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

项目总结/摘要 人类感染SARS-CoV-2的临床表现可以从非常轻微或无症状到严重 呼吸衰竭尽管免疫系统的各种细胞成分的过度活化已经被证实是免疫系统的一个重要组成部分。 在严重COVID-19疾病患者中观察到，决定对COVID-19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位点， 与严重的COVID-19显著相关，并根据其与 风险基因座，尽管这种方法不能准确地优先考虑致病基因。我们进行了第一次 在原代免疫细胞中进行全转录组关联研究（TWAS），以确定与 COVID-19严重程度。在这份R21提案中，我们将重点定义与以下方面相关的功能性COVID-19风险变体： 从我们的遗传分析中获得的前两个候选基因（OAS 1和IL 10 RB）。 在目标1中，我们将确定与OAS 1表达相关的功能性COVID-19风险eQTL， 经典单核细胞（NCM）。我们的TWAS研究显示，COVID-19严重程度与降低 干扰素诱导基因（OAS 1）在NCM中特异性表达。OAS 1编码寡腺苷酸合酶 降解病毒RNA并激活抗病毒反应的蛋白质家族，因此变体改变其表达 水平，特别是在NCM中，一种与COVID-19发病机制有关的细胞类型，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，以确定功能 在密集的OAS 1尼安德特人单倍体中具有>100个SNP的变体。简而言之，我们将执行（i）CRISPRi 测定与COVID-19风险SNP重叠的功能增强子，（ii）荧光素酶报告基因测定， 确定这种增强子中的功能变体，（iii）ChIP以确定增强子的等位基因特异性结合， 转录因子RXRα，预测其结合被COVID-19风险变体破坏，和（iv） HDR介导的COVID-19风险eQTL的优先编辑，以明确建立功能。 在目的2中，我们将确定与NK细胞中IL 10 RB表达相关的功能性COVID-19风险eQTL 和T细胞。我们的TWAS显示NK细胞中IL 10 RB表达的增加与NK细胞中IL 10的表达显著相关。 COVID-19的严重程度。IL 10 RB编码IL-10受体β，并且鉴于IL 10 RB的免疫调节作用， 10，可能NK细胞和T细胞中IL 10 RB的较高表达可增强它们的反应性 IL-10在这里，我们将进行如目标1中所述的实验研究，以定义功能性COVID-19- 预测干扰转录因子TCF 12和加塔-3的结合的风险变体， 在T细胞中调节NK细胞中IL 10 RB表达中的作用。 总的来说，R21项目中的功能研究将为遗传基础提供重要的机制性见解。 COVID-19的严重程度。

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.