Genetic and functional analyses of a novel locus associated with LDL-C and MI

项目摘要 该提案将促进主要研究者的职业目标，即促进对 脂质性状和心肌梗死（MI）的遗传基础，并将这种理解应用于预防 患者的疾病。根据他丰富的研究经验，该建议将培训他使用 互补方法-复杂心血管特征的人类遗传学，以及小鼠模型的使用 和胚胎干细胞用于与这些性状相关的基因的功能验证-以实现生物学上的 全基因组关联研究（genome-wide association studies，GWAS）这也将为他提供教学 统计遗传学、遗传学和基因组学方法、实验生物学和研究伦理学方面的培训。 最后，这将促进他向独立研究职位的过渡，并为他提供一个强大的科学 申请R 01级资助的基金会。 首席研究员将能够利用一个绝对独特的环境， 心血管疾病动物模型的强度（马萨诸塞州总医院心血管 研究中心）、心血管遗传学和基因组学（Broad Institute of MIT和哈佛，马萨诸塞州 人类遗传研究中心）和干细胞生物学（马萨诸塞州综合医院 医院心血管研究中心、哈佛干细胞研究所）完成拟开展的工作。 提出研究计划的理由是，发现新的和有效的治疗人类疾病的方法， 心血管疾病需要在人类中鉴定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的阶段，在这个阶段中，无偏的GWAS研究可以识别 常见疾病的新基因贡献者。在过去的两年里，超过50个新的和可复制的 基因组位点被发现与脂质水平、MI和2型糖尿病有关。 尽管这些观察结果可能很有希望，但仍需要做大量的工作来将新的关联转换为 治疗关键的一步是确定定位基因座中的致病DNA变异。许多基因座含有多个 基因;其他基因座不包含已知基因，表明它们可能反式作用于基因座外的基因。 因此，需要明确地鉴定由每个基因座调控的致病基因。最后还有一个 需要解决基因座中DNA变异影响致病基因的机制。简而言之，路径必须 从基因组定位到新的机制见解。 这项提案探索了这样一条新的低密度脂蛋白胆固醇（LDL-C）基因位点的途径。 C）和MI。在初步研究中，GWAS用于LDL-C和MI，并已鉴定出单核苷酸 染色体1p13.3上与LDL-C强相关的基因座中的多态性（SNP）-更多 比基因组中的任何其他位点都强烈-非常小的LDL（vsLDL）颗粒浓度和MI。 携带这些SNP的主要等位基因的两个拷贝的个体的LDL-C高16 mg/dl， 与次要等位基因纯合子相比，vsLDL水平升高，MI风险增加18%。 1p13.3上的相关基因组间隔跨越约100个碱基（kb），并含有四个基因-CELSR 2， PSRC 1、MYBPHL、SORT 1-与LDL-C调节无既定联系。 该提案的核心问题是：（1）哪些1p13.3 DNA变异导致LDL-C，vsLDL和MI，（2） 哪些1p13.3基因与LDL-C、vsLDL和MI相关，以及（3）DNA变异通过何种机制与LDL-C、vsLDL和MI相关， 影响基因功能？遗传精细定位、基因重测序、小鼠基因敲减和过表达， 人类胚胎干细胞的实验将用来回答这些问题。成功应该是 精确定义了一个或多个新致病基因和调节机制，从而提供了一种潜在的新的 治疗血脂异常和预防MI的靶点。

PROJECT SUMMARY This proposal will facilitate the principal investigator's career goals of advancing the understanding of the inherited basis for lipid traits and myocardial infarction (MI) and applying this understanding to the prevention of disease in patients. Building on his extensive research experience, the proposal will train him in the use of complementary approaches-human genetics of complex cardiovascular traits, and the use of mouse models and embryonic stem cells for functional validation of genes associated with these traits-to achieve a biological understanding of the results of genome-wide association studies (GWAS). It will also provide him with didactic training in statistical genetics, genetic and genomic methods, experimental biology, and research ethics. Finally, it will promote his transition to an independent research position and provide him a robust scientific foundation from which to apply for R01-level funding. The principal investigator will be able to take advantage of an absolutely unique environment with diverse strengths in animal models of cardiovascular disease (Massachusetts General Hospital Cardiovascular Research Center), cardiovascular genetics and genomics (Broad Institute of MIT and Harvard, Massachusetts General Hospital Center for Human Genetic Research), and stem cell biology (Massachusetts General Hospital Cardiovascular Research Center, Harvard Stem Cell Institute) to complete the proposed work. The rationale for the proposed research plan is that the discovery of new and effective treatments for human cardiovascular diseases requires the identification and validation in humans of novel disease mechanisms. Recently, studies of genomic variation entered a new phase, in which unbiased GWAS studies can identify novel genetic contributors to common diseases. In the last two years, more than fifty new and reproducible genomic loci were discovered contributing to lipid levels, MI, and type 2 diabetes. As promising as these observations may be, much work will be needed to convert novel associations into therapies. A key step is to identify the causal DNA variants in the mapped loci. Many loci contain multiple genes; other loci contain no known genes, suggesting that they may act in trans on genes outside of the loci. Thus, there is a need to unambiguously identify the causal gene(s) regulated by each locus. Finally, there is a need to address the mechanisms by which DNA variants in loci affect the causal genes. In short, a path must be blazed from genomic localization to new mechanistic insights. This proposal explores such a path for new genetic loci identified for low-density lipoprotein cholesterol (LDL- C) and MI. In preliminary studies, GWAS for LDL-C and MI and have identified single nucleotide polymorphisms (SNPs) in a locus on chromosome 1p13.3 that is robustly associated with LDL-C-more strongly than any other locus in the genome-very small LDL (vsLDL) particle concentration, and MI. Individuals who carry two copies of the major alleles of these SNPs have 16 mg/dl higher LDL-C, 40% higher vsLDL levels, and 18% greater risk of MI when compared with homozygotes for the minor alleles. The associated genomic interval on 1p13.3 spans ~100 kilobases (kb) and harbors four genes-CELSR2, PSRC1, MYBPHL, SORT1-with no established links to LDL-C regulation. The proposal's core questions are: (1) which 1p13.3 DNA variants contribute to LDL-C, vsLDL, and MI, (2) which 1p13.3 genes contribute to LDL-C, vsLDL, and MI, and (3) by what mechanisms do the DNA variants affect gene function? Genetic fine-mapping, gene resequencing, gene knockdown and overexpression in mice, and experiments in human embryonic stem cells will be used to answer these questions. Success should result in precise definition of a novel causal gene(s) and regulatory mechanism and thereby provide a potential new target for the treatment of dyslipidemia and prevention of MI.