Novel Mutations in SH2D1A Gene in X-linked Lymphoproliferative Syndrome, Diagnosed After B-Cell Non-Hodgkin Lymphoma.

BACKGROUND: X-linked lymphoproliferative disease type I (XLP I) is caused by mutations in the SH2D1A gene and characterized mainly by hypogammaglobulinemia and abnormal response to Epstein-Barr virus with a high predisposition to B-cell non-Hodgkin lymphoma development. OBSERVATIONS: In this article, we describe the experience of 2 centers in Belarus and in Russia that follow 3 male patients who were diagnosed with XLP I after lymphoma development and treatment. Three novel mutations c.51G>C (p.E17D), c.192G>T (p.W64C), and c.53insA (p.K18KfsX67) were found in 3 males patients with XLP I. Two of them did not have any signs of immunodeficiency before B-cell non-Hodgkin lymphoma development. CONCLUSIONS: We propose SH2D1A mutational screening be considered in male patients with or without hypogammaglobulinemia who received rituximab treatment for lymphoma and did not recover immunoglobulin G in a year after B-depleting therapy.

Phenotypic and functional characterisation of locally produced natural killer cells ex vivo expanded with the K562-41BBL-mbIL21 cell line.

We characterised the expansion, phenotype and functional activity of natural killer (NK) cells obtained for a clinical trial. Nineteen expansion procedures were performed to obtain NK cell products for 16 patients. NK cells were expanded ex vivo from haploidentical donor peripheral blood mononuclear cells in the presence of the locally generated feeder cell line K-562 with ectopic expression of 4-1BBL and mbIL-21. The median duration of expansion was 18 days (interquartile range 15-19). The median number of live cells yielded was 2.26 × 109 (range 1.6-3.4 × 109) with an NK content of 96.6% (range 95.1-97.9%). The median NK cell fold expansion was 171 (range 124-275). NK cell fold expansion depended on the number of seeded NK cells, the initial level of C-myc expression and the initial number of mature and immature NK cells. The majority of expanded NK cells had the phenotype of immature activated cells (NKG2A + , double bright CD56 + + CD16 + + , CD57-) expressing NKp30, NKp44, NKp46, NKG2D, CD69, HLA-DR and CD96. Despite the expression of exhaustion markers, expanded NK cells exhibited high cytolytic activity against leukaemia cell lines, high degranulation activity and cytokine production. There was a noted decrease in the functional activity of NK cells in tests against the patient's blasts.In conclusion, NK cells obtained by ex vivo expansion with locally generated K562-41BBL-mbIL21 cells had a relatively undifferentiated phenotype and enhanced cytolytic activity against cancer cell lines. Expansion of NK cells with feeder cells yielded a sufficient quantity of the NK cell product to reach high cell doses or increase the frequency of cell infusions for adoptive immunotherapy. Registered at clinicaltrials.gov as NCT04327037.

Decoding of exon splicing patterns in the human RUNX1-RUNX1T1 fusion gene.

The t(8;21) translocation is the most widespread genetic defect found in human acute myeloid leukemia. This translocation results in the RUNX1-RUNX1T1 fusion gene that produces a wide variety of alternative transcripts and influences the course of the disease. The rules of combinatorics and splicing of exons in the RUNX1-RUNX1T1 transcripts are not known. To address this issue, we developed an exon graph model of the fusion gene organization and evaluated its local exon combinatorics by the exon combinatorial index (ECI). Here we show that the local exon combinatorics of the RUNX1-RUNX1T1 gene follows a power-law behavior and (i) the vast majority of exons has a low ECI, (ii) only a small part is represented by "exons-hubs" of splicing with very high ECI values, and (iii) it is scale-free and very sensitive to targeted skipping of "exons-hubs". Stochasticity of the splicing machinery and preferred usage of exons in alternative splicing can explain such behavior of the system. Stochasticity may explain up to 12% of the ECI variance and results in a number of non-coding and unproductive transcripts that can be considered as a noise. Half-life of these transcripts is increased due to the deregulation of some key genes of the nonsense-mediated decay system in leukemia cells. On the other hand, preferred usage of exons may explain up to 75% of the ECI variability. Our analysis revealed a set of splicing-related cis-regulatory motifs that can explain "attractiveness" of exons in alternative splicing but only when they are considered together. Cis-regulatory motifs are guides for splicing trans-factors and we observed a leukemia-specific profile of expression of the splicing genes in t(8;21)-positive blasts. Altogether, our results show that alternative splicing of the RUNX1-RUNX1T1 transcripts follows strict rules and that the power-law component of the fusion gene organization confers a high flexibility to this process.

RUNX1T1/MTG8/ETO gene expression status in human t(8;21)(q22;q22)-positive acute myeloid leukemia cells.

The RUNX1-RUNX1T1 fusion gene, a product of the nonhomologous balanced translocation t(8;21)(q22;q22), is a complex genetic locus. We performed extensive bioinformatic analysis of transcription initiation as well as transcription termination sites in this locus and predicted a number of different RUNX1T1 transcripts. To confirm and quantify the RUNX1T1 gene expression, we analyzed samples from seven acute myeloid leukemia (AML) patients and from the Kasumi-1 cell line. We found variable activity of the four predicted RUNX1T1 promoters located downstream of the chromosome breakpoint. Nineteen alternative RUNX1T1 transcripts were identified by sequencing at least seventeen of which predictably can be translated into functional proteins. While the RUNX1T1 gene is not expressed in normal hematopoietic cells, it may participate in t(8;21)(q22;q22)-dependent leukemic transformation due to its multiple interactions in cell regulatory network particularly through synergistic or antagonistic effects in relation to activity of RUNX1-RUNX1T1 fusion gene.