Project Summary Inborn errors of immunity (IEI) are a heterogeneous group of disorders due to monogenic defects in the immune response that have a broad clinical spectrum including susceptibility to infection, autoimmunity, inflammation, and cancer susceptibility. There are more than 450 distinct IEI that have been discovered. Diagnosis of the precise genetic cause and mechanism (e.g., gain-of-function or loss-of-function) of IEI has led to enhanced care and treatment of patients through improved diagnosis, guidance for clinical monitoring based on genotype, and targeted therapies. While discovery of new IEI and testing for patients has rapidly grown, successful genetic diagnosis still only occurs for ~40% of the patients identified by expert clinicians as likely to have monogenic disease. However, IEI can also arise from de novo mutations in somatic cells, i.e., post- zygotic genetic changes in DNA sequence that may be missed by current sequencing approaches. This mosaic disease can phenocopy germline disease, particularly for disorders with a ‘dominant’ phenotype. We will address this gap through development of a strategy to detect and validate mosaic genetic variants. In Aim 1, we will use a custom capture sequencing assay to identify mosaicism in known and predicted IEI genes in patients who remain undiagnosed despite standard testing. In Aim 2, we will functionally validate mosaic variants using single cell sequencing techniques to correlate genotype with expression profile. The long-term goal of this application is to improve diagnosis of IEI to enable diagnosis and targeted therapy of patients with these disorders.