Mutations in Topoisomerase IIbeta result in B cell immunodeficiency

拓扑异构酶 IIbeta 突变导致 B 细胞免疫缺陷

• 批准号: 10786524
• 负责人: Lori Broderick
• 金额: $ 39.5万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-03-17 至 2024-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10786524
• 关键词: Affect; Antibody Diversity; B cell differentiation; B-Cell Development; B-Lymphocytes; Binding; Bioinformatics; Cell Compartmentation; Cell physiology; Cells; Chromatin Loop; Chromosomal Rearrangement; Chromosome abnormality; Collaborations; Collection; Communities; Critical Pathways; DNA; DNA Binding Domain; DNA Double Strand Break; DNA Repair; DNA Repair Gene; DNA biosynthesis; Data; Defect; Development; Disease; Dominant-Negative Mutation; Enzymes; Essential Genes; Failure; Generations; Genes; Genetic Transcription; Genetically Engineered Mouse; Genomics; Heterozygote; Human; Immune; Immunoglobulin Class Switching; Immunoglobulin Switch Recombination; Immunologic Deficiency Syndromes; Immunologics; Immunology; Impairment; In Vitro; Infection; Inherited; Investigation; Knock-in; Knowledge; Lead; Link; Lymphoid; Mediating; Metabolic; Modeling; Molecular; Mus; Mutant Strains Mice; Mutate; Mutation; Nonhomologous DNA End Joining; Pathway interactions; Patients; Pattern; Play; Proteins; Relaxation; Reporting; Research Personnel; Role; Saccharomyces cerevisiae; Scientist; Single-Gene Defect; Site; Stem Cell Development; Stress; Syndrome; System; T cell regulation; T-Cell Activation; T-Cell Development; Testing; Topoisomerase; Topoisomerase II; Transcription Process; V(D)J Recombination; autoreactivity; autosome; body system; central tolerance; congenital immunodeficiency; experimental study; functional disability; homologous recombination; human disease; in vivo; induced pluripotent stem cell; innovation; loss of function mutation; malformation; mouse model; mutant; negative affect; novel; prevent; progenitor; recruit; repaired; seal; stem cell model; therapeutic target

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.

项目概要/摘要 对罕见免疫缺陷患者的研究对于我们了解 B 细胞非常有启发 发育和人类疾病，揭示 B 细胞发育、中枢耐受和 防止感染。综合征性免疫缺陷，其中身体畸形常常 比潜在的免疫缺陷更明显，研究较少且不太完善 明白了。我们最近发现 TOP2B 突变是常染色体显性 B 综合征的基础 细胞免疫缺陷（霍夫曼综合征）。拓扑异构酶是松弛所需的必需基因 DNA 复制和基因转录过程中的拓扑应力，可防止游离态的产生 DNA 断裂。我们最近首次报道了 TOP2B 在单基因人类疾病中的作用， 并揭示了 TOP2B DNA 结合域中的杂合显性失活突变导致 对 B 细胞的发育和功能产生负面影响，但 T 细胞的激活不受影响。在这个新 研究人员建议，我们采用新颖的酿酒酵母模型，采取大胆的多物种方法，创新 我们组研发的基因工程小鼠，TOP2B突变患者最大集合 TOP2b 介导的免疫缺陷，以及新开发的患者衍生的诱导多能 干细胞（iPSC）在否则无法获得的人体系统中探索我们的假设。中央 假设酶功能丧失会导致基因转录错误，限制 V(D)J 重组，随后导致正常 B 细胞分化和功能失败。三个具体目标 建议检验这一假设。具体目标 1 将确定 TOP2B 中的突变如何产生 B 细胞 由于产生和重新密封双链断裂的能力出现故障而导致免疫缺陷。体外和 体内实验将检查患者相关的 TOP2B 突变如何导致同源蛋白受损 重组和非同源末端连接，破坏早期 B 细胞发育。基因组研究 相互作用将检查突变体 Top2b 如何影响染色质环挤出并损害基本 V(D)J 重组和类别转换的机制。具体目标 2 将决定突变如何发生 TOP2B 影响修复蛋白向双链断裂位点的募集，从而减少断裂 修复，并导致非同源末端连接的功效降低。 最后，具体目标 3 将描述 Top2b 酶功能如何调节 B 细胞发育。使用我们的 独特的小鼠模型，包括基因敲入和条件小鼠模型以及 iPSC，我们拥有独特的 了解这种 B 细胞免疫缺陷的分子机制的机会。