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.

项目摘要/摘要 对罕见免疫缺陷患者的研究对我们对B细胞的理解非常有启发性 发育和人类疾病，揭示了B细胞发育的机制，中央耐受性和 保护免受感染。综合征免疫缺陷，其中物理畸形通常是 比潜在的免疫缺陷更为明显，研究的频率较低，良好 理解。我们最近发现TOP2B中的突变是常染色体显性综合征B的基础 细胞免疫缺陷（霍夫曼综合征）。拓扑异构酶是放松所需的必要基因 DNA复制和基因转录过程中的拓扑应力，以防止自由产生 DNA断裂。我们最近报道了TOP2B在单基因人类疾病中的作用的第一个描述， 并揭示了TOP2B的DNA结合域中的杂合子，显性负突变导致 对B细胞的发育和功能的负面影响，而T细胞激活不影响。在这个新的中 调查员的建议，我们采用新颖的S. cerevisiae模型采用大胆的多种物种方法，创新 我们组开发的基因工程小鼠，这是患者最大的TOP2B突变集合 具有TOP2B介导的免疫缺陷以及新开发的患者诱导多能 干细胞（IPSC）在原本无法获得的人类系统中探索我们的假设。中央 假设是酶函数的丧失导致错误的基因转录，有限V（d）J 重组，随后正常B细胞​​分化和功能的失败。三个具体目标 建议检验这一假设。特定的目标1将确定TOP2B中的突变如何产生B细胞 通过故障创建和重新密封双链断裂的能力通过故障进行免疫缺陷。体外和 体内实验将检查TOP2B中与患者相关的突变如何导致同源性受损 重组和非同源末端连接，破坏了早期B细胞的发展。基因组研究 相互作用将检查突变体TOP2B如何影响染色质环的挤出并损害基本 V（d）J重组和班级切换的机制。具体目标2将决定如何突变 TOP2B会影响修复蛋白的募集到双链断裂部位，导致休息时间减少 维修，并导致非同源末端连接的功效降低。 最后，特定的AIM 3将描述TOP2B酶函数如何调节B细胞的发展。使用我们的 独特的鼠模型在内 了解该B细胞免疫缺陷的基础机制的机会。