Comprehensive functional genomic analysis of the multi-disease associated CDKN2A/B locus

多种疾病相关 CDKN2A/B 基因座的综合功能基因组分析

• 批准号: 10210579
• 负责人: TOREN FINKEL
• 金额: $ 61.39万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-30 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10210579
• 关键词: 9p21; Address; Age; Aging; Allelic Imbalance; Atherosclerosis; Binding; Binding Proteins; Biochemical Pathway; Biological; Biological Assay; Biological Process; CDKN2A gene; CRISPR/Cas technology; Cardiovascular Diseases; Cell Aging; Cell Cycle Progression; Cell Cycle Regulation; Cell Proliferation; Chromosomes; Cyclin-Dependent Kinase Inhibitor 2A; DNA; Disease; Disease Progression; Disease susceptibility; Drug Targeting; Electrophoretic Mobility Shift Assay; Elements; Endothelial Cells; Genes; Genetic; Genome; Genomic Segment; Genomics; Goals; Guillain Barré Syndrome; Human; In Vitro; Incidence; Lead; Left; Link; Linkage Disequilibrium; Longevity; Luciferases; Malignant Neoplasms; Maps; Mass Spectrum Analysis; Methods; Non-Insulin-Dependent Diabetes Mellitus; Nuclear Extract; Pathogenesis; Predisposition; Proteins; RNA Interference; Regulatory Element; Reporter; Role; Single Nucleotide Polymorphism; Smooth Muscle Myocytes; Specific qualifier value; Techniques; Untranslated RNA; Western Blotting; age related; base; cell type; chromatin immunoprecipitation; functional genomics; genetic regulatory protein; genome wide association study; genomic locus; macrophage; novel; risk variant

ABSTRACT The incidence of cardiovascular disease (CVD), Type 2 diabetes (T2D) and cancers all dramatically increase as a function of age. The underlying mechanisms of these diseases, which vary, are incompletely understood. Genome-wide association studies (GWAS) have identified many SNPs that are associated with these conditions. One of the strongest associations comes from the CDKN2A/B locus on chromosome 9p21.3 which has been associated with multiple age-related diseases, as well as overall human lifespan. Within this 200 kb locus, there are three encoded proteins, p16INK4a, p14ARF and p15INK4b, and one antisense non-coding RNA, the inhibitor of CDK4 (INK4) locus (AS/ANRIL). To date, it has not been firmly established which, if any, of these genes are the risk genes for the associated diseases. There are ~193 disease-associated, noncoding SNPs in linkage disequilibrium (LDs) across this 200 kb region, represented by 18 lead SNPs used for GWAS analysis. While the mechanisms underlying the contribution of these SNPs to specific diseases are not fully understood, a single genetic region associated with multiple different age-related diseases suggests that this locus may modulate these conditions by promoting aging itself, perhaps via induction of cellular senescence as a common mechanism. In this application, we propose to apply an experimental approach using high throughput techniques we have recently developed including Reel-seq and FREP/SDCP-MS, to systematically dissect this locus. We will first identify the disease-associated functional SNPs (fSNPs), as well as the regulatory elements across the 58 kb core region primarily associated with cardiovascular diseases using Reel-seq. Next, we will identify the regulatory proteins that specifically bind to all the fSNPs, as well as the regulatory elements, using FREP/SDCP- MS. A range of relevant cell types related to atherosclerosis will be used to generate the nuclear extract required for our screens. We will demonstrate the role of these regulatory proteins by confirming their direct effects on p16INK4a, p14ARF, p15INK4b and AS/ANRIL expression, and subsequently on cell cycle regulation and cellular senescence. A range of complementary techniques such as RNAi, CRISPR/cas9 gene editing, will be employed. Such analysis will provide the first in-depth understanding of this critical genomic region, as well as a unique strategy to uncover unifying biochemical pathways that simultaneously regulate atherosclerosis, as well as potentially multiple other age-related diseases.

抽象的 心血管疾病（CVD），2型糖尿病（T2D）和癌症的发生率都显着增加 年龄的功能。这些疾病的基本机制（不同）尚不完全理解。 全基因组关联研究（GWAS）已经确定了许多与这些条件相关的SNP。 最强的关联之一来自9p21.3染色体上的CDKN2A/B基因座 与多种与年龄有关的疾病以及人类的整体寿命相关。在这个200 kb的基因座中， 是三种编码的蛋白质，P16INK4A，P14ARF和P15INK4B，以及一个反义非编码RNA，抑制剂的抑制剂 CDK4（INK4）基因座（AS/Anril）。迄今为止，尚未确定它（如果有的话）是 相关疾病的风险基因。有〜193个与疾病相关的非编码SNP 在200 kb区域的不平衡（LDS），由18个用于GWAS分析的铅SNP代表。尽管 这些SNP对特定疾病的贡献的基础机制尚不完全了解，一个单一 与多种不同年龄相关疾病相关的遗传区域表明该基因座可能会调节 这些条件通过促进衰老本身，也许是通过诱导细胞衰老作为常见的 机制。在此应用程序中，我们建议使用高通量技术采用实验方法 我们最近开发了包括Reel-Seq和FREP/SDCP-MS，以系统地剖析该基因座。我们 将首先确定与疾病相关的功能SNP（FSNP）以及整个跨的调节元素 58 kb核心区域主要与心血管疾病相关。接下来，我们将确定 使用FREP/SDCP-专门结合所有FSNP以及调节元素的调节蛋白 多发性硬化症。一系列与动脉粥样硬化有关的相关细胞类型将用于产生所需的核提取物 对于我们的屏幕。我们将通过确认它们的直接影响来证明这些调节蛋白的作用 P16INK4A，P14ARF，P15INK4B和AS/ANRIL表达，随后在细胞周期调节和细胞上 衰老。将采用一系列互补技术，例如RNAi，CRISPR/CAS9基因编辑。 这种分析将为这个关键基因组区域以及独特的第一个深入了解 揭示同时调节动脉粥样硬化的统一生化途径的策略 潜在的多种其他与年龄有关的疾病。