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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：
10210295
负责人：
Christopher Andrew Henderson
金额：
$ 5.1万
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/10210295
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.

项目总结: