Massively scalable generation, discovery, and clinical (re-)classification of inborn errors of immunity using cutting-edge genome engineering 4011

Abstract Description Clinical next-generation sequencing (NGS) is a pillar of diagnosis for inborn errors of immunity (IEI). However, for each pathogenic variant identified, these methods yield hundreds of variants of uncertain significance (VUS), creating ambiguity in clinical management. Here, we present a high-throughput framework to generate genetic variants within the endogenous gene and map them with the clinically established functional readout. Gain-of-function (GOF) variants in PIK3CD and PIK3R1 lead to lymphoproliferation, autoimmunity, and infection—the Activated PI3K-Delta Syndrome (APDS). In primary human T cells from multiple healthy donors, we performed saturation CRISPR base-editor screening of PIK3CD and PIK3R1 coupled with a standard APDS-diagnostic flow cytometric assay to measure phosphorylated AKT and S6. We successfully detected most known pathogenic variants and found >100 variants which were clearly novel GOF. We individually validated 30 of these variants and show that many are sensitive to FDA-approved PI3KD inhibitors for APDS. These new functional annotations will be incorporated into public databases to reclassify variants, substantially broadening the population of previously undiagnosed patients who can immediately benefit from this precision medicine approach. This study is part of the Human Immune Variome Project, an effort to functionally classify variants across hundreds of genes implied in IEIs, to remove ambiguity related to clinical management and treatment. Funding Sources Supported by NIH/NCI R01CA280414; NIH/NCI R01CA266446; HICCC Human Tissue Immunology and Immunotherapy Initiative; NIH P30CA013696. Topic Categories Technological Innovations in Immunology (TECH)