Primary immunodeficiencies (PID) are caused by genetic mutations in the components of immune system. More than 300 subtypes of various PID have been described; and one major subgroup is classified as predominantly antibody de?ciency. Nave B cells produce low affinity IgM antibodies. To develop long-term immune protection against pathogens, B cells must generate high-affinity isotype-switched antibodies such as IgG. To achieve this goal, B cells undergo class switch recombination (CSR) and somatic hypermutation (SHM) in immunoglobulin (Ig) genes. Dysregulation of CSR can lead to PID such as Hyper IgM syndromes (HIGM). These are complex disorders of antibody deficiency that can be attributed to genetic mutations in various components of CSR/SHM such as activation-induced deaminase (AID), CD40, or ICOS. While many genetic mutations have been identified in PID patients with agammaglobulinemia or hypogammaglobulinemia, how these genetic mutations affect CSR process in a B cell intrinsic manner remains incompletely understood. Addressing such questions is highly significant, as dissection of the pathological mechanisms of antibody deficiencies will build the biological basis for new therapeutic strategies in PID. In this application, we propose to establish a novel approach to evaluate how genetic variants cause antibody deficiency in the context of CSR defects. Despite large numbers of mutations recently identified in PID patients, the molecular and clinical heterogeneity represents a challenge for establishing genotype-phenotype correlations. We anticipate that next generation sequencing (NGS) technique will identify increasing numbers of mutations in patients with antibody deficiencies. However, NGS does not reveal the biological significance of identified mutations. Validation of genetic variants as disease-causing mutations still requires functional assays to explain patient-specific cellular and tissue pathophysiology. The conventional gene-targeting approaches are insufficient to reveal the biological significance of these mutations efficiently. Thus, we propose to apply new genome-editing approaches to accomplish such goals. Given our strong expertise in the CSR model and our previous work on the role of phosphoinositide 3-kinase (PI3K) in controlling CSR, we plan to develop a model system to test the genetic variants identified in the components of PI3K pathway. Such approaches can be readily expanded to determine the contribution of other factors to defects in CSR in PID patients. In addition, we propose to establish a novel system for functional testing of genetic variants using primary B cells. If successful, our proposed studies will lead to a high impact in PID field that may substantively accelerate the conversion of genomic studies into translational applications for PID patients.
Relevance to public health. Our proposed studies offer a proof-of-principle for developing high throughput experimental systems to functionally validate genetic mutations identified in human patients. In addition, we expect that our studies will provide mechanistic insights into the signaling control of CSR, and reveal the connection between disease variants and molecular machinery required for CSR. Taken together, these studies may facilitate the diagnosis of primary antibody deficiencies and the development of therapeutic strategies in these disorders.