Experimental identification of functional GWAS variants linked to COVID-19 severity in immune cells

免疫细胞中与 COVID-19 严重程度相关的功能性 GWAS 变异的实验鉴定

• 批准号: 10741007
• 负责人: BENJAMIN JOACHIM SCHMIEDEL
• 金额: $ 22.88万
• 依托单位: LA JOLLA INSTITUTE FOR IMMUNOLOGY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-05-22 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10741007
• 关键词: Affect; Alleles; Antiviral Response; Automobile Driving; Binding; Biological Assay; COVID-19; COVID-19 impact; COVID-19 pathogenesis; COVID-19 risk; COVID-19 severity; COVID-19 susceptibility; CRISPR interference; Candidate Disease Gene; Cells; Clinical; Code; DNA Sequence; Data Set; Databases; Defect; Disease; Economics; Enhancers; Ethnic Origin; Exploratory/Developmental Grant; Future; Gene Expression; Gene Expression Profile; Genes; Genetic; Genetic Determinism; Genetic Markers; Genetic Polymorphism; Genetic Risk; Hospitalization; Human; IL10RB gene; Immune; Immune response; Immune system; Impairment; Individual; Inherited; Interferons; Interleukin-10; Link; Long COVID; Luciferases; Maps; Mediating; Meta-Analysis; Modeling; Molecular; Morbidity - disease rate; Natural Killer Cells; Pathogenesis; Pathway interactions; Patients; Population; Predisposition; Protein Family; Quantitative Trait Loci; Reporter; Reporting; Respiratory Failure; Risk; Role; SARS-CoV-2 infection; Severities; Severity of illness; Symptoms; T-Lymphocyte; Testing; Untranslated RNA; Vaccines; Validation; Variant; Work; acute infection; breakthrough infection; causal variant; cell type; disorder risk; epigenomics; exome sequencing; experimental study; genetic analysis; genome wide association study; genome-wide; genomic locus; immunoregulation; in vivo; insight; interleukin-10 receptor; model building; monocyte; mortality; novel; oligoadenylate; pandemic disease; promoter; response; risk variant; severe COVID-19; transcription factor; transcriptome; viral RNA

PROJECT SUMMARY/ABSTRACT The clinical presentation of SARS-CoV-2 infection in humans can range from very mild or no symptoms to severe respiratory failure. Although hyperactivation of various cellular components of the immune system have been observed in patients with severe COVID-19 illness, the host genetic factors that determine susceptibility to severe COVID-19 illness are not well understood. GWAS studies have reported several genetic loci that are significantly associated with severe COVID-19 and defined several target genes based on their proximity to the risk loci, although this approach does not accurately prioritize causal genes. We conducted one of the first transcriptome-wide association study (TWAS) in primary immune cells to define genes that are associated with COVID-19 severity. In this R21 proposal, we will focus on defining functional COVID-19 risk variants linked to top two candidate genes (OAS1 and IL10RB) from our genetic analyses. In Aim 1, we will determine the functional COVID-19 risk eQTLs associated with OAS1 expression in non- classical monocytes (NCM). Our TWAS study showed significant association of COVID-19 severity with reduced expression of interferon-inducible gene (OAS1) specifically in NCM. OAS1 encodes for oligoadenylate synthase family of proteins that degrade viral RNA and activate anti-viral responses, thus variants altering its expression levels, especially in NCM, a cell type which has been implicated in COVID-19 pathogenesis, are likely to have an important role in host immune responses. Here, we will undertake experimental studies to define the functional variant(s) in the dense OAS1 Neanderthal haploblock harboring >100 SNPs. Briefly, we will perform (i) CRISPRi assays to determine functional enhancers that overlap COVID-19 risk SNPs, (ii) luciferase reporter assays to determine the function variants in such enhancers, (iii) ChIP to determine allele-specific binding of the transcription factor RXRα the binding of which is predicted to be disrupted by COVID-19-risk variants, and (iv) HDR-mediated editing of prioritized COVID-19-risk eQTLs to definitively establish functionality. In Aim 2, we will determine the function COVID-19 risk eQTLs associated with IL10RB expression in NK cells and T cells. Our TWAS showed that increased expression of IL10RB in NK cells was significantly associated with COVID-19 severity. IL10RB encodes for IL-10 receptor beta, and given the immunomodulatory role of IL- 10, it is likely that the higher expression on the IL10RB in NK cells and T cells may enhance their responsiveness to IL-10. Here, we will perform experimental studies as described in Aim 1 to define the functional COVID-19- risk variants that are predicted to perturb the binding of the transcription factors TCF12 and GATA-3 and their role in the modulation of IL10RB expression in NK cells in T cells. Overall, functional studies in this R21 program will provide important mechanistic insights into the genetic basis of COVID-19 severity.

项目摘要/摘要 人类SARS-COV-2感染的临床表现范围从非常轻度或没有症状到严重 呼吸衰竭。尽管免疫系统的各种细胞成分的过度激活已经 在患有严重Covid-19疾病的患者中观察到，确定易感性的宿主遗传因素 严重的Covid-19疾病尚不清楚。 GWAS研究报告了几个遗传基因座 与严重的共vid-19显着相关，并根据其邻近定义了几个靶基因 风险基因座，尽管这种方法不能准确优先考虑因果基因。我们进行了第一个 一级免疫小球中的全转录组关联研究（TWA），以定义与与之相关的基因 COVID-19严重程度。在此R21提案中，我们将重点放在定义与链接到的功能性covid-19风险变体上 我们的遗传分析中的两个候选基因（OAS1和IL10RB）。 在AIM 1中，我们将确定与非OAS1表达相关的功能性covid-19风险eqtls 经典单核细胞（NCM）。我们的TWAS研究表明，COVID-19的严重程度与降低 干扰素诱导基因（OAS1）在NCM中的表达。 OAS1编码寡核苷酸合酶 蛋白质家族降解病毒RNA并激活抗病毒反应，从而改变其表达 水平，尤其是在NCM中，在COVID-19发病机理中已实现的细胞类型可能具有 在宿主免疫反应中的重要作用。在这里，我们将进行实验研究以定义功能 密集的OAS1尼安德特人单次斑块中的变体（S）携带> 100 SNP。简而言之，我们将表演（i）Crispri 确定重叠Covid-19风险SNP的功能增强器的测定，（ii）荧光素酶报告基因测定法 确定此类增强剂中的功能变异，（iii）芯片以确定等位基因特异性结合的结合 转录因子RXRα的结合被预测会被Covid-19风险变体破坏，并且（iv） HDR介导的优先级COVID-19风险EQTL的编辑，以确定建立功能。 在AIM 2中，我们将确定与NK细胞中与IL10RB表达相关的COVID-19风险EQTL 和T细胞。我们的TWA表明，NK细胞中IL10RB的表达增加显着相关 与COVID-19的严重程度。 IL10RB编码IL-10受体β，并给定IL-的免疫调节作用 10，NK细胞和T细胞中IL10RB上较高的表达可能会增强其响应能力 到IL-10。在这里，我们将进行AIM 1中所述的实验研究，以定义功能性covid-19-- 预测将扰动转录因子TCF12和GATA-3及其结合的风险变体 在T细胞中NK细胞中IL10RB表达的调节中的作用。 总体而言，该R21计划中的功能研究将为遗传基础提供重要的机械见解 Covid-19的严重程度。