Integrating lipid genotyes and phenotypes in iPS derived hepatocytes/adipocytes

描述（由申请人提供）： 人类心血管疾病新的有效治疗方法的发现需要识别和验证新的疾病机制。最近，基因组变异的研究进入了一个新阶段。其中无偏见的全基因组关联研究（GWAS）可以识别与常见疾病相关的新遗传位点。我们最近在 NHLBl 弗雷明汉心脏研究 (FHS) 和其他人群队列中描述了与血脂水平相关的 95 个基因座 - 低密度脂蛋白胆固醇 (LDL-C)、高密度脂蛋白胆固醇 (HDL-C) 或甘油三酯，这些基因座与心肌梗死 (Ml) 的风险密切相关。需要做大量的工作才能将新的关联转化为功能性见解，并最终降低 Ml 风险的治疗方法。关键一步是确定这些基因位点如何影响与脂质代谢相关的人体组织类型（主要是肝脏和脂肪）的表型。我们对患者的手术肝脏和脂肪组织样本中的基因型与基因表达进行了表达数量性状位点 (eQTL) 分析；从这项工作中，我们发现染色体 1p13-rs12740374 上的 LDL-C 和 M1 相关 SNP 与 S0RT1 基因的肝脏表达之间存在很强的关联。然而，这些研究受到组织稀缺和无法解决脂蛋白分泌等关键细胞表型的限制。因此，需要从特定基因型的患者中建立无限可再生的肝细胞和脂肪细胞来源。我们已经开发出通过外周血细胞 (PBC) 采集获得重编程诱导多能干 (IPS) 细胞的能力。我们还开发了一种新技术，利用体外转录的编码重编程因子的 RNA，在不进行任何基因组改变的情况下加速和扩大 IPS 的生成。最后，我们建立了将 iPS 细胞分化为功能性肝细胞和脂肪细胞的方案。我们的联盟由来自 FHS、哈佛干细胞研究所 (HSCI)、马萨诸塞州总医院 (MGH) 和哈佛医学院 (HMS) 的研究人员组成。我们拥有独特的优势，可以开发来自数百名 FHS 参与者的 IPS 细胞系和 iPS 衍生的肝细胞和脂肪细胞库，这些参与者拥有全基因组基因型数据以及许多心血管表型。我们建议利用这些资源：（1）开发有效的方案从血液样本中获取IPS细胞系，然后分化为功能性肝细胞和脂肪细胞； (2) 扩大这些方案的规模，以便能够从 Framingham 后代队列中约 400 名个体中高通量生成 IPS 细胞系、肝细胞和脂肪细胞； (3) 对这些肝细胞和脂肪细胞进行基因表达和代谢组学分析，从而能够对基因型与基因表达和代谢物水平以及现有表型数据（例如动脉粥样硬化的亚临床测量）进行综合统计分析。 （摘要完）

DESCRIPTION (provided by applicant): The discovery of new and effective treatments for human cardiovascular diseases 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 associate with blood lipid levels-LDL cholesterol (LDL-C), HDL cholesterol (HDL-C), or triglycerides, which are strongly associated with risk for myocardial infarction (Ml) In the NHLBl Framingham Heart Study (FHS) and other population cohorts. Much work will be needed to convert the novel associations into functional insights and, ultimately, therapies to reduce the risk of Ml. 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 a strong association between an LDL-C- and Ml-associated SNP on chromosome 1p13-rs12740374-and hepatic expression of the S0RT1 gene. However, these studies were limited by scarcity of tissue and the inability to address key cellular phenotypes such as lipoprotein secretion. Thus, there is a need to establish infinitely renewable sources of hepatocytes and adipocytes from patients of defined genotypes. We have developed the ability to obtain reprogrammed induced pluripotent stem (IPS) cells via peripheral blood cell (PBC) collection. We have also developed a novel technology to accelerate and scale up IPS generation, without any genomic alteration, using in vitro transcribed RNAs encoding reprogramming factors. Finally, we have established protocols to differentiate iPS cells into functional hepatocytes and adipocytes. Our consortium comprises investigators from the FHS, the Harvard Stem Cell Institute (HSCI), Massachusetts General Hospital (MGH), and Harvard Medical School (HMS). We are uniquely positioned to develop a library of IPS cell lines and iPS- derived hepatocytes and adipocytes from several hundred FHS participants, who have genome-wide genotype data as well as many cardiovascular phenotypes. We propose to leverage these resources to: (1) develop efficient protocols to obtain IPS cell lines from blood samples, followed by differentiation into functional hepatocytes and adipocytes; (2) scale up these protocols to enable high-throughput generation of IPS cell lines, hepatocytes, and adipocytes from ~400 Individuals In the Framingham Offspring Cohort; and (3) perform gene expression and metaboiomic profiling from these hepatocytes and adipocytes, enabling Integrative statistical analyses of genotypes with gene expression and metabolite levels, as well as existing phenotype data such as subclinical measures of atherosclerosis. (End of Abstract)