional reporter assays demonstrated that a construct harboring the HNF4 binding site significantly increased the promoter activity by 2.5-fold with a smaller effect noted for a USF1 construct. CONCLUSIONS: Here we describe, in a patient with resistant hypercholesterolemia, a novel PCSK9 gene rearrangement that enables upregulation of PCSK9 expression by allowing proximity to an active enhancer binding to HNF4A.

CARMAL Is a Long Non-coding RNA Locus That Regulates MFGE8 Expression.

Genome-wide association studies have identified several genetic loci linked to coronary artery disease (CAD) most of them located in non-protein coding regions of the genome. One such locus is the CAD Associated Region between MFGE8 and ABHD2 (CARMA), a ∼18 kb haplotype that was recently shown to regulate vicinal protein coding genes. Here, we further investigate the region by examining a long non-coding RNA gene locus (CARMAL/RP11-326A19.4/AC013565) abutting the CARMA region. Expression-genotype correlation analyses of public databases indicate that CARMAL levels are influenced by CAD associated variants suggesting that it might have cardioprotective functions. We found CARMAL to be stably expressed at relatively low levels and enriched in the cytosol. CARMAL function was investigated by several gene targeting approaches in HEK293T: inactive CRISPR fusion proteins, antisense, overexpression and inactivation by CRISPR-mediated knock-out. Modest increases in CARMAL (3-4×) obtained via CRISPRa using distinct single-guided RNAs did not result in consistent transcriptome effects. By contrast, CARMAL deletion or reduced CARMAL expression via CRISPRi increased MFGE8 levels, suggesting that CARMAL is contributing to reduce MFGE8 expression under basal conditions. While future investigations are required to clarify the mechanism(s) by which CARMAL acts on MFGE8, integrative bioinformatic analyses of the transcriptome of CARMAL deleted cells suggest that this locus may also be involved in leucine metabolism, splicing, transcriptional regulation and Shwachman-Bodian-Diamond syndrome protein function.

SGCG rs679482 Associates With Weight Loss Success in Response to an Intensively Supervised Outpatient Program.

Weight loss in response to energy restriction is highly variable, and identification of genetic contributors can provide insights into underlying biology. Leveraging 1000 Genomes imputed genotypes, we carried out genome-wide association study (GWAS) analysis in 551 unrelated obese subjects of European ancestry who participated in an intensively supervised weight loss program with replication of promising signals in an independent sample of 1,331 obese subjects who completed the program at a later date. By single nucleotide polymorphism-based and sib-pair analysis, we show that that weight loss is a heritable trait, with estimated heritability (h 2 = 0.49) within the range reported for obesity. We find rs679482, intronic to SGCG (sarcoglycan γ), highly expressed in skeletal muscle, to concordantly associate with weight loss in discovery and replication samples reaching GWAS significance in the combined meta-analysis (ß = -0.35, P = 1.7 × 10-12). Located in a region of open chromatin, rs679482 is predicted to bind DMRT2, and allele-specific transcription factor binding analysis indicates preferential binding of DMRT2 to rs679482-A. Concordantly, rs679482-A impairs native repressor activity and increases basal and DMRT2-mediated enhancer activity. These findings confirm that weight loss is a heritable trait and provide evidence by which a novel variant in SGCG, rs679482, leads to impaired diet response.

Publisher Correction: RIPK1 gene variants associate with obesity in humans and can be therapeutically silenced to reduce obesity in mice.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

RIPK1 gene variants associate with obesity in humans and can be therapeutically silenced to reduce obesity in mice.

Obesity is a major public health burden worldwide and is characterized by chronic low-grade inflammation driven by the cooperation of the innate immune system and dysregulated metabolism in adipose tissue and other metabolic organs. Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) is a central regulator of inflammatory cell function that coordinates inflammation, apoptosis and necroptosis in response to inflammatory stimuli. Here we show that genetic polymorphisms near the human RIPK1 locus associate with increased RIPK1 gene expression and obesity. We show that one of these single nucleotide polymorphisms is within a binding site for E4BP4 and increases RIPK1 promoter activity and RIPK1 gene expression in adipose tissue. Therapeutic silencing of RIPK1 in vivo in a mouse model of diet-induced obesity dramatically reduces fat mass, total body weight and improves insulin sensitivity, while simultaneously reducing macrophage and promoting invariant natural killer T cell accumulation in adipose tissue. These findings demonstrate that RIPK1 is genetically associated with obesity, and reducing RIPK1 expression is a potential therapeutic approach to target obesity and related diseases.

