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

从基因型到表型：CVD 中 ID3 SNP rs11574 的分子和功能表征

基本信息

批准号：
10022136
负责人：
Christopher Andrew Henderson
金额：
$ 3.4万
依托单位：
UNIVERSITY OF VIRGINIA
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-07-01 至 2023-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10022136
关键词：
Ablation Affect Affinity Alanine Alleles Amino Acid Substitution Amino Acids Animal Model Apolipoprotein E Arterial Fatty Streak Atherosclerosis Attenuated BHLH Protein Binding Biology CRISPR/Cas technology Cardiovascular Diseases Cardiovascular Pathology Cardiovascular system Cause of Death Cell Line Cell Lineage Cell Proliferation Cell model Cells Cellular biology Chromatin Clinical Code Coronary artery DNA Development Diet Differentiation Inhibitor Differentiation and Growth Disease Dominant-Negative Mutation Elements Event Family Future Gene Expression Genes Genetic Genetic Transcription Genotype Goals Hand Helix-Turn-Helix Motifs Heritability Heterozygote Homozygote Human Impairment In Vitro Inflammation Inflammatory Intervention Investments Knock-out Laboratories Lesion Link Lipids Mediating Minor Modeling Molecular Mus Mutate Mutation Myocardial Infarction Pathologic Processes Pathway interactions Phenotype Play Population Process Proliferating Proliferation Marker Promoter Regions Protein Isoforms Proteins Protocols documentation Reagent Regulation Research Proposals Risk Role Rupture Series Single Nucleotide Polymorphism Smooth Muscle Smooth Muscle Myocytes Stimulus TCF3 gene Testing Therapeutic Threonine Time Transgenic Mice Variant Vascular Smooth Muscle alpha Actin arterial lesion base burden of illness cardiovascular disorder risk carotid intima-media thickness cell growth cell type coronary artery calcification coronary event genetic variant genome editing in vivo indexing induced pluripotent stem cell inflammatory marker member molecular phenotype mutant novel oxidized low density lipoprotein prevent promoter response tool transcription factor treatment response treatment strategy vascular smooth muscle cell proliferation western diet

项目摘要

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.

项目概要：心血管疾病（CVD）及其并发症，包括心肌梗塞，是主要的疾病全世界的死亡原因。动脉粥样硬化是 CVD 的主要病理过程，受到以下因素的强烈调节：遗传因素，但其中许多遗传成分对斑块的机制和贡献的发展了解甚少。最近，在编码区发现了一个单核苷酸多态性（SNP）。 rs11574 处的分化基因抑制剂 3 (ID3) 在多项独立研究中被鉴定为与CVD有关。 rs11574 从主要等位基因 (G) 到次要等位基因 (A) 的突变改变了第 105 个等位基因 ID3 的氨基酸从丙氨酸变为苏氨酸，与 CVD 风险增加相关。 ID3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，次要的 rs11574 的等位基因降低了 ID3 结合和隔离 E12 的能力，增加了 E12 在启动子上的占据区域和增强转录。 ID3 调节血管平滑肌细胞 (VSMC) 的生长和体外和体内的分化。 VSMCs 在动脉粥样硬化病变的稳定中发挥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斑块破裂。对此的核心该过程是 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 Id3 的缺失显着增加动脉粥样硬化和临床关联表明rs11574在CVD中发挥作用；然而，这种编码的精确分子机制 SNP 改变 ID3 生物学，CVD 风险仍未知。本提案的研究旨在了解功能该 ID3 突变体对 VSMC 生物学的影响，并将检验 rs11574 的次要等位基因的假设导致 VSMC 响应致动脉粥样硬化刺激而发生有害变化。在目标 1 中，我建议创建使用 CRISPR/Cas9 构建各种突变型人类诱导多能干细胞 (iPSC) 系，包括完整的等位基因 ID3 系列（A/A、A/G、G/G）以及 ID3 和 E2A 基因的 2 个亚型 E12 和 E47 的敲除。我将这些突变 iPSC 系分化为 VSMC，并比较它们的表型差异基于基因型的增殖、炎症、基因表达和转录因子启动子占据。在目标 2 中，我将使用仅在 VSMC 中专门删除 Id3 的 VSMC 谱系追踪鼠标，以便更好地了解 Id3 在体内动物模型中对 VSMC 动脉粥样硬化的贡献。这些 Id3-/- 小鼠将用西方饮食或对照饮食喂养 12 或 24 周，并将与在相同条件下饲养的 Id3+/+ 小鼠进行比较谱系追踪背景以量化动脉粥样硬化的差异。拟议的研究将增强我们对 ID3 及其 rs11574 变体在动脉粥样硬化中的作用的了解，可能会发现新的未来临床干预的目标和途径。