B-cell precursors (BCP) arise from hematopoietic stem cells in bone marrow (BM). Identification and characterization of the different BCP subsets has contributed to the understanding of normal B-cell development. BCP first rearrange their immunoglobulin (Ig) heavy chain (IGH) genes to form the pre-B-cell receptor (pre-BCR) complex together with surrogate light chains. Appropriate signaling via this pre-BCR complex is followed by rearrangement of the Ig light chain genes, resulting in the formation, and selection of functional BCR molecules. Consecutive production, expression, and functional selection of the pre-BCR and BCR complexes guide the BCP differentiation process that coincides with corresponding immunophenotypic changes. We studied BCP differentiation in human BM samples from healthy controls and patients with a known genetic defect in V(D)J recombination or pre-BCR signaling to unravel normal immunophenotypic changes and to determine the effect of differentiation blocks caused by the specific genetic defects. Accordingly, we designed a 10-color antibody panel to study human BCP development in BM by flow cytometry, which allows identification of classical preB-I, preB-II, and mature B-cells as defined via BCR-related markers with further characterization by additional markers. We observed heterogeneous phenotypes associated with more than one B-cell maturation pathway, particularly for the preB-I and preB-II stages in which V(D)J recombination takes place, with asynchronous marker expression patterns. Next Generation Sequencing of complete IGH gene rearrangements in sorted BCP subsets unraveled their rearrangement status, indicating that BCP differentiation does not follow a single linear pathway. In conclusion, B-cell development in human BM is not a linear process, but a rather complex network of parallel pathways dictated by V(D)J-recombination-driven checkpoints and pre-BCR/BCR mediated-signaling occurring during B-cell production and selection. It can also be described as asynchronous, because precursor B-cells do not differentiate as full population between the different stages, but rather transit as a continuum, which seems influenced (in part) by V-D-J recombination-driven checkpoints.

• MeSH
• buněčná diferenciace genetika   imunologie   MeSH
• dítě MeSH
• lidé MeSH
• prekurzorové B-lymfoidní buňky imunologie   MeSH
• průtoková cytometrie metody   MeSH
• receptory antigenů B-buněk genetika   imunologie   MeSH
• syndromy imunologické nedostatečnosti genetika   imunologie   MeSH
• těžké řetězce imunoglobulinů genetika   MeSH
• V(D)J rekombinace genetika   imunologie   MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Porcine thymus contains three independent populations of cells that have rearranged immunoglobulin heavy chain VDJH genes. The first population can be found exclusively in medulla and it consists of existing mature B cells and plasma cells. The second consists of developing B cells characterized by the presence of selected VDJH rearrangement, similar to B cell lymphogenesis in the bone marrow. The third population is entirely unaffected by selection mechanism for productive VDJH rearrangement and represents T lineage cells that rearrange immunoglobulin genes. Transcription of unselected VDJH repertoire is not allowed in T cells. Sequence analysis of unselected VDJH repertoire from T cells also revealed important consequences for B cell lymphogenesis and selection of B cell repertoire. As far as we know, this is the first evidence that some species completely rearrange VDJH genes in T cells. Our results also support the finding that B cells actively develop in the thymus.

• MeSH
• B-lymfocyty imunologie   MeSH
• druhová specificita MeSH
• geny pro těžké řetězce imunoglobulinů genetika   MeSH
• lidé MeSH
• plod imunologie   MeSH
• podskupiny lymfocytů imunologie   MeSH
• prasata genetika   růst a vývoj   imunologie   MeSH
• T-lymfocyty imunologie   MeSH
• thymus růst a vývoj   imunologie   MeSH
• V(D)J rekombinace genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH

