http://repub.eur.nl/res/aut/22927/ List of Publications en http://repub.eur.nl/static-eur/img/logo.png http://repub.eur.nl http://repub.eur.nl/res/pub/39846/ 2013-04-08T00:00:00Z Background: Ataxia telangiectasia (AT) is a multisystem DNA-repair disorder caused by mutations in the ataxia telangiectasia mutated (ATM) gene. Patients with AT have reduced B- and T-cell numbers and a highly variable immunodeficiency. ATM is important for V(D)J recombination and immunoglobulin class-switch recombination (CSR); however, little is known about the mechanisms resulting in antibody deficiency severity. Objective: We sought to examine the immunologic mechanisms responsible for antibody deficiency heterogeneity in patients with AT. Methods: In this study we included patients with classical AT plus early-onset hypogammaglobulinemia (n = 3), classical AT (n = 8), and variant AT (late onset, n = 4). We studied peripheral B- and T-cell subsets, B-cell subset replication history, somatic hypermutation frequencies, CSR patterns, B-cell repertoire, and ATM kinase activity. Results: Patients with classical AT lacked ATM kinase activity, whereas patients with variant AT showed residual function. Most patients had disturbed naive B-cell and T-cell homeostasis, as evidenced by low cell numbers, increased proliferation, a large proportion CD21lowCD38lowanergic B cells, and decreased antigen receptor repertoire diversity. Impaired formation of T cell-dependent memory B cells was predominantly found in patients with AT plus hypogammaglobulinemia. These patients had extremely low naive CD4+T-cell counts, which were more severely reduced compared with those seen in patients with classical AT without hypogammaglobulinemia. Finally, AT deficiency resulted in defective CSR to distal constant regions that might reflect an impaired ability of B cells to undergo multiple germinal center reactions. Conclusion: The severity of the antibody deficiency in patients with AT correlates with disturbances in B- and T-cell homeostasis resulting in reduced immune repertoire diversity, which consequently affects the chance of successful antigen-dependent cognate B-T interaction. http://repub.eur.nl/res/pub/35023/ 2012-01-05T00:00:00Z Diamond-Blackfan anemia (DBA) is associated with developmental defects and profound anemia. Mutations in genes encoding a ribosomal protein of the small (eg, RPS19) or large (eg, RPL11) ribosomal subunit are found in more than half of these patients. The mutations cause ribosomal haploinsufficiency, which reduces overall translation efficiency of cellular mRNAs. We reduced the expression of Rps19 or Rpl11 in mouse erythroblasts and investigated mRNA polyribosome association, which revealed deregulated translation initiation of specific transcripts. Among these were Bag1, encoding a Hsp70 cochaperone, and Csde1, encoding an RNA-binding protein, and both were expressed at increased levels in erythroblasts. Their translation initiation is cap independent and starts from an internal ribosomal entry site, which appeared sensitive to knockdown of Rps19 or Rpl11. Mouse embryos lacking Bag1 die at embryonic day 13.5, with reduced erythroid colony forming cells in the fetal liver, and low Bag1 expression impairs erythroid differentiation in vitro. Reduced expression of Csde1 impairs the proliferation and differentiation of erythroid blasts. Protein but not mRNA expression of BAG1 and CSDE1 was reduced in erythroblasts cultured from DBA patients. Our data suggest that impaired internal ribosomal entry site-mediated translation of mRNAs expressed at increased levels in erythroblasts contributes to the erythroid phenotype of DBA. http://repub.eur.nl/res/pub/31032/ 2011-09-01T00:00:00Z Artemis deficiency is known to result in classical TB severe combined immunodeficiency (SCID) in case of Artemis null mutations, or Omenn's syndrome in case of hypomorphic mutations in the Artemis gene. We describe two unrelated patients with a relatively mild clinical TB SCID phenotype, caused by different homozygous Artemis splice-site mutations. The splice-site mutations concern either dysfunction of a 5′ splice-site or an intronic point mutation creating a novel 3′ splice-site, resulting in mutated Artemis protein with residual activity or low levels of wild type (WT) Artemis transcripts. During the first 10 years of life, the patients suffered from recurrent infections