Mutations in Topoisomerase IIbeta result in B cell immunodeficiency

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Project Summary/Abstract The study of rare immunodeficiency patients has been extremely instructive to our understanding of B cell development and human disease, revealing mechanisms of B cell development, central tolerance and protection from infection. Syndromic immunodeficiencies, in which the physical malformations are often more pronounced than the underlying immunologic defects, are less frequently studied and less well understood. We recently discovered that mutations in TOP2B underlie the autosomal dominant syndromic B cell immunodeficiency (Hoffman syndrome). Topoisomerases are essential genes required for relaxation of topological stress during DNA replication and gene transcription, acting to prevent the generation of free DNA breaks. We recently reported the first description of a role for TOP2B in a monogenetic human disease, and revealed that heterozygous, dominant negative mutations in the DNA-binding domain of TOP2B led to negative effects on development and function of B cells, while T cell activation was not affected. In this New Investigator proposal, we take a bold, multi-species approach using novel S. cerevisiae models, innovative genetically engineered mice developed by our group, the largest collection of TOP2B mutations in patients with TOP2b mediated immunodeficiency, as well as newly developed patient- derived induced pluripotent stem cells (iPSCs) to explore our hypotheses in an otherwise unobtainable human system. The central hypothesis is that loss of enzymatic function leads to erroneous gene transcription, limited V(D)J recombination, and subsequently the failure of normal B cell differentiation and function. Three specific aims are proposed to test this hypothesis. Specific Aim 1 will determine how mutations in TOP2B generate B cell immunodeficiency through malfunction in the ability to create and re- seal double strand breaks. In vitro and in vivo experiments will examine how patient-associated mutations in TOP2B lead to impaired homologous recombination and non-homologous end joining, disrupting early B cell development. Studies of genomic interactions will examine how mutant Top2b impacts chromatin loop extrusion and impairs fundamental mechanisms of V(D)J recombination and class switching. Specific Aim 2 will determine how mutations in TOP2B impact the recruitment of repair proteins to sites of double strand breaks, leading to reduced break repair, and resulting in reduced efficacy of nonhomologous end joining. Finally, Specific Aim 3 will delineate how Top2b enzymatic function modulates B cell development. Using our distinctive murine models including knock-in and conditional murine models, and iPSCs, we have a unique opportunity to understand the molecular mechanisms underlying this B cell immunodeficiency.