RESUMO
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.
Assuntos
HDL-Colesterol/genética , LDL-Colesterol/genética , Doença das Coronárias/genética , Frequência do Gene , Variação Genética , Triglicerídeos/sangue , 1-Alquil-2-acetilglicerofosfocolina Esterase/genética , 1-Alquil-2-acetilglicerofosfocolina Esterase/metabolismo , Adulto , Idoso , Alelos , Animais , População Negra/genética , HDL-Colesterol/sangue , LDL-Colesterol/sangue , Estudos de Coortes , Doença das Coronárias/sangue , Feminino , Estudos de Associação Genética , Código Genético , Humanos , Modelos Lineares , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Pessoa de Meia-Idade , Fenótipo , Análise de Sequência de DNA , Subtilisinas/genética , Subtilisinas/metabolismo , População Branca/genéticaRESUMO
BACKGROUND AND AIMS: 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. METHODS: 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. RESULTS: 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-/- carrying human ApoB-100 transgene) deficient in the LDL receptor (LDLR), Angptl3 silencing had minimum effect on LDL-C, suggesting the effect being linked to LDLR. This observation is supported by an additive effect on LDL-C between ANGPTL3 and PCSK9 siRNAs. ANGPTL3 gene deletion induced cellular long-chain TG and ApoB-100 accumulation with elevated LDLR and LDLR-related protein (LRP) 1 expression. Consistent with this, ANGPTL3 deficiency by gene deletion or silencing reduced nascent ApoB-100 secretion and increased LDL/VLDL uptake. CONCLUSIONS: 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.
Assuntos
Proteínas Semelhantes a Angiopoietina/metabolismo , LDL-Colesterol/sangue , Fígado/metabolismo , Proteína 3 Semelhante a Angiopoietina , Proteínas Semelhantes a Angiopoietina/deficiência , Proteínas Semelhantes a Angiopoietina/genética , Animais , Apolipoproteína B-100/genética , Apolipoproteína B-100/metabolismo , Biomarcadores/sangue , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas , Proteínas de Transferência de Ésteres de Colesterol/genética , Proteínas de Transferência de Ésteres de Colesterol/metabolismo , HDL-Colesterol/sangue , Regulação para Baixo , Edição de Genes/métodos , Células Hep G2 , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/sangue , Obesidade/genética , Pró-Proteína Convertase 9/genética , Pró-Proteína Convertase 9/metabolismo , Interferência de RNA , Receptores de LDL/deficiência , Triglicerídeos/sangueRESUMO
BACKGROUND: Familial 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 FINDINGS: In 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 3×10(9) 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/SIGNIFICANCE: Collectively, 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.