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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. !

摘要该项目旨在向儿科风湿病学领域的目标迈出重要一步，致力于30多年：确定实际的因果变异，赋予遗传风险，幼年特发性关节炎（JIA）。正如复杂性状的典型情况一样，我们发现，大多数遗传风险， JIA位于基因组的非编码区。这些区域富含H3 K4 me 1/H3 K27 ac 组蛋白标记增强子的表观遗传标记因此，JIA的遗传风险涉及到基因的调控，而不是改变蛋白质功能的氨基酸取代。而不是鉴于JIA主要是一种自身免疫性疾病，我们假设JIA的出现是因为白细胞受到遗传和表观遗传介导的干扰，削弱了他们的调节和协调能力，基因组中的转录。这种协调调节的丧失导致不适当的表达，在缺乏启动或维持炎症反应所需的正常外部信号的情况下，炎症反应。我们检验这一假设的一个重大障碍是JIA的实际因果变异是未知的。已知的JIA风险基因座超过38个，这些基因座包含超过18，000个变异。我们最近 48例JIA患者全基因组测序的完成，进一步增加了候选人的数量，基因座和变体。考虑到这些挑战，以及因果变量位于监管范围内（而不是监管范围内）的事实，蛋白质编码）区域，该领域迫切需要高通量方法来查询数万个确定它们对转录的影响，并鉴定这些变体发挥其作用的细胞。方面的影响. 在这项试点研究中，我们将介绍一个大规模平行报告基因检测（MPRA），在实验室开发的我们的合作者，博士帕迪斯Sabeti，作为一种识别潜在的因果变异，通过他们的能力，改变骨髓和淋巴细胞系中的基因表达。该检测试剂盒专门设计用于查询基因组非编码区内的遗传变异。我们将测试表现出强大的变体在所有已知的JIA相关风险基因座内与索引SNP的连锁不平衡，如从先前的研究（n= 7，312），检查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，我们预计将有显着的缩小了JIA潜在致病变异的范围，并将准备进行所需的研究，阐明特定等位基因与特定转录网络和细胞中的改变之间的因果关系功能协调发展的!