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.

项目总结/摘要 对罕见免疫缺陷患者的研究对我们理解B细胞的功能具有重要的指导意义。 发育和人类疾病，揭示了B细胞发育，中枢耐受和 保护免受感染。综合征性免疫缺陷，其中身体畸形往往是 比潜在的免疫缺陷更明显，研究得更少， 明白我们最近发现TOP 2 B突变是常染色体显性遗传综合征B的基础 细胞免疫缺陷（霍夫曼综合征）。拓扑异构酶是松弛神经所必需的基因。 拓扑压力在DNA复制和基因转录，以防止产生免费的 DNA断裂。我们最近报道了TOP 2B在单基因人类疾病中的作用的首次描述， 并揭示了TOP 2B的DNA结合域中的杂合显性负突变导致了 对B细胞的发育和功能有负面影响，而T细胞活化不受影响。在这个新 研究者建议，我们采取一个大胆的，多物种的方法使用新的S。酿酒模型，创新 我们小组开发的基因工程小鼠是患者中最大的TOP 2B突变集合 与TOP 2b介导的免疫缺陷，以及新开发的患者源性诱导多能 干细胞（iPSC）来探索我们的假设，否则无法获得人类系统。中央 假设酶功能的丧失导致错误的基因转录，限制V（D）J 重组，以及随后正常B细胞分化和功能的失败。三个具体目标 来检验这一假设。特定目标1将确定TOP 2 B中的突变如何产生B细胞 免疫缺陷通过功能障碍，创造和重新密封双链断裂。体外和 体内实验将研究TOP 2B中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，破坏早期B细胞发育。基因组研究 相互作用将研究突变体Top 2b如何影响染色质环挤出和损害基础 V（D）J复合和类转换机制。具体目标2将决定如何突变， TOP 2B影响修复蛋白在双链断裂位点的募集，导致断裂减少 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Top2 B酶功能如何调节B细胞发育。使用我们 独特的鼠模型，包括基因敲入和条件性鼠模型，以及iPSC，我们有一个独特的 有机会了解这种B细胞免疫缺陷的分子机制。