The effect of a novel recombination between the homeobox gene NKX2-5 and the TRD locus in T-cell acute lymphoblastic leukemia on activation of the NKK2-5 gene

Background and Objectives. The NK-like homeobox gene (NKX2-5/CSX) plays a crucial role in cardiac development but is not normally expressed in hematopoietic cells. Here, we describe for the first time a fusion between NKX2-5 and the T-cell receptor delta locus (TRD) resulting in NKX2-5 activation in a case of T-cell acute lymphoblastic leukemia (T-ALL). Design and Methods. Genomic DNA from a T-ALL patient with an atypical rearrangement, detected by Southern blotting, was analyzed by ligation-mediated polymerase chain reaction (PCR) with TRD-specific primers. Expression of NKX2-5 was analyzed by real-time quantitative PCR in the T-ALL case with the NKX2-5-TRD rearrangement, 18 other cases of T-ALL, three T-ALL derived cell lines, two non-hematopoietic cell lines, peripheral blood mononuclear cells from six healthy individuals and sorted thymocyte subsets. Results. Sequence analysis of ligation-mediated PCR products revealed a novel rearrangement between the third diversity segment of the TRD locus (TRDD3) and a region on chromosome 5q35.1 located 32 kb downstream of the NKX2-5/CSX gene. As a result of this recombination NKX2-5 was placed under influence of the TRD enhancer, resulting in strong ectopic NKX2-5 expression. High NKX2-5 expression was also found in the T-cell lines PEER and CCRF-CEM, which harbor an NKX2-5-BCL11B rearrangement, and in the embryonic kidney cell line 293. NKX2-5 was not expressed in any of the major thymocyte subsets, in normal peripheral blood mononuclear cells, or in the majority (17/18) of the other cases of T-ALL. Interpretation and Conclusions. Our finding of overexpression of yet another homeobox gene in TALL further supports the hypothesis that homeobox genes play an important role in malignant transformation of particular types of T-ALL

The effect of a novel recombination between the homeobox gene NKX2-5 and the TRD locus in T-cell acute lymphoblastic leukemia on activation of the NKX2-5 gene

Background and Objectives. The NK-like homeobox gene (NKX2-5/CSX) plays a crucial role in cardiac development but is not normally expressed in hematopoietic cells. Here, we describe for the first time a fusion between NKX2-5 and the T-cell receptor delta locus (TRD) resulting in NKX2-5 activation in a case of T-cell acute lymphoblastic leukemia (T-ALL). Design and Methods. Genomic DNA from a T-ALL patient with an atypical rearrangement, detected by Southern blotting, was analyzed by ligation-mediated polymerase chain reaction (PCR) with TRD-specific primers. Expression of NKX2-5 was analyzed by real-time quantitative PCR in the T-ALL case with the NKX2-5-TRD rearrangement, 18 other cases of T-ALL, three T-ALL derived cell lines, two non-hematopoietic cell lines, peripheral blood mononuclear cells from six healthy individuals and sorted thymocyte subsets. Results. Sequence analysis of ligation-mediated PCR products revealed a novel rearrangement between the third diversity segment of the TRD locus (TRDD3) and a region on chromosome 5q35.1 located 32 kb downstream of the NKX2-5/CSX gene. As a result of this recombination NKX2-5 was placed under influence of the TRD enhancer, resulting in strong ectopic NKX2-5 expression. High NKX2-5 expression was also found in the T-cell lines PEER and CCRF-CEM, which harbor an NKX2-5-BCL11B rearrangement, and in the embryonic kidney cell line 293. NKX2-5 was not expressed in any of the major thymocyte subsets, in normal peripheral blood mononuclear cells, or in the majority (17/18) of the other cases of T-ALL. Interpretation and Conclusions. Our finding of overexpression of yet another homeobox gene in T-ALL further supports the hypothesis that homeobox genes play an important role in malignant transformation of particular types of T-ALL.</p

The effect of a novel recombination between the homeobox gene NKX2-5 and the TRD locus in T-cell acute lymphoblastic leukemia on activation of the NKX2-5 gene

