A Novel Approach to Evaluate Genetic Variants in Primary Antibody Deficiency

评估原发性抗体缺乏症遗传变异的新方法

• 批准号: 9527609
• 负责人: Jing Hong Wang
• 金额: $ 23.33万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-01-15 至 2019-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9527609
• 关键词: Address; Affect; Affinity; Agammaglobulinemia; Antibodies; B-Lymphocytes; Biological; Biological Assay; Biological Models; CRISPR/Cas technology; Catalytic Domain; Cell Culture Techniques; Cells; Complex; DNA Sequence Alteration; Defect; Diagnosis; Disease; Dissection; Flow Cytometry; Functional disorder; Gene Targeting; Genes; Genetic Transcription; Genomics; Genotype; Goals; Human; Hyperimmunoglobulin M Syndrome; Immune; Immune system; Immunoglobulin Class Switching; Immunoglobulin G; Immunoglobulin Genes; Immunoglobulin M; Immunoglobulin Somatic Hypermutation; Immunoglobulin Switch Recombination; Lead; Lymphocyte; Modeling; Molecular; Mus; Mutation; PIK3CA gene; PTEN gene; Pathologic; Pathway interactions; Patients; Phenotype; Phosphatidylinositols; Phosphorylation; Phosphotransferases; Population; Process; Public Health; RNA; Reporting; Role; Signal Transduction; Structure of germinal center of lymph node; Subgroup; System; TNFRSF5 gene; Techniques; Testing; Tissues; Transcriptional Activation; Validation; Variant; Work; activation-induced cytidine deaminase; base; clinical heterogeneity; congenital immunodeficiency; disease-causing mutation; genetic variant; genome editing; insight; mouse model; mutant; next generation sequencing; novel; novel strategies; novel therapeutic intervention; pathogen; protein expression; therapeutic development

Project Summary/Abstract 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 deficiency. Naïve 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.

项目摘要/摘要 原发性免疫缺陷（PID）是由免疫成分中的基因突变引起的 系统。已经描述了300多个亚型的亚型；一个主要子组被归类为 主要是抗体效率。幼稚的B细胞产生低亲和力IgM抗体。长期发展 免疫保护病原体，B细胞必须产生高亲和力的同种型抗体，例如 IgG。为了实现这一目标，B细胞经历了类开关重组（CSR）和躯体超成名（SHM） 在免疫球蛋白（IG）基因中。 CSR的失调可能导致PID，例如高IgM综合征（HIGM）。 这些是抗体缺乏症的复杂疾病，可以归因于各种基因突变 CSR/SHM的成分，例如激活诱导的脱氨酶（AID），CD40或ICOS。虽然有很多通用 在agammaglobloblobloblemia或hardogammagamaglobloblobloinemia的PID患者中已经发现了突变，这些突变如何 遗传突变以B细胞的固有方式影响CSR过程。寻址 此类问题非常重要，因为对抗体缺陷的病理机制的解剖会 在PID中建立新的治疗策略的生物学基础。 在此应用中，我们建议建立一种新的方法来评估遗传变异的原因 CSR缺陷背景下的抗体缺乏。尽管最近在PID中发现了大量突变 患者，分子和临床异质性是建立基因型 - 表型的挑战 相关性。我们预计下一代测序（NGS）技术将确定越来越多的 抗体缺陷患者的突变。但是，NGS并未揭示 确定的突变。验证遗传变异作为引起疾病的突变仍然需要功能分析 解释患者特异性的细胞和组织病理生理学。传统的基因靶向方法是 不足以有效揭示这些突变的生物学意义。那，我们建议应用新的 基因组编辑的方法来实现此类目标。鉴于我们在CSR模型和我们的 先前关于磷酸肌醇3-激酶（PI3K）在控制CSR中的作用的工作，我们计划开发一个模型 测试在PI3K途径成分中鉴定出的遗传变异的系统。这样的方法可以是 很容易扩展，以确定其他因素对PID患者CSR缺陷的贡献。另外，我们 提出了使用原代B细胞来建立一种新型系统，用于对遗传变异的功能测试。如果成功， 我们提出的研究将导致对PID领域的影响很大，这可能会实质上加速 基因组研究对PID患者的转化应用。