Off-target effects of CRISPRa on interleukin-6 expression.

Inactive fusion variants of the CRISPR-Cas9 system are increasingly being used as standard methodology to study transcription regulation. Their ability to readily manipulate the native genomic loci is particularly advantageous. In this work, we serendipitously uncover the key cytokine IL6 as an off-target of the activating derivative of CRISPR (CRISPRa) while studying RP11-326A19.4, a novel long-non coding RNA (lncRNA). Increasing RP11-326A19.4 expression in HEK293T cells via CRISPRa-mediated activation of its promoter region induced genome-wide transcriptional changes, including upregulation of IL6, an important cytokine. IL6 was increased in response to distinct sgRNA targeting the RP11-326A19.4 promoter region, suggesting specificity. Loss of the cognate sgRNA recognition sites failed to abolish CRISPRa mediated activation of IL6 however, pointing to off-target effects. Bioinformatic approaches did not reveal predicted off-target binding sites. Off-target activation of IL6 was sustained and involved low level activation of known IL6 regulators. Increased IL6 remained sensitive to further activation by TNFα, consistent with the existence of independent mechanisms. This study provides experimental evidence that CRISPRa has discrete, unpredictable off-targeting limitations that must be considered when using this emerging technology.

Regulation of MFGE8 by the intergenic coronary artery disease locus on 15q26.1.

BACKGROUND AND AIMS: A recently identified locus for coronary artery disease (CAD) tagged by rs8042271 is in a region of tight linkage disequilibrium (LD) between 2 genes (MFGE8, ABHD2) previously linked to atherosclerosis. Here we have explored the regulatory framework of this region to identify its functional relationship to CAD. METHODS: The CAD Associated Region between MFGE8 and ABHD2 (CARMA) was investigated by bioinformatic approaches and transcriptional reporter assays to prioritize target genes and identify putative causal variants. Findings were integrated with publicly available gene expression datasets. MFGE8 silencing was performed in cell models relevant to CAD. RESULTS: The regulatory potential of CARMA is disseminated sparsely over the entire region. CARMA contains multiple eQTL that regulate MFGE8 in coronary artery and coronary artery smooth muscle cell (CoSMC). SNPs that predict the expression of MFGE8 in artery are concordantly associated with higher risk of CAD (pval = 0.0014). Targeting CARMA by CRISPR/Cas9 in a cellular model increased MFGE8 expression. MFGE8 silencing was found to reduce CoSMC and monocyte (THP-1) but not endothelial cell proliferation. CONCLUSIONS: These findings support a mechanistic link between a GWAS identified CAD risk locus and atherosclerosis. The intergenic locus CARMA regulates MFGE8 in a haplotype dependent manner. Individuals genetically susceptible to increased MFGE8 expression exhibit greater CAD risk. Suppressing MFGE8 expression reduced SMC and THP-1 proliferation. These data support an atherogenic contribution of CARMA/MFGE8 that may be linked to cell proliferation and/or improved survival of CAD relevant cell types.

Genome-wide gene-based analyses of weight loss interventions identify a potential role for NKX6.3 in metabolism.

Hundreds of genetic variants have been associated with Body Mass Index (BMI) through genome-wide association studies (GWAS) using observational cohorts. However, the genetic contribution to efficient weight loss in response to dietary intervention remains unknown. We perform a GWAS in two large low-caloric diet intervention cohorts of obese participants. Two loci close to NKX6.3/MIR486 and RBSG4 are identified in the Canadian discovery cohort (n = 1166) and replicated in the DiOGenes cohort (n = 789). Modulation of HGTX (NKX6.3 ortholog) levels in Drosophila melanogaster leads to significantly altered triglyceride levels. Additional tissue-specific experiments demonstrate an action through the oenocytes, fly hepatocyte-like cells that regulate lipid metabolism. Our results identify genetic variants associated with the efficacy of weight loss in obese subjects and identify a role for NKX6.3 in lipid metabolism, and thereby possibly weight control.

Cholesteryl ester transfer protein (CETP) expression protects against diet induced atherosclerosis in SR-BI deficient mice.

