RESUMEN
BACKGROUND: Genome-wide association studies have identified hundreds of loci associated with lipid levels. However, the genetic mechanisms underlying most of these loci are not well-understood. Recent work indicates that changes in the abundance of alternatively spliced transcripts contribute to complex trait variation. Consequently, identifying genetic loci that associate with alternative splicing in disease-relevant cell types and determining the degree to which these loci are informative for lipid biology is of broad interest. METHODS: We analyze gene splicing in 83 sample-matched induced pluripotent stem cell (iPSC) and hepatocyte-like cell lines (n=166), as well as in an independent collection of primary liver tissues (n=96) to perform discovery of splicing quantitative trait loci (sQTLs). RESULTS: We observe that transcript splicing is highly cell type specific, and the genes that are differentially spliced between iPSCs and hepatocyte-like cells are enriched for metabolism pathway annotations. We identify 1384 hepatocyte-like cell sQTLs and 1455 iPSC sQTLs at a false discovery rate of <5% and find that sQTLs are often shared across cell types. To evaluate the contribution of sQTLs to variation in lipid levels, we conduct colocalization analysis using lipid genome-wide association data. We identify 19 lipid-associated loci that colocalize either with an hepatocyte-like cell expression quantitative trait locus or sQTL. Only 2 loci colocalize with both a sQTL and expression quantitative trait locus, indicating that sQTLs contribute information about genome-wide association studies loci that cannot be obtained by analysis of steady-state gene expression alone. CONCLUSIONS: These results provide an important foundation for future efforts that use iPSC and iPSC-derived cells to evaluate genetic mechanisms influencing both cardiovascular disease risk and complex traits in general.
Asunto(s)
Empalme Alternativo , Estudio de Asociación del Genoma Completo , Humanos , Estudio de Asociación del Genoma Completo/métodos , Empalme del ARN , Sitios de Carácter Cuantitativo , LípidosRESUMEN
OBJECTIVE: The noncoding single-nucleotide polymorphism rs12740374 has been hypothesized to be the causal variant responsible for liver-specific modulation of SORT1(sortilin 1) expression (ie, expression quantitative trait locus) and, by extension, the association of the SORT1 locus on human chromosome 1p13 with low-density lipoprotein cholesterol levels and coronary heart disease. The goals of this study were to compare 3 different hepatocyte models in demonstrating that the rs12740374 minor allele sequence is responsible for transcriptional activation of SORT1 expression. APPROACH AND RESULTS: We found that although primary human hepatocytes of varied rs12740374 genotypes strongly replicated the SORT1 expression quantitative trait locus observed previously in whole-liver samples, a population cohort of induced pluripotent stem cell-derived hepatocyte-like cells poorly replicated the expression quantitative trait locus. In primary human hepatocytes from multiple individuals heterozygous at rs12740374, we used CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-associated 9) to specifically target the rs12740374 minor allele sequence ex vivo, resulting in a reproducible reduction in SORT1 expression. We generated a locus-humanized transgenic mouse with a bacterial artificial chromosome bearing the human SORT1 locus with the rs12740374 minor allele. In this mouse model, we used CRISPR-Cas9 to target the rs12740374 minor allele sequence in the liver in vivo, resulting in a substantial reduction of hepatic SORT1 expression. CONCLUSIONS: The rs12740374 minor allele sequence enhances SORT1 expression in hepatocytes. CRISPR-Cas9 can be used in primary human hepatocytes ex vivo and locus-humanized mice in vivo to interrogate the function of noncoding regulatory regions. Induced pluripotent stem cell-derived hepatocyte-like cells experience limitations that prevent faithful modelling of some hepatocyte expression quantitative trait loci.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Aterosclerosis/metabolismo , Hepatocitos/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/genética , Animales , Aterosclerosis/genética , Aterosclerosis/patología , Sistemas CRISPR-Cas , Células Cultivadas , Modelos Animales de Enfermedad , Edición Génica/métodos , Regulación de la Expresión Génica , Predisposición Genética a la Enfermedad , Hepatocitos/patología , Heterocigoto , Humanos , Células Madre Pluripotentes Inducidas/patología , Ratones Transgénicos , Fenotipo , Polimorfismo de Nucleótido Simple , Sitios de Carácter Cuantitativo , Transcripción GenéticaRESUMEN
Abetalipoproteinemia (ABL) is an inherited disorder of lipoprotein metabolism resulting from mutations in microsomal triglyceride transfer protein (MTTP). In addition to expression in the liver and intestine, MTTP is expressed in cardiomyocytes, and cardiomyopathy has been reported in several ABL cases. Using induced pluripotent stem cells (iPSCs) generated from an ABL patient homozygous for a missense mutation (MTTPR46G), we show that human hepatocytes and cardiomyocytes exhibit defects associated with ABL disease, including loss of apolipoprotein B (apoB) secretion and intracellular accumulation of lipids. MTTPR46G iPSC-derived cardiomyocytes failed to secrete apoB, accumulated intracellular lipids, and displayed increased cell death, suggesting intrinsic defects in lipid metabolism due to loss of MTTP function. Importantly, these phenotypes were reversed after the correction of the MTTPR46G mutation by CRISPR/Cas9 gene editing. Together, these data reveal clear cellular defects in iPSC-derived hepatocytes and cardiomyocytes lacking MTTP activity, including a cardiomyocyte-specific regulated stress response to elevated lipids.
