The immunophenotypic and immunogenotypic B-cell differentiation arrest in bone marrow of RAG-deficient SCID patients corresponds to residual recombination activities of mutated RAG proteins

Abstract

AbstractThe protein products of the recombination activating genes (RAG1and RAG2) initiate the formation of immunoglobulin (Ig) and T-cell receptors, which are essential for B- and T-cell development, respectively. Mutations in the RAGgenes result in severe combined immunodeficiency disease (SCID), generally characterized by the absence of mature B and T lymphocytes, but presence of natural killer (NK) cells. Biochemically, mutations in the RAGgenes result either in nonfunctional proteins or in proteins with partial recombination activity. The mutated RAGgenes of 9 patients from 7 families were analyzed for their recombination activity using extrachromosomal recombination substrates, rearrangement of endogenous Ig loci in RAGgene–transfected nonlymphoid cells, or the presence of Ig gene rearrangements in bone marrow (BM). Recombination activity was virtually absent in all 6 patients with mutations in the RAG core domains, but partial activity was present in the other 3 RAG-deficient patients, 2 of them having Omenn syndrome with oligoclonal T lymphocytes. Using 4-color flow cytometry, we could define the exact stage at which B-cell differentiation was arrested in the BM of 5 RAG-deficient SCID patients. In 4 of 5 patients, the absence of recombination activity was associated with a complete B-cell differentiation arrest at the transition from cytoplasmic (Cy) Igμ− pre–B-I cells to CyIgμ+ pre–B-II cells. However, the fifth patient showed low frequencies of precursor B cells with CyIgμ and surface membrane IgM, in line with the partial recombination activity of the patient's mutatedRAG gene and the detection of in-frame Ig gene rearrangements in BM.