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

项目总结： 心血管疾病(CVD)及其并发症，包括心肌梗死，是 全球范围内的死因。动脉粥样硬化是心血管疾病的主要病理过程，受到多种因素的强烈调控。 可遗传因素，但许多这些遗传成分对斑块的机制和贡献 人们对发展知之甚少。最近，一个单核苷酸多态(SNP)的编码区 位于rs11574的分化抑制基因3(Id3)在几项独立研究中被鉴定为 与心血管疾病有关。Rs11574从主等位基因(G)突变为次要等位基因(A)改变了第105位 ID3的氨基酸从丙氨酸变为苏氨酸，并与心血管疾病风险增加有关。ID3函数 作为bHLH转录因子的显性负调控因子，在我们实验室的研究表明，次要的 Rs11574等位基因降低了ID3与E12结合和隔离的能力，增加了E12在启动子上的占有率 区域和增强转录。ID3调节血管平滑肌细胞的生长和增殖 在体外和体内均有分化。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。这是关键所在 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ID3缺失显著增加动脉粥样硬化，并与临床相关 表明rs11574在心血管疾病中起作用；然而，这种编码的确切分子机制 SNP改变ID3生物学和心血管疾病风险尚不清楚。这项建议中的研究试图理解功能 该ID3突变对VSMC生物学的影响，并将检验rs11574的次要等位基因的假设 对动脉粥样硬化刺激的反应导致VSMC的有害变化。在目标1中，我建议创造 含全等位基因的CRISPR/Cas9在不同突变型人诱导多能干细胞(IPSC)系中的应用 ID3系列(A/A、A/G、G/G)以及ID3和E2A基因的两个亚型E12和E47的敲除。我 将把这些突变的IPSC系区分为VSMC，并比较它们在 基于基因的增殖、炎症、基因表达和转录因子启动子的占有率。 在目标2中，我将使用仅在VSMC中删除ID3的VSMC血统追踪小鼠，以便更好地 在活体动物模型中了解ID3在VSMCs动脉粥样硬化中的作用。这些ID3-/-小鼠将 用西方或对照饲料喂养12或24周，并与在相同条件下饲养的ID3/小鼠进行比较 血统追踪背景，以量化动脉粥样硬化的差异。拟议的研究将加强 我们对ID3及其rs11574变异在动脉粥样硬化中的作用的理解，可能会发现新的 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.