From genotype to phenotype: Molecular and functional characterization of the ID3 SNP rs11574 in CVD

项目概要： 心血管疾病（CVD）及其并发症，包括心肌梗死，是主要的 全球范围内的死因。动脉粥样硬化是CVD的主要病理过程， 遗传因素，但机制和贡献的许多这些遗传成分的斑块 发展知之甚少。最近，在编码区的单核苷酸多态性（SNP）， 在几项独立的研究中鉴定了rs 11574处的基因分化抑制因子3（ID 3）， 与CVD有关。rs 11574从主要等位基因（G）突变为次要等位基因（A）改变了第105位 ID 3的氨基酸从丙氨酸变为苏氨酸，并且与CVD的风险增加相关。ID 3函数 作为bHLH转录因子的显性负调节因子，我们实验室的研究表明， rs 11574的等位基因降低ID 3结合和隔离E12的能力，增加E12在启动子区的占有率 区域和增强转录。ID 3调节血管平滑肌细胞（VSMC）生长， 在体外和体内的分化。血管平滑肌细胞在动脉粥样硬化病变的稳定中起着不可或缺的作用 因为它们有助于斑块内纤维帽的形成并防止其破裂。这方面的核心 这一过程是VSMC增殖、调节炎症和调节成熟VSMC标志物的能力 基因表达。 Apoe-/-或Ldlr-/-小鼠中Id 3的缺失显著增加动脉粥样硬化和临床相关性 表明rs 11574在CVD中起作用;然而，这种编码的精确分子机制 SNP改变ID 3生物学和CVD风险仍然未知。本提案中的研究旨在了解 这一ID 3突变体对VSMC生物学的影响，并将检验rs 11574的次要等位基因 导致VSMC响应致动脉粥样硬化刺激的有害变化。在目标1中，我建议创建 使用CRISPR/Cas9的各种突变的人诱导多能干细胞（iPSC）系，包括完整的等位基因 一系列ID 3（A/A、A/G、G/G）以及ID 3和E2 A基因的2种同种型E12和E47的敲除。我 将这些突变的iPSC系分化成VSMC，并比较它们的表型差异， 增殖、炎症、基因表达和基于基因型的转录因子启动子占用。 在目标2中，我将使用仅在VSMC中特异性缺失Id 3的VSMC谱系追踪小鼠，以便更好地 了解Id 3对体内动物模型中VSMC动脉粥样硬化的贡献。这些Id 3-/-小鼠将 喂食西方或对照饮食12或24周，并将与在相同条件下饲养的Id 3 +/+小鼠进行比较。 谱系追踪背景以量化动脉粥样硬化的差异。拟议的研究将加强 我们对ID 3及其rs 11574变异体在动脉粥样硬化中的作用的理解， 未来临床干预的目标和途径。

PROJECT SUMMARY: Cardiovascular disease (CVD) and its complications, including myocardial infarction, are the leading cause of death worldwide. Atherosclerosis, the primary pathological process of CVD, is strongly regulated by heritable factors, yet the mechanisms and contributions of many of these genetic components to plaque development are poorly understood. Recently, a single nucleotide polymorphism (SNP) in the coding region of the gene inhibitor of differentiation 3 (ID3) at rs11574 was identified in several independent studies to be associated with CVD. Mutation of rs11574 from the major allele (G) to the minor allele (A) changes the 105th amino acid of ID3 from an alanine to a threonine and is associated with an increased risk of CVD. ID3 functions as a dominant negative regulator of bHLH transcription factors, and studies in our laboratory show that the minor allele of rs11574 reduces the ability of ID3 to bind to and sequester E12, increasing E12 occupancy at promoter regions and enhancing transcription. ID3 regulates vascular smooth muscle cell (VSMC) growth and differentiation both in vitro and in vivo. VSMCs play an integral role in the stabilization of atherosclerotic lesions as they contribute to the formation of the fibrous cap within plaques and prevent their rupture. Central to this process is the ability of VSMCs to proliferate, to regulate inflammation, and to modulate mature VSMC marker gene expression. Deletion of Id3 in Apoe-/- or Ldlr-/- mice significantly increases atherosclerosis, and clinical associations indicate that rs11574 plays a role in CVD; however, the precise molecular mechanisms through which this coding SNP alters ID3 biology and CVD risk remain unknown. Studies in this proposal seek to understand the functional consequences of this ID3 mutant on VSMC biology and will test the hypothesis that the minor allele of rs11574 contributes to detrimental changes in VSMCs in response to atherogenic stimuli. In Aim 1, I propose the creation of various mutant human induced pluripotent stem cell (iPSC) lines using CRISPR/Cas9 including the full allelic series of ID3 (A/A, A/G, G/G) as well as knockouts of ID3 and the 2 isoforms of the E2A gene, E12 and E47. I will differentiate these mutant iPSC lines into VSMCs and compare them for phenotypic differences in proliferation, inflammation, gene expression, and transcription factor promoter occupancy based upon genotype. In Aim 2, I will use a VSMC lineage tracing mouse with Id3 specifically deleted only in VSMCs in order to better understand the contribution of Id3 to atherosclerosis in VSMCs in an in vivo animal model. These Id3-/- mice will be fed either a Western or control diet for 12 or 24 weeks and will be compared to Id3+/+ mice bred on the same lineage tracing background in order to quantify differences in atherosclerosis. The proposed studies will enhance our understanding of the role ID3 and its variants at rs11574 play in atherosclerosis and may identify novel targets and pathways for future clinical intervention.