Connecting the gap between GWAS and functional targets for lupus susceptibility

连接 GWAS 和狼疮易感性功能目标之间的差距

基本信息

批准号：
10433444
负责人：
Swapan K. Nath
金额：
$ 26.22万
依托单位：
OKLAHOMA MEDICAL RESEARCH FOUNDATION
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-05-05 至 2024-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10433444
关键词：
Address Affect African ancestry Age Alleles Antigen-Antibody Complex Asian ancestry Autoimmune Diseases B-Lymphocytes Biological Assay CRISPR/Cas technology Caucasians Cell Line Cells Clinical Clustered Regularly Interspaced Short Palindromic Repeats Data Deposition Development Diagnosis Diagnostic Disease Dizygotic Twins Drug Targeting Enhancers Epigenetic Process Ethnic Origin Etiology Future Gene Expression Genes Genetic Genetic Predisposition to Disease Genomics Hispanic ancestry Immune Individual Intervention Kidney Knowledge Lead Light Link Linkage Disequilibrium Lupus Lupus Erythematosus Meta-Analysis Methods Molecular Monozygotic twins Nucleic Acid Regulatory Sequences Organ Pathogenesis Pathologic Patients Plant Roots Predisposition Prevalence Production Proteins Raji Cell Recurrence Regulatory Element Research Resolution Risk Running Siblings Single Nucleotide Polymorphism Single Nucleotide Polymorphism in Regulatory Sequence Skin Susceptibility Gene Systemic Lupus Erythematosus T cell response Techniques Testing Time Translations Untranslated RNA Validation Variant Woman Work base causal variant cell type clinical application curative treatments effective therapy effectiveness validation follow-up gene interaction genome wide association study genome-wide high reward high risk insight multiorgan damage next generation next generation sequencing novel novel therapeutics pathogenic autoantibodies prevent public health relevance response risk variant therapeutic target

项目摘要

ABSTRACT Systematic lupus erythematosus (SLE or lupus) is a currently incurable autoimmune disease, characterized by abnormal immune cell (e.g., B-lymphocytes) response and the production of numerous pathogenic autoantibodies, culminating in multi-organ damage (e.g., kidneys, skin). While the etiology of SLE is incompletely understood, a substantial genetic contribution is well established. Several genome-wide association studies (GWAS) have identified over 100 SLE predisposing loci (p<5x10-8), mostly single nucleotide polymorphisms (SNPs). Most of these SNPs do not directly alter protein products, and previous work from us and others have shown that many such SNPs are enriched within cis regulatory elements (cRE) (i.e., enhancers and silencers) and likely to modulate gene expression. However, pinpointing the causal, predisposing SNPs within cREs and deciphering their precise mechanisms represent major obstacles to progress in the field. Consequently, this knowledge gap has severely hindered the translation of GWAS findings into clinical applications. Hence, there is a profound need for unbiased, comprehensive, and high-throughput approaches to address the mechanistic link between hundreds of potential regulatory SNPs (rSNPs) and SLE susceptibility. We hypothesize that SLE- predisposing rSNPs aberrantly induce cRE activities that influence the expression of target genes in unstimulated and/or stimulated B-cells. To systematically delineate rSNPs and their impact on target genes, we propose to establish a high-throughput experimental pipeline to determine and validate rSNPs underlying GWAS loci. In Aim 1, we will apply the high-throughput technique “Self-Transcribing Active Regulatory Region-sequencing” (STARR-seq) to functionally quantify the regulatory activities of hundreds of SNP-containing regions simultaneously. Using Raji cells (B-lymphocyte) in both unstimulated and stimulated conditions, we will apply STARR-seq to evaluate 756 selected rSNPs within 79 distinct GWAS loci for SLE susceptibility. In Aim 2a, we will apply next-generation (NG)-Capture-C to detect SNP-specific effects on cis interactions with endogenous, cognate target genes in Raji cells, needing no strong a priori hypothesis of target genes and functional consequences. We will evaluate the same set of 756 SNPs in Aim 1 and Aim 2a. We anticipate this two-prong tandem strategy will bridge the gap between GWAS-derived rSNPs and mechanistic links to their target genes. To validate the effectiveness and accuracy of the proposed methods, Aim 2b will use CRISPR-based (epi)genetic editing of a selected rSNP to validate the allele-specific functional effects on the endogenous target gene(s) in isogenic cells. Collectively, the proposed unbiased approaches for discovering and validating SLE “causal” SNPs is high risk/high reward and may lead to breakthroughs in the understanding of SLE etiology and intervention strategies. Discovery of rSNPs, cREs, and their target genes will significantly advance our knowledge of SLE genetics, and yield directions for future in-depth mechanistic research on the contribution of rSNPs and target genes in lupus susceptibility, which could define future therapeutic targets derived from GWAS.

抽象的系统性红斑狼疮（SLE或狼疮）是目前无法治愈的自身免疫性疾病，其特征是异常免疫核管（例如B淋巴细胞）反应和许多致病性的产生自身抗体，最终导致多器官损伤（例如肾脏，皮肤）。而SLE的病因不完全理解，实质性的遗传贡献已得到很好的确定。几项全基因组关联研究（GWAS）已经确定了100多个SLE诱发局部（P <5x10-8），主要是单核苷酸多态性（SNP）。这些SNP中的大多数都不会直接改变蛋白质产品，而我们和其他人的先前工作都有表明许多这样的SNP富含在顺式调节元件（CRE）中（即增强剂和消音器）并可能调节基因表达。但是，指出了Cres中的因果，易感的SNP，解密其精确机制代表了该领域进步的主要障碍。因此，这个知识差距严重阻碍了GWAS发现转化为临床应用。因此，那里是对无偏，全面和高通量方法的巨大需求，以解决机理数百个潜在的调节SNP（RSNP）与SLE敏感性之间的联系。我们假设sle- 诱发的RSNP异常诱导CRE活性，影响靶基因在未刺激中的表达和/或刺激的B细胞。为了系统地描绘RSNP及其对目标基因的影响，我们建议建立高通量实验管道，以确定和验证GWAS基因座的基础RSNP。在 AIM 1，我们将采用高通量技术“自我传输主动调节区域” （starr-seq）在功能上量化数百个含SNP区域的调节活动在未刺激和刺激的条件下使用Raji细胞（B淋巴细胞），我们将应用 starr-seq评估756个在79个不同的GWAS基因座中选择的RSNP，以实现SLE敏感性。在AIM 2A中，我们将应用下一代（NG）-CAPTURE-C来检测SNP特异性对CIS与内源性相互作用的影响 Raji细胞中的同源靶基因，不需要强的靶基因和功能的先验假设结果。我们将在AIM 1和AIM 2A中评估相同的756个SNP。我们期待这两个统计串联策略将弥合GWAS衍生的RSNP与目标基因的机械链接之间的差距。为了验证拟议方法的有效性和准确性，AIM 2B将使用基于CRISPR的方法（EPI）选定的RSNP的遗传编辑，以验证等位基因特异性功能对内源靶标的作用等生细胞中的基因。总的来说，提议的无偏见方法发现和验证SLE “因果” SNP是高风险/高奖励，可能会导致对SLE病因的理解和干预策略。发现RSNP，CRE及其靶基因将大大推动我们的了解SLE遗传学的知识，并产生方向，以实现未来的深入的机械研究狼疮易感性中的RSNP和靶基因，这些基因可以定义从GWAS得出的未来治疗靶标。