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：
10444974
负责人：
Christopher Andrew Henderson
金额：
$ 4.85万
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 2023-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10444974
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 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 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 dietary control differentiation protocol 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.

项目摘要：心血管疾病（CVD）及其并发症，包括心肌梗塞，是领先的全球死亡原因。动脉粥样硬化是CVD的主要病理过程，受到强烈调节可遗传的因素，但是许多这些遗传成分对斑块的机制和贡献发展知之甚少。最近，在编码区域中的单个核苷酸多态性（SNP）在几项独立研究中发现了分化3（ID3）的基因抑制剂（ID3）是与CVD相关。 RS11574的突变从主要等位基因（G）到小等位基因（a）改变了第105 ID3的氨基酸从丙氨酸到苏氨酸，与CVD风险增加有关。 ID3功能作为BHLH转录因子的主要负调节因子，我们的实验室研究表明，未成年人 RS11574等位基因降低ID3与E12结合和隔离的能力，增加了启动子的E12占用率区域和增强转录。 ID3调节血管平滑肌细胞（VSMC）生长和体外和体内分化。 VSMC在动脉粥样硬化病变的稳定中起着不可或缺的作用由于它们有助于在斑块中形成纤维帽并防止其破裂。核心过程是VSMC增殖，调节炎症和调节成熟VSMC标记的能力基因表达。 apoE-/ - 或ldlr - / - 小鼠中ID3的删除显着增加了动脉粥样硬化和临床关联表明RS11574在CVD中起作用；但是，该编码的精确分子机制 SNP改变了ID3生物学，CVD风险仍然未知。该提案中的研究试图了解功能该ID3突变体对VSMC生物学的后果，并将检验以下假设：RS11574的未成年人等位基因响应动脉粥样硬化刺激而导致VSMC的有害变化。在AIM 1中，我提出了创作使用CRISPR/CAS9（包括完整的等位基因） ID3（A/A，A/G，G/G）以及ID3的敲除和E2A基因E12和E47的2个同工型的系列。我将把这些突变的IPSC线区分为VSMC，并将其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有率。在AIM 2中，我将使用仅在VSMC中专门删除的ID3的VSMC谱系跟踪鼠标以更好了解体内动物模型中ID3对VSMC的动脉粥样硬化的贡献。这些ID3 - / - 老鼠将喂食西方或对照饮食12或24周，将与在同一繁殖的ID3+/+小鼠进行比较谱系追踪背景是为了量化动脉粥样硬化的差异。拟议的研究将增强我们对RS11574在动脉粥样硬化中发挥作用的角色ID3及其变体的理解，并可能识别出新颖的未来临床干预的目标和途径。