High-throughput screening and stem cell modeling of causal eQTL variants

因果 eQTL 变异的高通量筛选和干细胞建模

• 批准号: 8826773
• 负责人: Kiran Musunuru
• 金额: $ 31.4万
• 依托单位: HARVARD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-04-01 至 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8826773
• 关键词: 1p13; Adipocytes; Adipose tissue; Affect; Alleles; Binding Sites; Biological Assay; Biology; CCAAT-Enhancer-Binding Proteins; Cardiovascular Diseases; Cause of Death; Cell Line; Cell model; Cholesterol; Chromosomes; Complex; DNA; DNA Sequence; Disease; Family; Gene Expression; Genes; Genetic; Genetic Transcription; Genetic study; Genomics; Genotype; Health; Hepatic; Hepatocyte; High Density Lipoprotein Cholesterol; Histocompatibility Testing; Human; Human Genetics; Human Genome; LDL Cholesterol Lipoproteins; Lead; Linkage Disequilibrium; Lipids; Lipoproteins; Liver; Measures; Methodology; Minor; Molecular; Operative Surgical Procedures; Patients; Phase; Phenotype; Pluripotent Stem Cells; Quantitative Trait Loci; Reporter; Reporter Genes; Research Personnel; Risk; Single Nucleotide Polymorphism; Site; Stem cells; Technology; Tissue Sample; Triglycerides; Untranslated RNA; Validation; Variant; Work; base; blood lipid; cardiovascular disorder risk; disorder risk; effective therapy; gene function; genome editing; genome wide association study; genomic variation; high throughput screening; human tissue; innovation; insight; lipid metabolism; next generation; next generation sequencing; novel; nuclease; prevent; research study; success

DESCRIPTION (provided by applicant): The discovery of new and effective treatments for human cardiovascular disease (CVD) requires the identification and validation of novel disease mechanisms. Recently, studies of genomic variation entered a new phase, in which unbiased genome-wide association studies (GWAS) can identify novel genetic loci associated with common diseases. We have recently described 95 loci associated with blood lipid levels LDL cholesterol (LDL-C), HDL cholesterol (HDL-C), or triglycerides (TG), which are strongly associated with risk for CVD. Much work will be needed to convert the novel associations into functional insights and, ultimately, therapies to reduce the risk of CVD. A key step is to determine how these genetic loci affect phenotypes in human tissue types relevant to lipid metabolism, principally liver and adipose. We have performed expression quantitative trait locus (eQTL) analyses of genotype vs. gene expression in surgical liver and adipose tissue samples from patients; from this work, we found strong associations between a number of lipid-associated tag single nucleotide polymorphisms (SNPs) and either hepatic or adipose expression of nearby genes. These observations suggest that causal SNPs in linkage disequilibrium (LD) with the tag SNPs directly influence the expression of causal genes that are responsible for changes in blood lipid levels in humans. Identifying these causal SNPs and causal genes would lead to insights into the molecular mechanisms by which the DNA variants drive phenotypic changes in liver and adipose and, ultimately, affect the risk of disease. Our general strategy is to combine several innovations to identify casual SNPs. We will: (1) perform high- throughput screening of candidate SNPs in eQTL loci for alteration of reporter gene expression in the appropriate tissue type, using a novel massively parallel reporter assay (MPRA), to prioritize SNPs for further study; (2) use human genome editing with cutting-edge TAL effector nuclease (TALEN) technology to alter each high-priority SNP in human pluripotent stem cells (hPSCs), so as to generate isogenic cell lines that differ only at the SNP; (3) differentiate the isogenic hPSCs into the appropriate tissue type; and (4) measure nearby gene expression to confirm that the SNP is truly causal for the eQTL. We propose to implement this general strategy for 57 lipid-associated loci with eQTLs in human liver and adipose. Success in completing this project will not only provide fresh new insights into the biology of lipid metabolism, but will also establish a new methodological paradigm by which investigators can determine which DNA sequence variants identified in next-generation human genetic studies underlie the genetic basis of complex phenotypes.

描述（由申请人提供）：发现人类心血管疾病（CVD）的新有效治疗方法需要鉴定和验证新型疾病机制。最近，对基因组变异的研究进入了一个新阶段，在该阶段中，公正的全基因组关联研究（GWAS）可以鉴定出与常见疾病相关的新型遗传基因座。我们最近描述了与血脂水平LDL胆固醇（LDL-C），HDL胆固醇（HDL-C）或甘油三酸酯（TG）相关的95个基因座，它们与CVD的风险密切相关。将新型关联转化为功能见解，并最终需要降低CVD风险的疗法需要大量工作。关键步骤是确定这些遗传基因座如何影响与脂质代谢，主要是肝脏和脂肪有关的人体组织类型的表型。我们已经对来自患者的手术肝和脂肪组织样品进行了基因型与基因表达的表达定量性状基因座（EQTL）分析。从这项工作中，我们发现了许多与脂质相关的TAG单核苷酸多态性（SNP）与附近基因的肝或脂肪表达之间的密切关联。这些观察结果表明，与TAG SNP的连锁不平衡（LD）中的因果SNP直接影响导致人类血脂水平变化的因果基因的表达。鉴定这些因果SNP和因果基因将导致对DNA变异驱动肝脏和脂肪中表型变化的分子机制的见解，并最终影响疾病的风险。我们的一般策略是结合几项创新以识别休闲SNP。我们将：（1）使用新型的大规模平行报告基因测定法（MPRA）对eqtl基因座中候选SNP进行高吞吐量筛查，以改变适当组织类型中的报告基因表达，以优先使用SNP进行进一步研究； （2）将人类基因组编辑与尖端的TAL效应核酸酶（TALEN）技术使用人类多能干细胞（HPSC）中的每个高优先级SNP改变，以便产生仅在SNP处不同的等源细胞系； （3）将等源性HPSC区分为适当的组织类型； （4）测量附近的基因表达，以确认SNP是EQTL的真正因果关系。我们建议在人肝脏和脂肪中对57个脂质相关基因座的57个相关基因座实施这种一般策略。完成该项目的成功不仅将为脂质代谢的生物学提供新的新见解，而且还将建立一个新的方法论范式，研究人员可以通过该范式确定在下一代人遗传研究中鉴定的哪些DNA序列变体是复杂表型的遗传基础。