Genetic Etiology of Agammaglobulinemia with Absent B Cells

E2 A是一种广泛表达的转录因子，对早期B细胞发育至关重要。的基因 E2 A（TCF 3）通过选择性剪接编码两个独特的碱性螺旋环螺旋（bHLH）蛋白E12和E47。 E12和E47两者可以作为同源二聚体和作为彼此或与组织特异性结合的异源二聚体起作用。 转录因子虽然涉及E2 A的体细胞突变和易位见于白血病和白血病患者， 淋巴瘤，该基因的生殖系突变尚未报道。我们最近发现了4名患者 B细胞数量明显减少，无丙种球蛋白血症， E47的bHLH结构域的碱性区域中的杂合氨基酸取代（E555 K），DNA 蛋白质的结合片段。这些患者中存在的少量B细胞具有不寻常的 表型特征为E2 A的下游靶标CD 19的表达增加， 缺乏B细胞抗原受体。骨髓分析显示前B细胞数量减少， CD 19表达增加和酪氨酸磷酸化增加。表达载体研究 表明突变蛋白是稳定的并且定位于细胞核。突变导致显性 在电泳迁移率变动分析（EMSA）和荧光素酶报告基因中的负效应， 鼠M增强子。为了更好地了解控制早期B细胞发育的事件， 将检查E47突变的功能后果。在具体目标1中，我们将确定 E47中的E555 K突变改变了其与重要伴侣二聚化或结合关键DNA序列的能力 作为同源二聚体或异源二聚体，使用EMSA和染色质免疫沉淀（ChIP）。ChIP检测 将使用代表已转染的B细胞分化的各个阶段的B细胞系进行 用E47野生型或突变型表达载体。在特定目标2中，我们将创建一个敲入小鼠品系 E555 K突变的小鼠，并将这些小鼠与E2 A-/-小鼠和E47-/-小鼠以及野生型小鼠进行比较。 小鼠这些小鼠中B谱系细胞的数量和表型、VDJ重组状态和CD 4 +/CD 8 + T细胞亚群。 将检测活化标记的表达。在具体目标3中，我们将分析功能 突变体E47在体内和体外系统中的后果。RNA-seq将用于分析这些改变 在野生型与突变型小鼠的B细胞前体的转录组中。基因调控区 通过ChIP或RNA-seq鉴定的基因将通过报告构建体验证。最后，转录组分析 将对患者的常见淋巴前体进行检测。E555 K突变的鉴定 代表第一种常染色体显性形式的无丙种球蛋白血症。它符合最近的观察结果 表明从头突变并不罕见，并且可能发生在基因组的特定位点。的 建议的研究结果将加强我们对早期B细胞需求的理解 发展，并可能建议用于治疗自身免疫性疾病和癌症的途径。

E2A is a broadly expressed transcription factor that is essential for early B cell development. The gene for E2A, TCF3, encodes 2 unique basic helix loop helix (bHLH) proteins, E12 and E47, by alternative splicing. Both E12 and E47 can function as homodimers and as heterodimers with each other or with tissue specific transcription factors. Although somatic mutations and translocations involving E2A are seen in leukemias and lymphomas, germline mutations in this gene have not been reported. We have recently identified 4 patients with markedly reduced numbers of B cells and agammaglobulinemia who have exactly the same de novo heterozygous amino acid substitution (E555K) in the basic region of the bHLH domain of E47, the DNA binding segment of the protein. The small number of B cells present in these patients have an unusual phenotype characterized by the increased expression of CD19, a downstream target of E2A, and the absence of a B cell antigen receptor. Bone marrow analysis shows reduced numbers of pro-B cells, increased expression of CD19 and increased tyrosine phosphorylation. Studies with expression vectors indicate that the mutant protein is stable and localizes to the nucleus. The mutation causes a dominant negative effect in electrophoretic mobility shift assays (EMSAs) and luciferase reporters when binding the murine m enhancer. To better understand events that control early B cell development, the proposed studies will examine the functional consequences of the E47 mutation. In Specific Aim 1, we will determine if the E555K mutation in E47 alters its ability to dimerize with essential partners or to bind critical DNA sequences as a homodimer or heterodimer using EMSAs, and chromatin immunoprecipitation (ChIP). The ChIP assays will be done using B cell lines representing various stages of B cell differentiation that have been transfected with E47 wild type or mutant expression vectors. In Specific Aim 2, we will create a knock-in strain of mice with the E555K mutation, and compare these mice with E2A-/- mice and E47-/- mice as well as wild type mice. The number and phenotype of B lineage cells in these mice, the VDJ recombination status and the expression of activation markers will be examined. In Specific Aim 3 we will analyze the functional consequences of mutant E47 in in vivo and in vitro systems. RNA-seq will be used to analyze the alterations in the transcriptome of B cell precursors from wild type versus mutant mice. Regulatory regions of genes identified by ChIP or RNA-seq will be verified by reporter contructs. Finally, transcriptome analysis of common lymphoid precursors from the patients will be performed. The identification of the E555K mutation represents the first autosomal dominant form of agammaglobulinemia. It conforms to recent observations indicating that de novo mutations are not rare and are likely to occur at specific sites in the genome. The results of the proposed studies will enhance our understanding of the requirements for early B cell development and may suggest pathways that be used to treat autoimmune disease and cancer.