Low-frequency coding DNA sequence variants in the proprotein convertase subtilisin/kexin type 9 gene (PCSK9) lower plasma low-density lipoprotein cholesterol (LDL-C), protect against risk of coronary heart disease (CHD), and have prompted the development of a new class of therapeutics. It is uncertain whether the PCSK9 example represents a paradigm or an isolated exception. We used the "Exome Array" to genotype >200,000 low-frequency and rare coding sequence variants across the genome in 56,538 individuals (42,208 European ancestry [EA] and 14,330 African ancestry [AA]) and tested these variants for association with LDL-C, high-density lipoprotein cholesterol (HDL-C), and triglycerides. Although we did not identify new genes associated with LDL-C, we did identify four low-frequency (frequencies between 0.1% and 2%) variants (ANGPTL8 rs145464906 [c.361C>T; p.Gln121*], PAFAH1B2 rs186808413 [c.482C>T; p.Ser161Leu], COL18A1 rs114139997 [c.331G>A; p.Gly111Arg], and PCSK7 rs142953140 [c.1511G>A; p.Arg504His]) with large effects on HDL-C and/or triglycerides. None of these four variants was associated with risk for CHD, suggesting that examples of low-frequency coding variants with robust effects on both lipids and CHD will be limited.

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.

BackgroundFamilial hypercholesterolemia (FH) is an autosomal codominant disorder caused by mutations in the low-density lipoprotein receptor (LDLR) gene. Homozygous FH patients (hoFH) have severe hypercholesterolemia leading to life threatening atherosclerosis in childhood and adolescence. Mice with germ line interruptions in the Ldlr and Apobec1 genes (Ldlr−/−Apobec1−/−) simulate metabolic and clinical aspects of hoFH, including atherogenesis on a chow diet.Methods/Principal FindingsIn this study, vectors based on adeno-associated virus 8 (AAV8) were used to deliver the gene for mouse Ldlr (mLDLR) to the livers of Ldlr−/−Apobec1−/− mice. A single intravenous injection of AAV8.mLDLR was found to significantly reduce plasma cholesterol and non-HDL cholesterol levels in chow-fed animals at doses as low as 3×109 genome copies/mouse. Whereas Ldlr−/−Apobec1−/− mice fed a western-type diet and injected with a control AAV8.null vector experienced a further 65% progression in atherosclerosis over 2 months compared with baseline mice, Ldlr−/−Apobec1−/− mice treated with AAV8.mLDLR realized an 87% regression of atherosclerotic lesions after 3 months compared to baseline mice. Immunohistochemical analyses revealed a substantial remodeling of atherosclerotic lesions.Conclusions/SignificanceCollectively, the results presented herein suggest that AAV8-based gene therapy for FH may be feasible and support further development of this approach. The pre-clinical data from these studies will enable for the effective translation of gene therapy into the clinic for treatment of FH.

Background:ACAT-related enzyme 2 required for viability 1 (ARV1) is a putative lipid transporter of the endoplasmic reticulum that is conserved across eukaryotic species. The ARV1 protein contains a conserved N-terminal cytosolic zinc ribbon motif known as the ARV1 homology domain, followed by multiple transmembrane regions anchoring it in the ER. Deletion of ARV1 in yeast results in defective sterol trafficking, aberrant lipid synthesis, ER stress, membrane disorganization and hypersensitivity to fatty acids (FAs). We sought to investigate the role of Arv1 in mammalian lipid metabolism.Methods:Homologous recombination was used to disrupt the Arv1 gene in mice. Animals were examined for alterations in lipid and lipoprotein levels, body weight, body composition, glucose tolerance and energy expenditure.Results:Global loss of Arv1 significantly decreased total cholesterol and high-density lipoprotein cholesterol levels in the plasma. Arv1 knockout mice exhibited a dramatic lean phenotype, with major reductions in white adipose tissue (WAT) mass and body weight on a chow diet. This loss of WAT is accompanied by improved glucose tolerance, higher adiponectin levels, increased energy expenditure and greater rates of whole-body FA oxidation.Conclusions:This work identifies Arv1 as an important player in mammalian lipid metabolism and whole-body energy homeostasis.

Genome-wide association studies (GWAS) have identified 95 loci in the human genome that harbor common variants associated with plasma lipid traits. Of the 95 loci, 17 harbor genes known to cause monogenic lipid disorders and collectively a third of them contain genes with characterized roles in lipid metabolism. Therefore in the majority of loci the causal genes are unknown. We selected 32 genes, not previously implicated in lipid metabolism and representing a total of 26 loci, to test for their ability to modify plasma lipid concentrations upon somatic overexpression in vivo. We utilized adeno-associated virus serotype 8 (AAV8) to overexpress the selected genes specifically in the livers of both C57BL/6 mice and in an appropriate humanized mouse model (either mice expressing human apolipoprotein A-I for HDL loci or Apobec1-knockout, Ldlr haploinsufficient mice expressing human apolipoprotein B-100 for triglyceride and LDL loci). Approximately half of the genes tested reproducibly affected plasma lipids. For 13 of the interrogated loci the lipid-associated variants also correlated with expression variations of the respective genes in liver (liver expression quantitative trait loci-eQTLs). We demonstrate a causal role for 7 of these 13 genes. The overexpression of these 7 genes not only affected the predicted lipid class, but additionally exerted its effect in the predicted direction in 6 of 7 cases (Tmem57, Slc39a8, Ppp1r3b, Vkorc1, Tbkbp1 and Ube2l3). Additionally for a subset of the examined genes we proceeded to develop small interfering RNA (siRNA) nanoparticles that were particularly targeted to the liver. We were able to obtain robust knockdown for a significant number of genes and, in several cases, observe reciprocal effects on plasma lipids from our overexpression and knockdown studies. This work has identified several novel lipid regulators, whose further investigation can uncover novel mechanisms and pathways controlling plasma lipids.

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