Identifying Causal Variants in Juvenile Arthritis Using a Massively Parallel Reporter Assay

使用大规模并行报告基因检测识别幼年关节炎的致病变异

基本信息

批准号：
9767028
负责人：
JAMES N JARVIS
金额：
$ 17.55万
依托单位：
STATE UNIVERSITY OF NEW YORK AT BUFFALO
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-09-01 至 2021-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9767028
关键词：
Alleles Amino Acid Substitution Area Autoimmune Diseases Biological Assay CD4 Positive T Lymphocytes Cell Differentiation process Cell Line Cell physiology Cells Childhood Chronic Childhood Arthritis Complex Data Disease Elements Enhancers Epigenetic Process Exploratory/Developmental Grant Funding Gene Expression Gene Expression Regulation Genes Genetic Genetic Diseases Genetic Risk Genetic Transcription Genetic Variation Genetic study Genome Genomic Segment Genomics Goals HL60 Histones Impairment Inflammation Mediators Inflammatory Response Institutes Laboratories Lead Leukocytes Link Linkage Disequilibrium Luciferases Lymphoid Cell Mediating Methods Molecular Myeloid Cells NIH Program Announcements Natural Immunity Nature New Hampshire New York Open Reading Frames Pathogenesis Pathway interactions Patients Pilot Projects Proteins Publishing Regulation Regulatory Element Reporter Rheumatology Role Signal Transduction Single Nucleotide Polymorphism Stimulus T-Lymphocyte Testing Touch sensation Transcriptional Regulation Universities Untranslated RNA Variant Vermont Work adaptive immunity causal variant computerized tools design disorder risk environmental change epigenome experimental study genetic variant genome sequencing genome-wide high reward high risk immune function indexing insight interest monocyte neutrophil new technology novel novel strategies rheumatologist risk variant sensor trait transcriptome whole genome

项目摘要

Abstract This project aims to take an important step toward a goal that the field of pediatric rheumatology has been working towards for more than 30 years: identifying the actual causal variants that confer genetic risk in juvenile idiopathic arthritis (JIA). As is typical for complex traits, we have found that most of the genetic risk for JIA resides within non-coding regions of the genome. These regions are enriched for H3K4me1/H3K27ac histone marks, epigenetic signatures of enhancers. Thus, genetic risk for JIA involves changes in the regulation of genes, rather than amino acid substitutions that alter the function(s) of proteins. Rather than viewing JIA as primarily an autoimmune disease, we hypothesize that JIA emerges because leukocytes suffer genetically and epigenetically-mediated perturbations that blunt their capacity to regulate and coordinate transcription across the genome. This loss of coordinate regulation leads to inappropriate expression of inflammatory mediators in the absence of the normal external signals typically required to initiate or sustain an inflammatory response. A significant impediment to our testing that hypothesis is that the actual causal variants for JIA are unknown. There are more than 38 known risk loci for JIA, and these loci contain more than 18,000 variants. Our recent completion of whole genome sequencing on 48 patients with JIA has further added to the number of candidate loci and variants. Given these challenges, and the fact that the causal variants lie within regulatory (rather than protein-coding) regions, the field is in urgent need of a high throughput method to query tens of thousands of variants, determine their effects on transcription, and identify the cells in which these variants exert their effects. In this pilot study, we will introduce a massively parallel reporter assay (MPRA), developed in the laboratory of our collaborator, Dr. Pardis Sabeti, as a way of identifying potential causal variants through their capacity to alter gene expression in myeloid and lymphoid cell lines. This assay was specifically designed to query genetic variants within non-coding regions of the genome. We will test variants that demonstrate strong linkage disequilibrium with index SNPs within all known JIA associated risk loci as identified from previous studies (n= 7,312), examining their effects on gene expression. WE will then use luciferase assays to corroborate the results from the MPRA. At the end of this 2-year pilot study, we expect to have significantly narrowed the list of potential causal variants in JIA and will be prepared to undertake the studies required to elucidate a causal link between specific alleles and alterations in specific transcriptional networks and cellular functions. !

抽象的该项目旨在朝着一个重要的一步朝着小儿风湿病领域迈出的目标致力30多年：确定赋予遗传风险的实际因果变体青少年特发性关节炎（JIA）。正如复杂性状的典型特征一样，我们发现大多数的遗传风险 Jia位于基因组的非编码区域内。这些区域富含H3K4ME1/H3K27AC 组蛋白标记，增强子的表观遗传特征。因此，JIA的遗传风险涉及变化调节基因，而不是改变蛋白质功能的氨基酸取代。而不是我们将JIA视为一种自身免疫性疾病，我们假设贾出现了，因为白细胞受苦在遗传和表观遗传介导的扰动上钝化其调节和坐标的能力整个基因组的转录。坐标调节的损失导致不适当的表达在没有普通外部信号的情况下，炎症调解人通常需要发起或维持炎症反应。我们检验的一个重大障碍是假设是，JIA的实际因果变异尚不清楚。 JIA有38多个已知的风险基因座，这些基因座包含18,000多个变体。我们最近完成48例JIA患者的整个基因组测序已进一步增加了候选人的数量基因座和变体。鉴于这些挑战以及因果变体位于监管中的事实（而不是蛋白质编码区域，该领域迫切需要高通量的方法来查询数万个变体，确定它们对转录的影响，并确定这些变体发挥其施加的细胞效果。在这项试点研究中，我们将引入实验室中开发的大规模平行记者测定法（MPRA）我们的合作者Pardis Sabeti博士是通过其能力来识别潜在因果变体的一种方式改变髓样和淋巴样细胞系中的基因表达。该测定专门设计用于查询基因组非编码区域内的遗传变异。我们将测试证明强大的变体与以前的所有已知JIA相关风险基因座中与索引SNP的连锁不平衡研究（n = 7,312），检查了它们对基因表达的影响。然后，我们将使用荧光素酶测定证实了MPRA的结果。在这项为期两年的试点研究结束时，我们预计会有明显的缩小了JIA潜在因果变异的列表，并准备好进行所需的研究阐明特定等位基因和特定转录网络和细胞的变化之间的因果关系功能。呢