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

• 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
• 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

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）是一种目前无法治愈的自身免疫性疾病，其特征是 免疫细胞（例如 B 淋巴细胞）反应异常以及大量致病菌的产生 自身抗体，最终导致多器官损伤（例如肾脏、皮肤）。虽然 SLE 的病因尚不完全 众所周知，遗传因素的显着贡献已得到证实。多项全基因组关联研究 (GWAS) 已鉴定出超过 100 个 SLE 易感位点 (p<5x10-8)，大部分是单核苷酸多态性 （SNP）。大多数这些 SNP 不会直接改变蛋白质产物，我们和其他人之前的工作已经证明 研究表明，许多此类 SNP 在顺式调控元件 (cRE)（即增强子和沉默子）中富集 并可能调节基因表达。然而，查明 cRE 中的因果性、易感性 SNP 和 破译其精确机制是该领域取得进展的主要障碍。因此，这 知识差距严重阻碍了 GWAS 研究结果转化为临床应用。因此，有 迫切需要公正、全面和高通量的方法来解决机械问题 数百个潜在的监管 SNP (rSNP) 与 SLE 易感性之间的联系。我们假设 SLE- 易感 rSNP 异常诱导 cRE 活性，从而影响未受刺激的靶基因的表达 和/或刺激的 B 细胞。为了系统地描述 rSNP 及其对靶基因的影响，我们建议 建立高通量实验流程来确定和验证 GWAS 位点的 rSNP。在 目标1，我们将应用高通量技术“自转录活性调节区测序” (STARR-seq) 对数百个含有 SNP 的区域的调控活动进行功能量化 同时地。在未刺激和刺激条件下使用 Raji 细胞（B 淋巴细胞），我们将应用 STARR-seq 评估 79 个不同 GWAS 位点内的 756 个选定的 rSNP 的 SLE 易感性。在目标 2a 中，我们 将应用下一代 (NG)-Capture-C 来检测 SNP 对与内源性顺式相互作用的特异性影响， Raji 细胞中的同源靶基因，不需要靶基因和功能的强先验假设 结果。我们将评估目标 1 和目标 2a 中的同一组 756 个 SNP。我们预计这将分为两方面 串联策略将弥合 GWAS 衍生的 rSNP 与其目标基因的机械联系之间的差距。 为了验证所提出方法的有效性和准确性，Aim 2b 将使用基于 CRISPR 的 对选定的 rSNP 进行（表观）遗传编辑，以验证对内源靶标的等位基因特异性功能效应 同基因细胞中的基因。总的来说，提出的用于发现和验证 SLE 的公正方法 “因果”SNP 具有高风险/高回报，可能会导致对 SLE 病因学和认识的突破。 干预策略。 rSNP、cRE 及其靶基因的发现将显着推进我们的研究 SLE 遗传学知识，并为未来深入机制研究的贡献提供方向 rSNP 和狼疮易感性中的靶基因，可以定义来自 GWAS 的未来治疗靶点。