http://repub.eur.nl/res/aut/3608/ List of Publications en http://repub.eur.nl/static-eur/img/logo.png http://repub.eur.nl 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. http://repub.eur.nl/res/pub/35577/ 2007-02-03T00:00:00Z We analyzed the phenotype of cells derived from SCID patients with different mutations in the Artemis gene. Using clonogenic survival assay an increased sensitivity was found to X-rays (2-3-fold) and bleomycin (2-fold), as well as to etoposide, camptothecin and methylmethane sulphonate (up to 1.5-fold). In contrast, we did not find increased sensitivity to cross-linking agents mitomycin C and cis-platinum. The kinetics of DSB repair assessed by pulsed-field gel electrophoresis and γH2AX foci formation after ionizing irradiation, indicate that 15-20% of DSB are not repaired in Artemis-deficient cells. In order to get a better understanding of the repair defect in Artemis-deficient cells, we studied chromosomal damage at different stages of the cell cycle. In contrast to AT cells, Artemis-deficient cells appear to have a normal G1/S-block that resulted in a similar frequency of dicentrics and translocations, however, frequency of acentrics fragments was found to be 2-4-fold higher compared to normal fibroblasts. Irradiation in G2resulted in a higher frequency of chromatid-type aberrations (1.5-3-fold) than in normal cells, indicating that a fraction of DSB requires Artemis for proper repair. Our data are consistent with a function of Artemis protein in processing of a subset of complex DSB, without G1cell cycle checkpoint defects. This type of DSB can be induced in high proportion and persist through S-phase and in part might be responsible for the formation of chromatid-type exchanges in G1-irradiated Artemis-deficient cells. Among different human radiosensitive fibroblasts studied for endogenous (in untreated samples) as well as X-ray-induced DNA damage, the ranking order on the basis of higher incidence of spontaneously occurring chromosomal alterations and induced ones was: ligase 4 ≥ AT > Artemis. This observation implicates that in human fibroblasts following exposure to ionizing radiation a lower risk might be created when cells are devoid of endogenous damage. http://repub.eur.nl/res/pub/8414/ 2006-01-01T00:00:00Z V(D)J recombination of Ig and TCR loci is a stepwise process during which site-specific DNA double-strand breaks (DSBs) are made by RAG1/RAG2, followed by DSB repair by nonhomologous end joining. Defects in V(D)J recombination result in SCID characterized by absence of mature B and T cells. A subset of T-B-NK+ SCID patients is sensitive to ionizing radiation, and the majority of these patients have mutations in Artemis. We present a patient with a new type of radiosensitive T-B-NK+ SCID with a defect in DNA ligase IV (LIG4). To date, LIG4 mutations have only been described in a radiosensitive leukemia patient and in 4 patients with a designated LIG4 syndrome, which is associated with chromosomal instability, pancytopenia, and developmental and growth delay. The patient described here shows that a LIG4 mutation can also cause T-B-NK+ SCID without developmental defects. The LIG4-deficient SCID patient had an incomplete but severe block in precursor B cell differentiation, resulting in extremely low levels of blood B cells. The residual D(H)-J(H) junctions showed extensive nucleotide deletions, apparently caused by prolonged exonuclease activity during the delayed D(H)-J(H) ligation process. In conclusion, different LIG4 mutations can result in either a developmental defect with minor immunological abnormalities or a SCID picture with normal development. http://repub.eur.nl/res/pub/8235/ 2003-01-01T00:00:00Z Severe combined immunodeficiency disease (SCID) can be immunologically classified by the absence or presence of T, B, and natural killer (NK) cells. About 30% of T(-)B(-)NK(+) SCID patients carry mutations in the recombination activating genes (RAG). Some T(-)B(-)NK(+) SCID patients without RAG gene mutations are sensitive to ionizing radiation, and several of these radiosensitive (RS) SCID patients were recently shown to have large deletions or truncation mutations in the Artemis gene, implying a role for Artemis in DNA double-strand break (dsb) repair. We identified 5 RS-SCID patients without RAG gene mutations, 4 of them with Artemis gene mutations. One patient had a large genomic deletion, but the other 3 patients carried simple missense mutations in conserved amino acid residues in the SNM1 homology domain of the Artemis protein. Extrachromosomal V(D)J recombination assays showed normal and precise signal joint formation, but inefficient coding joint formation in fibroblasts of these patients, which could be complemented by the wild-type Artemis gene. The cells containing the missense mutations in the SNM1 homology domain had the same recombination phenotype as the cells with the large deletion, indicating that these amino acid residues are indispensable for Artemis function. Immunogenotyping and immunophenotyping of bone marrow samples of 2 RS-SCID patients showed the absence of complete V(H)-J(H) gene rearrangements and consequently a complete B-cell differentiation arrest at the pre-B-cell receptor checkpoint-that is, at the transition from CyIgmu(-) pre-B-I cells to CyIgmu(+) pre-B-II cells. The completeness of this arrest illustrates the importance of Artemis at this stage of lymphoid differentiation. http://repub.eur.nl/res/pub/3197/ 2002-01-01T00:00:00Z The end-joining pathway of DNA double-strand break (DSB) repair is necessary for proper V(D)J recombination and repair of DSB caused by ionizing radiation. This DNA repair pathway can either use short stretches of (micro)homology near the DNA ends or use no homology at all (direct end-joining). We designed assays to determine the relative efficiencies of these (sub)pathways of DNA end-joining. In one version, a DNA substrate is linearized in such a way that joining on a particular microhomology creates a novel restriction enzyme recognition site. In the other one, the DSB is made by the RAG1 and RAG2 proteins. After PCR amplification of the junctions, the different end-joining modes can be discriminated by restriction enzyme digestion. We show that inactivation of the 'classic' end-joining factors (Ku80, DNA-PK(CS), ligase IV and XRCC4) results in a dramatic increase of microhomology-directed joining of the linear substrate, but very little decrease in overall joining efficiency. V(D)J recombination, on the other hand, is severely impaired, but also shows a dramatic shift towards microhomology use. Interestingly, two interstrand cross-linker-sensitive cell lines showed decreased microhomology-directed end-joining, but without an effect on V(D)J recombination. These results suggest that direct end-joining and microhomology-directed end-joining constitute genetically distinct DSB repair pathways.