Investigating Functional Mechanisms of Noncoding Genetic Variations in the Human Fc Gamma Receptor Locus 2260728

Abstract Introduction Genetic variations in the low-affinity Fc Gamma Receptor (FcγR) region have been associated with various diseases and treatment efficacies. Using a high-throughput in vitro screen called Regulatory-Element Sequencing (Reel-Seq) we have identified over 400 candidate functional Single Nucleotide Polymorphisms (fSNPs) from the genomic region. In this study, we aim to identify regulatory function of these candidate non-coding fSNPs in THP-1 monocytes. Methods At each candidate FcγR fSNP position, we introduced random mutation in the THP-1 monocyte line using CRISPR-Cas9 based genome editing and generated over 30 knockout clones per fSNP site. We stimulated these THP-1 knockout clones with various treatments including Interferon and Lipopolysaccharide (LPS). Flow cytometry and qPCR were performed to measure the changes in cell-surface FcγR protein abundances and FcγR transcript abundances. Antibody-Dependent Cellular Phagocytosis (ADCP) assays are being conducted on selected clones to evaluate whether normal phagocytosis function has changed through the alteration of FcγR abundances. Results We have observed significant changes in cell-surface FcγR abundance in these THP-1 knockout cells based on flow cytometry analysis. Detailed analysis of stimulated cells is ongoing, and we will share the full results at the meeting. Conclusion Noncoding genetic variants identified by our in vitro screen may play important roles in monocyte function, through the regulation of cell-surface FcγR abundance. Funding Source NIH NIAID #1R01AI187318-01, NCSU CVM Internal Grant 2022, CMI-EID Associate member Professional Development Scholarship 2023 Topic Categories Immune Response Regulation: Molecular Mechanisms (IRM)