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Prepublished online as a Blood First Edition Paper on October 24, 2002; DOI 10.1182/blood-2002-01-0187.
IMMUNOBIOLOGY
From the Department of Immunology, Erasmus
MC/University Medical Center Rotterdam, The Netherlands;
Department of Cell Biology and Genetics, Erasmus MC/University Medical
Center Rotterdam, The Netherlands; Department of
Pediatrics, Division of Immunology and Infectious Diseases, Erasmus
MC/University Medical Center Rotterdam, The Netherlands;
Department of Pediatrics, Leiden University Medical Center, The
Netherlands; Department of Pediatrics, University Medical Center
Nijmegen - St Radboud, The Netherlands; Department of
Radiation Genetics and Chemical Mutagenesis, Leiden University Medical
Center, The Netherlands; Department of Radiation Oncology,
Erasmus MC/University Medical Center Rotterdam, The
Netherlands; and Department of Molecular Cell Genetics, the
Ludwik Rydygier University of Medical Sciences, Bydgoszcz,
Poland.
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 Severe combined immunodeficiency disease (SCID) is
clinically characterized by opportunistic infections, protracted
diarrhea, and failure to thrive.1 Patients generally die
within the first year of life unless treated with bone marrow (BM)
transplantation. Other treatment options include enzyme substitution in
case of adenosine deaminase-deficient SCID and gene therapy in case of common Although SCID consists of a heterogeneous group of diseases, it is
immunologically characterized by the absence or dysfunctioning of T
lymphocytes. SCID can be subdivided based on the additional presence or
absence of B lymphocytes and natural killer (NK) cells in
peripheral blood (PB). A number of
T During the process of immunoglobulin (Ig) and T-cell receptor (TCR)
gene recombination in differentiating lymphocytes, the RAG proteins
introduce DNA dsb's, which are repaired via nonhomologous end joining
(NHEJ).5 NHEJ requires active DNA-dependent protein kinase
(DNA-PK), which consists of Ku70, Ku80, and the catalytic subunit
(DNA-PKcs). The DNA-PK protein complex functions as a DNA
damage sensor, with Ku70 and Ku80 forming a heterodimer that binds to
DNA ends, while DNA-PKcs has serine and threonine protein kinase activity.6 In the final phase, the 2 broken DNA
ends are ligated by the DNA ligase IV-XRCC4 complex.7,8
However, it was shown that a number of radiosensitive (RS)
T We studied 5 RS-SCID patients (from 4 families) without RAG
gene mutations and found that 4 of them had mutations in the
Artemis gene. Immunophenotyping and immunogenotyping of BM
samples from 2 RS-SCID patients with Artemis gene mutations
provided additional information about the role of Artemis during
precursor B-cell differentiation.
Cell samples
BM samples from T All cell samples were obtained according to the informed consent
guidelines of the medical ethics committees of the Erasmus MC/University Medical Center Rotterdam and the Leiden University Medical Center.
Sensitivity of fibroblasts to ionizing radiation
Messenger RNA and DNA isolation and cDNA reaction DNA was extracted from granulocytes or fibroblasts using the QIAamp Blood kit (Qiagen, Chatsworth, CA).14 Total RNA was isolated from BMMCs or fibroblasts using the GenElute Mammalian RNA kit (Sigma-Aldrich, St Louis, MO). Complementary DNA was prepared from mRNA as described before, using random hexamers and Superscript reverse transcriptase (Life Technologies, Paisley, United Kingdom).15PCR amplification and analysis of Ig gene rearrangements Polymerase chain reaction (PCR) was performed as described previously.15 In each 100-µL PCR reaction, 0.1 to 1 µg cDNA, 10 to 12.5 pmol of 5' and 3' oligonucleotides, and 1 unit of AmpliTaq gold polymerase (Applied Biosystems, Foster City, CA) were used. Oligonucleotides for amplification of the Artemis and Ig heavy chain (IGH) gene rearrangements (VH-JH and DH-JH) were published before.10,16,17 PCR conditions were 7 minutes at 95°C, followed by 45 seconds at 94°C, 90 seconds at 55°C to 60°C, 2 minutes at 72°C for 40 cycles, followed by a final extension step (7 minutes at 72°C). DNA from BMMCs was analyzed for rearrangements of DH1, DH2, DH3, DH4, DH5, DH6, DH7, VH1/7, VH2, VH3, VH4, VH5, and VH6 to JH.13Fluorescent sequencing reaction and analysis PCR products of Artemis were purified using QIAquick PCR purification kit (Qiagen). Cloned IGH gene rearrangements were isolated via GenElute Plasmid MiniPrep Kit (Sigma-Aldrich). A 2 to 9 µL template was sequenced with 5 µL BigDye terminator mix (Applied Biosystems) using 3.3 to 6.6 pmol sequencing primers. All sequencing was performed as described before16 and run on an ABI Prism 377 fluorescent sequencer (Applied Biosystems).Western blot analysis of proteins involved in DNA dsb repair Protein samples from fibroblasts were separated on an 11% polyacrylamide gel and analyzed by Western blotting. The expression of Mre11 (rabbit polyclonal antibody [Ab] no. 2244),18 XRCC4 (rabbit polyclonal Ab NIH14), Ku70 (goat polyclonal Ab C19, Santa Cruz Biotechnology, Santa Cruz, CA) and Ku80 (goat polyclonal Ab C20, Santa Cruz