Comprehensive functional genomic analysis of the multi-disease associated CDKN2A/B locus
Comprehensive functional genomic analysis of the multi-disease associated CDKN2A/B locus
批准号:
10491270
负责人:
TOREN FINKEL
金额:
$61.73万
依托单位国家:
美国
项目类别:
财政年份:
2021
资助国家:
美国
项目状态:
未结题
起止时间:
2021-09-30 至 2026-06-30
关键词:
9p21AddressAgeAgingAllelic ImbalanceAtherosclerosisBindingBinding ProteinsBiochemical PathwayBiologicalBiological AssayBiological ProcessCDKN2A geneCRISPR/Cas technologyCardiovascular DiseasesCell AgingCell Cycle ProgressionCell Cycle RegulationCell ProliferationChromosomesCyclin-Dependent Kinase Inhibitor 2ADNADiseaseDisease ProgressionDisease susceptibilityDrug TargetingElectrophoretic Mobility Shift AssayElementsEndothelial CellsGenesGeneticGenomeGenomic SegmentGenomicsGoalsGuillain Barré SyndromeHumanIn VitroIncidenceLeadLeftLinkLinkage DisequilibriumLongevityLuciferasesMalignant NeoplasmsMapsMass Spectrum AnalysisMethodsNon-Insulin-Dependent Diabetes MellitusNuclear ExtractPathogenesisPredispositionProteinsRNA InterferenceRegulatory ElementReporterRoleSingle Nucleotide PolymorphismSmooth Muscle MyocytesSpecific qualifier valueTechniquesUntranslated RNAWestern Blottingage relatedbasecell typechromatin immunoprecipitationfunctional genomicsgenetic regulatory proteingenome wide association studygenomic locusmacrophagenovelrisk variant
中文摘要
摘要
心血管疾病(CVD)、2型糖尿病(T2D)和癌症的发病率都随着
这是年龄的作用。这些疾病的潜在机制各不相同,但尚不完全清楚。
全基因组关联研究已经确定了许多与这些疾病相关的SNPs。
最强的关联之一来自染色体9p21.3上的CDKN2A/B基因座,它已经被
与多种与年龄有关的疾病有关,以及人类的整体寿命。在这个200kb的基因座内,
是p16INK4a、p14ARF和p15INK4b三种编码蛋白,以及一种反义非编码RNA。
CDK4(INK4)基因座(AS/ANRIL)。到目前为止,还没有确定这些基因中的哪些是
相关疾病的风险基因。连锁中有大约193个与疾病相关的非编码SNPs
这一200kb区域的不平衡(LDS)由18个铅SNPs代表,用于GWAs分析。而当
这些SNP导致特定疾病的潜在机制尚不完全清楚,单一的
与多种不同年龄相关疾病相关的遗传区域表明,该基因座可能调节
这些条件通过促进衰老本身,可能是通过诱导细胞衰老作为一种常见的
机制。在此应用程序中,我们建议应用一种使用高吞吐量技术的实验方法
我们最近开发了包括Reel-seq和FREP/SDCP-MS在内的系统剖析该基因座的方法。我们
将首先确定与疾病相关的功能性SNPs(FSNPs),以及
58kb的核心区主要与心血管疾病有关。接下来,我们将确定
利用FREP/SDCP与所有fSNPs以及调控元件特异结合的调控蛋白-
一系列与动脉粥样硬化相关的细胞类型将被用来产生所需的核提取液
在我们的屏幕上。我们将通过确认这些调节蛋白对基因的直接影响来证明它们的作用
P16INK4a、p14ARF、p15INK4b和AS/ANRIL的表达,进而对细胞周期调控和细胞
衰老。将采用一系列补充技术,如RNAi、CRISPR/Cas9基因编辑。
这种分析将提供对这一关键基因组区域的首次深入了解,以及独特的
发现同时调节动脉粥样硬化的统一生化途径以及
可能存在多种其他与年龄相关的疾病。
英文摘要
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.
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会议论文
Comprehensive functional genomic analysis of the multi-disease associated CDKN2A/B locus
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批准号:10672975
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项目类别:
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资助金额:$59.92万
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