Genetic Etiology of Agammaglobulinemia with Absent B Cells

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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.