Asunto(s)
Apolipoproteínas B/metabolismo , Proteínas Portadoras/metabolismo , Hepatocitos/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Miocitos Cardíacos/metabolismo , Estrés Fisiológico , Abetalipoproteinemia/metabolismo , Edición Génica , Humanos , FenotipoRESUMEN
Efforts to identify pharmaceuticals to treat heritable metabolic liver diseases have been hampered by the lack of models. However, cells with hepatocyte characteristics can be produced from induced pluripotent stem cells (iPSCs). Here, we have used hepatocyte-like cells generated from homozygous familial hypercholesterolemia (hoFH) iPSCs to identify drugs that can potentially be repurposed to lower serum LDL-C. We found that cardiac glycosides reduce the production of apolipoprotein B (apoB) from human hepatocytes in culture and the serum of avatar mice harboring humanized livers. The drugs act by increasing the turnover of apoB protein. Analyses of patient medical records revealed that the treatment of patients with cardiac glycosides reduced serum LDL-C levels. These studies highlight the effectiveness of using iPSCs to screen for potential treatments for inborn errors of hepatic metabolism and suggest that cardiac glycosides could provide an approach for reducing hepatocyte production of apoB and treating hypercholesterolemia.
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Glicósidos Cardíacos/uso terapéutico , Evaluación Preclínica de Medicamentos , Hepatocitos/citología , Hipercolesterolemia/tratamiento farmacológico , Células Madre Pluripotentes Inducidas/citología , Animales , Apolipoproteínas B/metabolismo , Glicósidos Cardíacos/farmacología , LDL-Colesterol/sangre , Células Hep G2 , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Homocigoto , Humanos , Hipercolesterolemia/sangre , Hipolipemiantes/química , Hipolipemiantes/farmacología , Hipolipemiantes/uso terapéutico , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/metabolismo , Ratones , Ratones Endogámicos NOD , Proteolisis/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Bibliotecas de Moléculas Pequeñas/uso terapéuticoRESUMEN
Genome-wide association studies have struggled to identify functional genes and variants underlying complex phenotypes. We recruited a multi-ethnic cohort of healthy volunteers (n = 91) and used their tissue to generate induced pluripotent stem cells (iPSCs) and hepatocyte-like cells (HLCs) for genome-wide mapping of expression quantitative trait loci (eQTLs) and allele-specific expression (ASE). We identified many eQTL genes (eGenes) not observed in the comparably sized Genotype-Tissue Expression project's human liver cohort (n = 96). Focusing on blood lipid-associated loci, we performed massively parallel reporter assays to screen candidate functional variants and used genome-edited stem cells, CRISPR interference, and mouse modeling to establish rs2277862-CPNE1, rs10889356-DOCK7, rs10889356-ANGPTL3, and rs10872142-FRK as functional SNP-gene sets. We demonstrated HLC eGenes CPNE1, VKORC1, UBE2L3, and ANGPTL3 and HLC ASE gene ACAA2 to be lipid-functional genes in mouse models. These findings endorse an iPSC-based experimental framework to discover functional variants and genes contributing to complex human traits.
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Sitios Genéticos , Variación Genética , Hepatocitos/citología , Células Madre Pluripotentes Inducidas/citología , Lípidos/sangre , Animales , Secuencia de Bases , Estudios de Cohortes , Perfilación de la Expresión Génica , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Hepatocitos/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Metabolismo de los Lípidos/genética , Hígado/metabolismo , Ratones , Especificidad de Órganos/genética , Polimorfismo de Nucleótido Simple/genética , Sitios de Carácter Cuantitativo/genéticaRESUMEN
Despite the recognized role of the ATP-binding Cassette Transporter A1 (ABCA1) in high-density lipoprotein (HDL) metabolism, our understanding of ABCA1 deficiency in human hepatocytes is limited. To define the functional effects of human hepatocyte ABCA1 deficiency, we generated induced pluripotent stem cell (iPSC)-derived hepatocyte-like cells (HLCs) from Tangier disease (TD) and matched control subjects. Control HLCs exhibited robust cholesterol efflux to apolipoprotein A-I (apoA-I) and formed nascent HDL particles. ABCA1-deficient HLCs failed to mediate lipid efflux or nascent HDL formation, but had elevated triglyceride (TG) secretion. Global transcriptome analysis revealed significantly increased ANGPTL3 expression in ABCA1-deficient HLCs. Angiopoietin-related protein 3 (ANGPTL3) was enriched in plasma of TD relative to control subjects. These results highlight the required role of ABCA1 in cholesterol efflux and nascent HDL formation by hepatocytes. Furthermore, our results suggest that hepatic ABCA1 deficiency results in increased hepatic TG and ANGPTL3 secretion, potentially underlying the elevated plasma TG levels in TD patients.
