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

项目摘要/摘要 人类感染SARS-CoV-2的临床表现可从非常轻微或没有症状到严重不等。 呼吸衰竭。尽管免疫系统的各种细胞成分的过度激活一直是 在重症新冠肺炎患者中观察，决定易感性的宿主遗传因素 严重的新冠肺炎疾病尚不清楚。Gwas的研究报告了几个遗传位点，它们是 与严重的新冠肺炎显著相关，并根据它们与新冠肺炎的亲缘关系定义了几个靶基因 风险基因，尽管这种方法不能准确地确定因果基因的优先顺序。我们进行了最早的 在原代免疫细胞中进行转录组范围关联研究(TWAS)以确定与 新冠肺炎的严重性。在此R21提案中，我们将重点定义与以下各项相关的功能性新冠肺炎风险变量 从我们的遗传分析中获得的前两个候选基因(OAS1和IL10RB)。 在目标1中，我们将确定与OAS1表达相关的功能性新冠肺炎风险eQTL 经典单核细胞(NCM)。我们的三叉戟研究表明，新冠肺炎的严重程度与 干扰素诱导基因(OAS1)在NCM中的特异性表达OAS1编码寡腺苷合成酶 一类蛋白质家族，能降解病毒RNA并激活抗病毒反应，从而改变其表达的变体 水平，特别是在神经母细胞瘤中，一种已被卷入新冠肺炎发病机制的细胞类型，可能具有 在宿主免疫反应中起着重要作用。在这里，我们将进行实验研究，以定义功能 含有&gt；100个SNPs的密集OAS1尼安德特人单倍体中的变异(S)。简而言之，我们将执行(I)CRISPRI 确定与新冠肺炎风险SNPs重叠的功能增强子的分析，(Ii)荧光素酶报告分析 确定这种增强子中的功能变体，(Iii)芯片确定等位基因特异性结合 转录因子RxRα，其结合被预测会被新冠肺炎风险变异体破坏，以及(Iv) 以人类发展报告为媒介编辑优先的新冠肺炎风险eQTL，以明确建立功能。 在目标2中，我们将确定与NK细胞中IL10RB表达相关的功能新冠肺炎风险eQTL 和T细胞。我们的TWAS研究表明，NK细胞中IL10Rb的表达增加与 新冠肺炎的严重性。IL-10Rb编码IL-10受体β，具有免疫调节作用。 IL 10Rb在NK细胞和T细胞上的高表达可能增强了它们的反应性 致IL-10。在这里，我们将进行目标1中描述的实验研究，以定义功能性新冠肺炎- 预测会扰乱转录因子TCF12和GATA-3以及它们的结合的风险变体 IL 10Rb在调节T细胞NK细胞表达中的作用 总体而言，R21计划中的功能研究将为遗传学基础提供重要的机械学见解 新冠肺炎的严重性。

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.