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

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.