Asunto(s)
Transportador 1 de Casete de Unión a ATP/metabolismo , Lipoproteínas HDL/metabolismo , Transportador 1 de Casete de Unión a ATP/genética , Proteína 3 Similar a la Angiopoyetina , Proteínas Similares a la Angiopoyetina/sangre , Proteínas Similares a la Angiopoyetina/genética , Proteínas Similares a la Angiopoyetina/metabolismo , Apolipoproteína A-I/metabolismo , Diferenciación Celular , Células Cultivadas , Colesterol/metabolismo , Hepatocitos/citología , Hepatocitos/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/citología , Enfermedad de Tangier/metabolismo , Enfermedad de Tangier/patología , Transcriptoma , Triglicéridos/metabolismoRESUMEN
RATIONALE: An efficient and reproducible source of genotype-specific human macrophages is essential for study of human macrophage biology and related diseases. OBJECTIVE: To perform integrated functional and transcriptome analyses of human induced pluripotent stem cell-derived macrophages (IPSDMs) and their isogenic human peripheral blood mononuclear cell-derived macrophage (HMDM) counterparts and assess the application of IPSDM in modeling macrophage polarization and Mendelian disease. METHODS AND RESULTS: We developed an efficient protocol for differentiation of IPSDM, which expressed macrophage-specific markers and took up modified lipoproteins in a similar manner to HMDM. Like HMDM, IPSDM revealed reduction in phagocytosis, increase in cholesterol efflux capacity and characteristic secretion of inflammatory cytokines in response to M1 (lipopolysaccharide+interferon-γ) activation. RNA-Seq revealed that nonpolarized (M0) as well as M1 or M2 (interleukin-4) polarized IPSDM shared transcriptomic profiles with their isogenic HMDM counterparts while also revealing novel markers of macrophage polarization. Relative to IPSDM and HMDM of control individuals, patterns of defective cholesterol efflux to apolipoprotein A-I and high-density lipoprotein-3 were qualitatively and quantitatively similar in IPSDM and HMDM of patients with Tangier disease, an autosomal recessive disorder because of mutations in ATP-binding cassette transporter AI. Tangier disease-IPSDM also revealed novel defects of enhanced proinflammatory response to lipopolysaccharide stimulus. CONCLUSIONS: Our protocol-derived IPSDM are comparable with HMDM at phenotypic, functional, and transcriptomic levels. Tangier disease-IPSDM recapitulated hallmark features observed in HMDM and revealed novel inflammatory phenotypes. IPSDMs provide a powerful tool for study of macrophage-specific function in human genetic disorders as well as molecular studies of human macrophage activation and polarization.
Asunto(s)
Técnicas de Cultivo de Célula , Células Madre Pluripotentes Inducidas/citología , Macrófagos/metabolismo , Enfermedad de Tangier/patología , Transcriptoma , Transportador 1 de Casete de Unión a ATP/deficiencia , Transportador 1 de Casete de Unión a ATP/genética , Transportador 1 de Casete de Unión a ATP/fisiología , Adulto , Anciano , Animales , Antígenos de Diferenciación/análisis , Secuencia de Bases , Diferenciación Celular , Células Cultivadas , Colesterol/metabolismo , Cuerpos Embrioides/citología , Femenino , Genotipo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Inflamación , Interferón gamma/farmacología , Lipopolisacáridos/farmacología , Activación de Macrófagos/efectos de los fármacos , Macrófagos/citología , Macrófagos/efectos de los fármacos , Masculino , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Fagocitosis , Fenotipo , ARN Mensajero/genética , Alineación de Secuencia , Homología de Secuencia de Ácido Nucleico , Enfermedad de Tangier/genética , Enfermedad de Tangier/metabolismo , Adulto JovenRESUMEN
As increasing numbers of vertebrate genomes are sequenced, comparative genomics offers tremendous promise to unveil mechanisms of transcriptional gene regulation on a large scale. However, the challenge of analysing immense amounts of sequence data and relating primary sequence to function is daunting. Several teleost species occupy crucial niches in the world of comparative genomics, as experimental model organisms of wide utility and living roadmaps of molecular evolution. Extant species have evolved after a teleost-specific genome duplication, and offer the opportunity to examine the evolution of thousands of duplicate gene pairs. Transgenesis in zebrafish is being increasingly employed to functionally examine non-coding sequences, from fish and mammals. Here, we discuss current approaches to the study of gene regulation in teleosts, and the promise of future research.