necessitating antibiotic prophylaxis and intravenous immunoglobulins. Both mutations resulted in increased ionizing radiation sensitivity and insufficient variable, diversity and joining (V(D)J) recombination, causing B-lymphopenia and exhaustion of the naive T-cell compartment. The patient with the novel 3′ splice-site had progressive granulomatous skin lesions, which disappeared after stem cell transplantation (SCT). We showed that an alternative approach to SCT can, in principle, be used in this case; an antisense oligonucleotide (AON) covering the intronic mutation restored WT Artemis transcript levels and non-homologous end-joining pathway activity in the patient fibroblasts. http://repub.eur.nl/res/pub/26120/ 2011-06-02T00:00:00Z A girl presented during childhood with a single course of extensive chickenpox and moderate albeit recurrent pneumonia in the presence of idiopathic CD4+T lymphocytopenia (ICL). Her clinical condition remained stable over the past 10 years without infections, any granulomatous disease, or autoimmunity. Immunophenotyping demonstrated strongly reduced naive T and B cells with intact proliferative capacity. Antibody reactivity on in vivo immunizations was normal. T-cell receptor-Vβ repertoire was polyclonal with a very low content of T-cell receptor excision circles (TRECs). Kappa-deleting recombination excision circles (KRECs) were also abnormal in the B cells. Both reflect extensive in vivo proliferation. Patient-derived CD34+hematopoietic stem cells could not repopulate RAG2-/-IL2Rγc-/-mice, indicating the lymphoid origin of the defect. We identi-fied 2 novel missense mutations in RAG1 (p.Arg474Cys and p.Leu506Phe) resulting in reduced RAG activity. This report gives the first genetic clue for ICL and extends the clinical spectrum of RAG mutations from severe immune defects to an almost normal condition. http://repub.eur.nl/res/pub/28582/ 2010-04-04T00:00:00Z V(D)J rearrangement in lymphoid cells involves repair of double-strand breaks (DSBs) through non-homologous end joining (NHEJ). Defects in this process lead to increased radiosensitivity and severe combined immunodeficiency (RS-SCID). Here, a SCID patient, M3, is described with a T-B+NK+phenotype but without causative mutations in CD3δ, ε, ζ or IL7Rα, genes specifically involved in T cell development. Clonogenic survival of M3 fibroblasts showed an increased sensitivity to the DSB-inducing agents ionizing radiation and bleomycin, as well as the crosslinking compound, mitomycin C. We did not observe inactivating mutations in known NHEJ genes and results of various DSB-repair assays in G1M3 cells were indistinguishable from those obtained with normal cells. However, we found increased chromosomal radiosensitivity at the G2phase of the cell cycle. Checkpoint analysis indicated functional G1/S and intra-S checkpoints after irradiation but impaired activation of the "early" G2/M checkpoint. Together these results indicate a novel class of RS-SCID patients characterized by the specific absence of T lymphocytes and associated with defects in G2-specific DSB repair. The pronounced G2/M radiosensitivity of the RS-SCID patient described here, suggests a defect in a putative novel and uncharacterized factor involved in cellular DNA damage responses and T cell development. http://repub.eur.nl/res/pub/19346/ 2009-01-05T00:00:00Z Radiosensitive T-B- severe combined immunodeficiency (RS-SCID) is caused by defects in the nonhomologous end-joining (NHEJ) DNA repair pathway, which results in failure of functional V(D)J recombination. Here we have identified the first human RS-SCID patient to our knowledge with a DNA-PKcs missense mutation (L3062R). The causative mutation did not affect the kinase activity or DNA end-binding capacity of DNA-PKcs itself; rather, the presence of long P-nucleotide stretches in the immunoglobulin coding joints indicated that it caused insufficient Artemis activation, something that is dependent on Artemis interaction with autophosphorylated DNA-PKcs. Moreover, overall end-joining activity was hampered, suggesting that Artemis-independent DNA-PKcs functions were also inhibited. This study demonstrates that the presence of DNA-PKcs kinase activity is not sufficient to rule out a defect in this gene during diagnosis and treatment of RS-SCID patients. Further, the data suggest that residual DNA-PKcs activity is indispensable in humans.