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。近年来，对基因组变异的研究进入了一个新的阶段。其中无偏全基因组关联研究可以识别与常见疾病相关的新的遗传基因座。我们最近在NHLBl Framingham心脏研究(FHS)和其他人群队列中描述了95个与血脂水平相关的基因--低密度脂蛋白(LDL-C)、高密度脂蛋白(HDL-C)或甘油三酯，它们与心肌梗死(ML)的风险密切相关。要将新的关联转化为功能洞察力，并最终转化为降低ML风险的疗法，还需要做大量的工作。关键的一步是确定这些遗传位点如何影响与脂肪代谢相关的人类组织类型的表型，主要是肝脏和脂肪。我们对来自患者的外科肝脏和脂肪组织样本中的基因型和基因表达进行了表达数量性状基因座(EQTL)分析；从这项工作中，我们发现染色体1p13-rs12740374上的一个与低密度脂蛋白-C和M1相关的SNP与S0RT1基因的肝脏表达有很强的相关性。然而，这些研究受到组织稀缺和无法解决诸如脂蛋白分泌等关键细胞表型的限制。因此，有必要建立来自特定基因型别患者的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采集外周血细胞(PBC)获得重编程诱导多能干细胞(IPS)的能力。我们还开发了一种新技术，利用编码重编程因子的体外转录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细胞分化为有功能的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)、马萨诸塞州综合医院(MGH)和哈佛医学院(HMS)的研究人员组成。我们有得天独厚的优势，可以开发来自数百名FHS参与者的IPS细胞系和iPS来源的肝细胞和脂肪细胞的文库，这些参与者拥有全基因组的基因数据以及许多心血管表型。我们建议利用这些资源：(1)开发高效的方案，从血液样本中获得IPS细胞系，然后分化为有功能的肝细胞和脂肪细胞；(2)扩大这些方案的规模，以高通量地从Framingham子代队列中的~400个个体获得IPS细胞系、肝细胞和脂肪细胞；以及(3)从这些肝细胞和脂肪细胞中执行基因表达和代谢组谱分析，从而能够根据基因表达和代谢物水平对基因型进行综合统计分析，以及现有的表型数据，如动脉粥样硬化的亚临床指标。 (摘要结束)

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)