OBJECTIVE: To determine whether expression of the human CETP transgene protects against diet-induced atherosclerosis in SR-BI deficient mice. METHODS AND RESULTS: SR-BI deficient (-/-) mice were crossed with CETP transgenic (CETPtg) mice to produce a colony of SR-BI(-/-) x CETPtg mice in a C57Bl/6 background. Age and sex matched groups of genetically modified and wild-type C57Bl/6 mice were fed a high fat, high cholesterol diet for 22 weeks. In both wild-type and SR-BI(-/-) mice, expression of the CETP transgene reduced the cholesterol content and increased the density of lipoprotein particles in the HDL density range. In SR-BI(-/-) x CETPtg mice, CETP activity inversely correlated with total plasma cholesterol levels and shifted the buoyant HDL typical of SR-BI deficiency toward a more normal density HDL particle. Atherosclerosis at the level of the aortic arch was evident in both male and female SR-BI deficient mice but occurred to a greater extent in the females. Expression of CETP markedly attenuated the development of atherosclerosis in SR-BI deficient mice fed an atherogenic diet (P<0.003). CONCLUSIONS: Expression of the human CETP transgene protects SR-BI deficient mice from atherosclerosis, consistent with a role for CETP in remodeling HDL and providing an alternative pathway for the selective uptake of HDL-CE by the liver.

Whole-Gene Duplication of PCSK9 as a Novel Genetic Mechanism for Severe Familial Hypercholesterolemia.

BACKGROUND: Familial hypercholesterolemia (FH) is a common genetic disorder of severely elevated low-density lipoprotein (LDL) cholesterol, characterized by premature atherosclerotic cardiovascular disease. Although copy number variations (CNVs) are a large-scale mutation-type capable of explaining FH cases, they have been, to date, assessed only in the LDLR gene. Here, we performed novel CNV screening in additional FH-associated genes using a next-generation sequencing-based approach. METHODS: In 704 patients with FH, we sequenced FH-associated genes APOB, PCSK9, LDLRAP1, APOE, STAP1, LIPA, and ABCG5/8 using our LipidSeq targeted next-generation sequencing panel. Bioinformatic tools were applied to LipidSeq data for CNV screening, and identified CNVs were validated using whole-exome sequencing and microarray-based copy number analyses. RESULTS: We identified a whole-gene duplication of PCSK9 in 2 unrelated Canadian FH index cases; this PCSK9 CNV was also found to cosegregate with affected status in family members. Features in affected individuals included severely elevated LDL cholesterol levels that were refractory to intensive statin therapy, pronounced clinical stigmata, premature cardiovascular events, and a plasma PCSK9 of approximately 5000 ng/mL in 1 index case. We found no CNVs in APOB, LDLRAP1, APOE, STAP1, LIPA, and ABCG5/8 in our cohort of 704 FH individuals. CONCLUSIONS: Here, we report the first description of a CNV affecting the PCSK9 gene in FH. This finding is associated with a profound FH phenotype and the highest known plasma PCSK9 level reported in a human. This finding also has therapeutic relevance, as elevated PCSK9 levels may limit the efficacy of high-dose statin therapy and also PCSK9 inhibition.

Functional Validation of a Common Nonsynonymous Coding Variant in ZC3HC1 Associated With Protection From Coronary Artery Disease.

BACKGROUND: Although virtually all coronary artery disease associated single-nucleotide polymorphisms identified by genome-wide association studies (GWAS) are in noncoding regions of the genome, a common polymorphism in ZC3HC1 (rs11556924), resulting in an arginine (Arg) to histidine (His) substitution in its encoded protein, NIPA (Nuclear Interacting Partner of Anaplastic Lyphoma Kinase) is linked to a protection from coronary artery disease. NIPA plays a role in cell cycle progression, but the functional consequences of this polymorphism have not been established. METHODS AND RESULTS: Here we demonstrate that total ZC3HC1 expression in whole blood is similar across genotypes, despite expression being slightly biased toward the risk allele in heterozygotes. At the protein level, the protective His363 NIPA variant exhibits increased phosphorylation of a critical serine residue (Ser354) and higher protein expression as compared with the Arg363 variant. Binding experiments indicate that neither SKP1 (S-phase kinase-associated protein 1) nor CCNB1 binding were affected by the polymorphism. Despite similar nuclear distribution, NIPA His363 exhibits greater nuclear mobility. NIPA suppression results in a modest reduction of proliferation in vascular smooth muscle cells, but given low proliferative capacity, a significant effect of the variant was not noted. By contrast, we demonstrate that the protective variant reduces cell proliferation in HeLa cells. CONCLUSIONS: These findings extend the genetic association between rs11556924 and coronary artery disease risk by characterizing its effects on the encoded protein, NIPA. The resulting amino acid change Arg363His is associated with increased expression and nuclear mobility, as well as lower rates of cell growth in HeLa cells, further supporting a role for cell proliferation in atherosclerosis and its clinical consequences.