BACKGROUND: The endonuclease ARTEMIS, which is encoded by the DCLRE1C gene, is a component of the nonhomologous end-joining pathway and participates in hairpin opening during the V(D)J recombination process and repair of a subset of DNA double-strand breaks. Patients with ARTEMIS deficiency usually present with severe combined immunodeficiency (SCID) and cellular radiosensitivity, but hypomorphic mutations can cause milder phenotypes (leaky SCID). OBJECTIVE: We sought to correlate the functional effect of human DCLRE1C mutations on phenotypic presentation in patients with ARTEMIS deficiency. METHODS: We studied the recombination and DNA repair activity of 41 human DCLRE1C mutations in Dclre1c(-/-) v-abl kinase-transformed pro-B cells retrovirally engineered with a construct that allows quantification of recombination activity by means of flow cytometry. For assessment of DNA repair efficacy, resolution of γH2AX accumulation was studied after ionizing radiation. RESULTS: Low or absent activity was detected for mutations causing a typical SCID phenotype. Most of the patients with leaky SCID were compound heterozygous for 1 loss-of-function and 1 hypomorphic allele, with significant residual levels of recombination and DNA repair activity. Deletions disrupting the C-terminus result in truncated but partially functional proteins and are often associated with leaky SCID. Overexpression of hypomorphic mutants might improve the functional defect. CONCLUSIONS: Correlation between the nature and location of DCLRE1C mutations, functional activity, and the clinical phenotype has been observed. Hypomorphic variants that have been reported in the general population can be disease causing if combined in trans with a loss-of-function allele. Therapeutic strategies aimed at inducing overexpression of hypomorphic alleles might be beneficial.

• MeSH
• alely MeSH
• B-lymfocyty fyziologie   účinky záření   MeSH
• dítě MeSH
• dospělí MeSH
• fenotyp MeSH
• heterozygot MeSH
• histony metabolismus   MeSH
• ionizující záření MeSH
• jaderné proteiny genetika   MeSH
• kojenec MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• mutace genetika   MeSH
• mutační analýza DNA MeSH
• novorozenec MeSH
• onkogenní proteiny v-abl genetika   metabolismus   MeSH
• oprava DNA genetika   MeSH
• předškolní dítě MeSH
• těžká kombinovaná imunodeficience genetika   MeSH
• tolerance záření genetika   MeSH
• transformované buněčné linie MeSH
• V(D)J rekombinace genetika   MeSH
• Check Tag
• dítě MeSH
• dospělí MeSH
• kojenec MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• novorozenec MeSH
• předškolní dítě MeSH
• Publikační typ
• časopisecké články MeSH
• Research Support, N.I.H., Extramural MeSH

The ETV6-RUNX1 fusion gene, found in 25% of childhood acute lymphoblastic leukemia (ALL) cases, is acquired in utero but requires additional somatic mutations for overt leukemia. We used exome and low-coverage whole-genome sequencing to characterize secondary events associated with leukemic transformation. RAG-mediated deletions emerge as the dominant mutational process, characterized by recombination signal sequence motifs near breakpoints, incorporation of non-templated sequence at junctions, ∼30-fold enrichment at promoters and enhancers of genes actively transcribed in B cell development and an unexpectedly high ratio of recurrent to non-recurrent structural variants. Single-cell tracking shows that this mechanism is active throughout leukemic evolution, with evidence of localized clustering and reiterated deletions. Integration of data on point mutations and rearrangements identifies ATF7IP and MGA as two new tumor-suppressor genes in ALL. Thus, a remarkably parsimonious mutational process transforms ETV6-RUNX1-positive lymphoblasts, targeting the promoters, enhancers and first exons of genes that normally regulate B cell differentiation.

• MeSH
• fúzní onkogenní proteiny genetika   MeSH
• genetická variace * MeSH
• genová knihovna MeSH
• genová přestavba genetika   MeSH
• homeodoménové proteiny genetika   MeSH
• lidé MeSH
• molekulární sekvence - údaje MeSH
• pre-B-buněčná leukemie genetika   MeSH
• protein PEBP2A2 genetika   MeSH
• regulace genové exprese u nádorů genetika   MeSH
• rekombinace genetická genetika   MeSH
• sekvence nukleotidů MeSH
• sekvenční analýza DNA MeSH
• sekvenční delece genetika   MeSH
• transkripční faktory bHLH genetika   MeSH
• transkripční faktory genetika   MeSH
• tumor supresorové geny MeSH
• V(D)J rekombinace genetika   MeSH
• variabilita počtu kopií segmentů DNA genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH