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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

基本信息

批准号：
10672975
负责人：
TOREN FINKEL
金额：
$ 59.92万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
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/10672975
关键词：
9p21.3 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 Macrophage Malignant Neoplasms Maps Mass Spectrum Analysis Methods Non-Insulin-Dependent Diabetes Mellitus Nuclear Extract Pathogenesis Predisposition Proteins Qualifying RNA Interference Regulatory Element Reporter Role Single Nucleotide Polymorphism Smooth Muscle Myocytes Specific qualifier value Techniques Untranslated RNA Western Blotting age related cell type chromatin immunoprecipitation functional genomics genetic regulatory protein genome wide association study genomic locus novel risk variant

项目摘要

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) 和癌症的发病率均急剧增加年龄的函数。这些疾病的潜在机制各不相同，目前尚不完全清楚。全基因组关联研究 (GWAS) 已识别出许多与这些病症相关的 SNP。最强烈的关联之一来自染色体 9p21.3 上的 CDKN2A/B 基因座，该基因座已被证实与多种与年龄相关的疾病以及人类的整体寿命有关。在这个 200 kb 的位点内，有是三种编码蛋白，p16INK4a、p14ARF 和 p15INK4b，以及一种反义非编码 RNA，即 CDK4 (INK4) 基因座 (AS/ANRIL)。迄今为止，尚未确定这些基因中的哪一个（如果有的话）是相关疾病的风险基因。有约 193 个与疾病相关的非编码 SNP 连锁这个 200 kb 区域的不平衡 (LD)，以用于 GWAS 分析的 18 个先导 SNP 为代表。尽管这些 SNP 对特定疾病的作用机制尚不完全清楚，单一的与多种不同年龄相关疾病相关的遗传区域表明该位点可能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一种常见的机制。在此应用中，我们建议采用使用高通量技术的实验方法我们最近开发了 Reel-seq 和 FREP/SDCP-MS 来系统地剖析这个基因座。我们首先将确定与疾病相关的功能性 SNP (fSNP)，以及整个系统的调控元件使用 Reel-seq 获得主要与心血管疾病相关的 58 kb 核心区域。接下来，我们将识别使用 FREP/SDCP- 特异性结合所有 fSNP 的调节蛋白以及调节元件多发性硬化症。一系列与动脉粥样硬化相关的细胞类型将用于生成所需的核提取物对于我们的屏幕。我们将通过确认这些调节蛋白的直接影响来证明它们的作用 p16INK4a、p14ARF、p15INK4b 和 AS/ANRIL 表达，以及随后的细胞周期调节和细胞衰老。将采用一系列互补技术，例如 RNAi、CRISPR/cas9 基因编辑。此类分析将首次深入了解这一关键基因组区域，以及独特的揭示同时调节动脉粥样硬化的统一生化途径的策略，以及潜在的多种其他年龄相关疾病。