A Novel Approach to Evaluate Genetic Variants in Primary Antibody Deficiency

项目总结/摘要 原发性免疫缺陷症（PID）是由免疫系统中的基因突变引起的。 系统已经描述了各种PID的300多种亚型;并且一个主要的亚组被分类为 主要是抗体缺乏。幼稚B细胞产生低亲和力IgM抗体。发展长期 为了对病原体进行免疫保护，B细胞必须产生高亲和力的同种型转换抗体， IgG。为了实现这一目标，B细胞经历类别转换重组（CSR）和体细胞超突变（SHM）。 免疫球蛋白（IG）基因。CSR的失调可导致PID，如高IgM综合征（HIGM）。 这些是抗体缺乏的复杂疾病，可归因于各种基因突变。 CSR/SHM的组分，如活化诱导的脱氨酶（AID）、CD 40或ICOS。虽然许多基因 已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，这些突变是如何发生的 基因突变以B细胞内在方式影响CSR过程仍然不完全清楚。解决 这些问题是非常重要的，因为对抗体缺乏的病理机制的剖析将 为PID的新治疗策略建立生物学基础。 在本申请中，我们提出建立一种新的方法来评估遗传变异如何导致 在CSR缺陷的背景下抗体缺乏。尽管最近在PID中发现了大量的突变， 患者，分子和临床异质性代表了建立基因型-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。然而，NGS并没有揭示出 识别突变。遗传变异作为致病突变的验证仍然需要功能测定 来解释患者特异性细胞和组织病理生理学。传统的基因靶向方法有 不足以有效地揭示这些突变的生物学意义。因此，我们建议采用新的 基因组编辑方法来实现这些目标。鉴于我们在企业社会责任模式方面的强大专业知识， 在前人关于磷脂酰肌醇3-激酶（PI 3 K）在控制CSR中的作用的基础上，我们计划建立一个模型， 系统来测试在PI 3 K途径的组分中鉴定的遗传变体。这些方法可以是 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 提出建立一种使用原代B细胞进行遗传变体功能测试的新系统。如果成功， 我们提出的研究将在PID领域产生很大的影响，这可能会大大加速 将基因组研究转化为PID患者的翻译应用。

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.