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

摘要 心血管疾病（CVD）、2型糖尿病（T2 D）和癌症的发病率都急剧增加， 年龄的函数。这些疾病的潜在机制各不相同，尚未完全了解。 全基因组关联研究（GWAS）已经确定了许多与这些疾病相关的SNP。 最强的关联之一来自染色体9p21.3上的CDKN 2A/B基因座， 与多种与年龄有关的疾病以及人类的总体寿命有关。在这个200 kb的基因座内， 是三个编码蛋白，p16 INK 4a，p14 ARF和p15 INK 4 b，和一个反义非编码RNA， CDK 4（INK 4）基因座（AS/ANRIL）。到目前为止，还没有确定这些基因中的哪一个（如果有的话）是基因。 相关疾病的风险基因。有~193个与疾病相关的非编码SNP连锁 在该200 kb区域上的不平衡（LD），由用于GWAS分析的18个前导SNP表示。而 这些SNP对特定疾病的作用机制尚未完全了解， 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，也许通过诱导细胞衰老作为一种常见的 机制在这个应用中，我们提出了一个实验性的方法，使用高通量技术 我们最近开发了包括Reel-seq和FREP/SDCP-MS在内的方法来系统地剖析该基因座。我们 将首先识别疾病相关的功能性SNP（fSNP）以及整个基因组中的调节元件 使用Reel-seq的主要与心血管疾病相关的58 kb核心区域。接下来，我们将确定 使用FREP/SDCP，特异性结合所有fSNP的调节蛋白，以及调节元件。 女士一系列与动脉粥样硬化相关的相关细胞类型将用于产生所需的核提取物 for our screens屏幕.我们将通过证实这些调节蛋白对细胞凋亡的直接影响来证明它们的作用。 p16 INK 4a、p14 ARF、p15 INK 4 b和AS/ANRIL表达，以及随后对细胞周期调控和细胞凋亡的影响。 衰老将采用一系列互补技术，如RNAi，CRISPR/cas9基因编辑。 这样的分析将提供对这一关键基因组区域的第一次深入了解，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 潜在的多种与年龄相关的疾病。

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.