Background and Objectives. The NK-like homeobox gene (NKX2-5/CSX) plays a crucial role in cardiac development but is not normally expressed in hematopoietic cells. Here, we describe for the first time a fusion between NKX2-5 and the T-cell receptor delta locus (TRD) resulting in NKX2-5 activation in a case of T-cell acute lymphoblastic leukemia (T-ALL). Design and Methods. Genomic DNA from a T-ALL patient with an atypical rearrangement, detected by Southern blotting, was analyzed by ligation-mediated polymerase chain reaction (PCR) with TRD-specific primers. Expression of NKX2-5 was analyzed by real-time quantitative PCR in the T-ALL case with the NKX2-5-TRD rearrangement, 18 other cases of T-ALL, three T-ALL derived cell lines, two non-hematopoietic cell lines, peripheral blood mononuclear cells from six healthy individuals and sorted thymocyte subsets. Results. Sequence analysis of ligation-mediated PCR products revealed a novel rearrangement between the third diversity segment of the TRD locus (TRDD3) and a region on chromosome 5q35.1 located 32 kb downstream of the NKX2-5/CSX gene. As a result of this recombination NKX2-5 was placed under influence of the TRD enhancer, resulting in strong ectopic NKX2-5 expression. High NKX2-5 expression was also found in the T-cell lines PEER and CCRF-CEM, which harbor an NKX2-5-BCL11B rearrangement, and in the embryonic kidney cell line 293. NKX2-5 was not expressed in any of the major thymocyte subsets, in normal peripheral blood mononuclear cells, or in the majority (17/18) of the other cases of T-ALL. Interpretation and Conclusions. Our finding of overexpression of yet another homeobox gene in T-ALL further supports the hypothesis that homeobox genes play an important role in malignant transformation of particular types of T-ALL.</p

Beyond Autoantibodies: Biologic Roles of Human Autoreactive B Cells in Rheumatoid Arthritis Revealed by RNA?Sequencing

Objective:To obtain the comprehensive transcriptome profile of human citrulline?specific B cells from patients with rheumatoid arthritis (RA). Methods:Citrulline? and hemagglutinin?specific B cells were sorted by flow cytometry using peptide–streptavidin conjugates from the peripheral blood of RA patients and healthy individuals. The transcriptome profile of the sorted cells was obtained by RNA?sequencing, and expression of key protein molecules was evaluated by aptamer?based SOMAscan assay and flow cytometry. The ability of these proteins to effect differentiation of osteoclasts and proliferation and migration of synoviocytes was examined by in vitro functional assays. Results:Citrulline?specific B cells, in comparison to citrulline?negative B cells, from patients with RA differentially expressed the interleukin?15 receptor ? (IL?15R?) gene as well as genes related to protein citrullination and cyclic AMP signaling. In analyses of an independent cohort of cyclic citrullinated peptide–seropositive RA patients, the expression of IL?15R? protein was enriched in citrulline?specific B cells from the patients’ peripheral blood, and surprisingly, all B cells from RA patients were capable of producing the epidermal growth factor ligand amphiregulin (AREG). Production of AREG directly led to increased migration and proliferation of fibroblast?like synoviocytes, and, in combination with anti–citrullinated protein antibodies, led to the increased differentiation of osteoclasts. Conclusion:To the best of our knowledge, this is the first study to document the whole transcriptome profile of autoreactive B cells in any autoimmune disease. These data identify several genes and pathways that may be targeted by repurposing several US Food and Drug Administration–approved drugs, and could serve as the foundation for the comparative assessment of B cell profiles in other autoimmune diseases

Identification of a new cluster of T-cell receptor delta recombining elements

Within the human T-cell receptor δ (TCRD) gene we have identified a new cluster of seven δ recombining elements (δRec2.1–2.7), located 2·6–5·2 kilobases downstream of the Vδ2 gene segment. The δRec2 elements are isolated recombining signal sequences (RSS), which were shown to rearrange with the Dδ3 and Jδ1 segments of the TCRD gene as well as with the ψJα of the TCRA gene. Rearrangements involving the δRec2 elements were found in all peripheral blood (PB) samples from 10 healthy individuals, although their frequency was about 100-fold lower than that of classical δRec rearrangements. The total frequency of δRec2 rearrangements was lower in PB T lymphocytes, as compared with thymocytes, suggesting that they are deleted during T-cell development. The decrease of the frequency of the δRec2-Dδ3 rearrangements was most prominent: 11 times lower in PB T lymphocytes than in thymocytes. Since the δRec2-Jδ1 rearrangements contained the Dδ3 segment in the junctional region, we assume that they are derived from the δRec2-Dδ3 rearrangements. In contrast, the majority of δRec2-ψJα rearrangements did not contain the Dδ3 segment, indicating that they are single step rearrangements. The δRec2-Jδ1 and δRec2-ψJα rearrangements seem to be T-lineage specific, but the δRec2-Dδ3 rearrangements were also found at very low frequencies in B lymphocytes and natural killer cells. Our results suggest that δRec2 rearrangements are transient steps in the recombinatorial process of the TCRAD locus and are probably deleted by subsequent Vα-Jα rearrangements. We hypothesize, that in a similar manner to the classical δRec rearrangements, the δRec2 rearrangements might also contribute to T-cell differentiation towards the TCR-αβ lineage