A Novel Approach to Evaluate Genetic Variants in Primary Antibody Deficiency

项目摘要/摘要 原发性免疫缺陷是由免疫成分的基因突变引起的。 系统。已描述了300多种不同的PID亚型；其中一个主要亚型被归类为 主要是fi抗体。幼稚的B细胞产生低亲和力的IgM抗体。发展长远 对病原体的免疫保护，B细胞必须产生高亲和力的同型转换抗体，如 免疫球蛋白。为了实现这一目标，B细胞经历了类开关重组(CSR)和体细胞超突变(SHM) 免疫球蛋白(Ig)基因。CSR调节失调可导致PID，如高IgM综合征(HIGM)。 这些是复杂的抗体缺乏症，可归因于各种不同类型的基因突变 CSR/SHM的组成部分，如激活诱导脱氨酶(AID)、CD40或ICOS。虽然许多基因 已经在无丙种球蛋白血症或低丙种球蛋白血症的PID患者中发现了突变，这些突变是如何 基因突变对CSR过程的影响是以B细胞的内在方式进行的，目前尚不完全清楚。寻址 这些问题非常重要，因为剖析抗体缺陷的病理机制将 为产后出血的新治疗策略奠定生物学基础。 在这个应用中，我们建议建立一种新的方法来评估基因变异是如何导致 在企业社会责任缺陷的背景下，抗体缺陷。尽管最近在PID中发现了大量突变 患者，分子和临床的异质性是建立基因-表型的一个挑战 关联性。我们预计下一代测序(NGS)技术将识别越来越多的 抗体缺陷患者的突变。然而，NGS并未揭示其生物学意义。 已确定的突变。确认基因变异为致病突变仍需进行功能分析 解释患者特定的细胞和组织病理生理学。传统的基因打靶方法是 不足以有效地揭示这些突变的生物学意义。因此，我们建议应用新的 实现这些目标的基因组编辑方法。鉴于我们在企业社会责任模式方面的强大专业知识和我们的 关于磷脂酰肌醇3-激酶(PI3K)在控制CSR中的作用的先前工作，我们计划开发一个模型 系统来检测在PI3K途径的组成部分中发现的遗传变异。这种方法可以是 易于扩展以确定其他因素对PID患者CSR缺陷的贡献。此外，我们 建议建立一种利用原代B细胞进行基因变异功能测试的新系统。如果成功， 我们提出的研究将在PID领域产生很大影响，可能会实质性地加速 产后抑郁患者翻译应用的基因组研究。

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.