Genetic Etiology of Agammaglobulinemia with Absent B Cells

描述（由申请人提供）：E2A是一种广泛表达的转录因子，对早期B细胞发育至关重要。E2A基因TCF3通过选择性剪接编码2种独特的基本螺旋环螺旋（bHLH）蛋白E12和E47。E12和E47都可以作为同二聚体和异二聚体相互作用或与组织特异性转录因子相互作用。虽然涉及E2A的体细胞突变和易位见于白血病和淋巴瘤，但该基因的种系突变尚未报道。我们最近发现了4例B细胞数量明显减少和无球蛋白血症的患者，他们在E47的bHLH结构域的基本区域（DNA结合片段）具有完全相同的从头杂合氨基酸取代（E555K）。这些患者中存在的少量B细胞具有不寻常的表型，其特征是CD19 （E2A的下游靶点）的表达增加，以及B细胞抗原受体的缺失。骨髓分析显示前b细胞数量减少，CD19表达增加，酪氨酸磷酸化增加。通过表达载体的研究表明，突变蛋白是稳定的，并定位于细胞核。当结合小鼠m增强子时，该突变在电泳迁移率转移试验（emsa）和荧光素酶报告中引起主要的负面影响。为了更好地了解控制早期B细胞发育的事件，拟议的研究将检查E47突变的功能后果。在Specific Aim 1中，我们将使用emsa和染色质免疫沉淀（ChIP）确定E47中的E555K突变是否会改变其与基本伴侣二聚体或将关键DNA序列结合为同二聚体或异二聚体的能力。ChIP试验将使用代表B细胞分化不同阶段的B细胞系进行，这些B细胞系已被E47野生型或突变型表达载体转染。在Specific Aim 2中，我们将创建具有E555K突变的小鼠敲入株，并将这些小鼠与E2A-/-小鼠和E47-/-小鼠以及野生型小鼠进行比较。观察小鼠B系细胞的数量和表型、VDJ重组状态和激活标记物的表达。在特异性目标3中，我们将分析突变体E47在体内和体外系统中的功能后果。RNA-seq将用于分析野生型与突变型小鼠B细胞前体转录组的变化。通过ChIP或RNA-seq鉴定的基因的调控区域将通过报告基因进行验证。最后，将对来自患者的常见淋巴样前体进行转录组分析。E555K突变的鉴定代表了第一个常染色体显性形式的无球蛋白血症。这与最近的观察结果一致，表明新生突变并不罕见，而且可能发生在基因组的特定位点。拟议的研究结果将增强我们对早期B细胞发育需求的理解，并可能提出用于治疗自身免疫性疾病和癌症的途径。

DESCRIPTION (provided by applicant): 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.