Biotechnology), DNA-PKcs (rabbit polyclonal Ab no. 2129), ligase IV (goat polyclonal Ab T20, Santa Cruz Biotechnology), ATM (gift from Dr S. Jackson, Wellcome Trust and Cancer Research, Cambridge, United Kingdom), and NBS1 (p95NBS1, Calbiochem, San Diego, CA) proteins was analyzed.Analysis of NHEJ via transfection of linearized DNA constructs Linearized DNA constructs with homologous ends (ATCAGC sequence) were transfected into fibroblasts of RS-SCID patients and fibroblasts of a patient with a mutation in DNA ligase IV (180BR) as described before.19 Newly formed junctions were PCR amplified, and the relative use of a particular microhomology was assayed by digestion with the restriction enzyme BstXI (Figure 2).19Extrachromosomal VDJ recombination assay For analysis of signal and coding joint formation in the different RS-SCID fibroblasts with Artemis mutations, 2 µg RAG1 and 2 µg RAG2 expression vectors together with 1 µg of the recombination substrate pGG49 or pGG51,20 each containing 2 recombination signal sequence (RSS) elements, were transfected into fibroblasts using Fugene Transfection Reagent (Roche Diagnostics, Indianapolis, IN) as described before.13 Transfected cells were cultured for 2 days at 37°C and 5% CO2 before isolation of extrachromosomal DNA. Upon V(D)J recombination, the sequence between the RSS elements is deleted, after which coding (pGG51) or signal joint (pGG49) formation can be detected by PCR analysis of the newly formed junctions (Figure 3). DNA recovered from these transfection experiments was resuspended in 20 µL H2O, of which 1 µL was used for PCR analysis. First, DNA was predigested with ClaI to reduce the signal derived from nonrecombined V(D)J substrate. To amplify the coding or signal joints, a nested PCR of 2 × 25 cycles was performed. The first round of PCR was performed with oligonucleotides NV09F and DG147 (Table 1) in a total volume of 50 µL. Subsequently, 1 µL of this PCR reaction was used as a template for the second round of PCR using oligonucleotides NV08F and FM30, of which FM30 was radioactively labeled with 32P-adenosine
triphosphate (ATP). The PCR conditions were the same as
described before.13 PCR fragments were separated on a 6% polyacrylamide gel, and products were visualized by
phosphorimaging.13
Artemis complementation studies To determine whether the recombination defect could be complemented, either wild-type (wt) Artemis or a mutant Artemis expression construct was cotransfected with the RAG1 and RAG2 expression vectors and the coding joint recombination substrate pGG51 into fibroblasts of the RS-SCID patients with Artemis gene mutations. The wt Artemis expression construct was made by performing a PCR with the DG238 and DG239 oligonucleotides (Table 1) on Hela cDNA. This 2.1-kb PCR product was digested with XhoI and MluI and cloned into the XhoI and MluI sites of plasmid pMS127B.21 The point mutations of patients Artemis-2 and Artemis-3.1 were cloned by performing this same PCR on cDNA generated from the fibroblasts of these patients. This 2.1-kb fragment was digested with NsiI and EcoRV, after which the 500-bp fragment containing the point mutations was exchanged for the NsiI-EcoRV part of the wt construct.Flow cytometric analysis of BM samples from RS-SCID patients with Artemis gene mutations Fifty microliter aliquots of thawed BMMCs (10 × 106 cells per milliliter) were incubated for 10 minutes at room temperature with combinations of optimally titrated monoclonal antibody (MAb): 50 µL fluorescein isothiocyanate (FITC)-conjugated MAb, 50 µL phycoerythrin (PE)-conjugated MAb, 50 µL peridinin chlorophyll protein (PerCP) cyanin (CY) 5.5-conjugated MAb, and 50 µL allophycocyanin (APC)-conjugated MAb were used to detect membrane-bound antigens. After incubation, the cells were washed and further processed depending on the type of quadruple labeling.22,23Quadruple labelings for membrane-bound antigens were directly analyzed by flow cytometry using FACSCalibur (Becton Dickinson, San Jose, CA). For quadruple labelings involving intracellular staining of cytoplasmic (Cy) CD79a, CyIgµ, CyVpreB,24 and intranuclear staining of terminal deoxynucleotidyl transferase (TdT), we first performed the membrane labelings, followed by permeabilization of the BM cells using IntraPrep Permeabilization Reagent (Immunotech, Marseille, France) and subsequent intracellular staining.25,26
Patient characteristics, Western blot analysis, and disease-causing Artemis gene mutations We analyzed material from 32 T BNK+ SCID patients for the
presence of RAG gene mutations, both at the genomic and at
the transcriptional level. We could not detect mutations in the
RAG genes in 23 patients (72%). From 13 TBNK+ SCID patients without
RAG mutations, fibroblasts were available and analyzed for
sensitivity to ionizing radiation in a clonogenic survival assay.
Fibroblasts from 5 T
Patient Artemis-1 showed a homozygous deletion of exons 10, 11, and 12 of the Artemis gene. At the mRNA level, exon 9 was coupled to exon 13, resulting in a frameshift and premature stop at codon 269 in the SNM1 homology domain (numbering according to Moshous et al).10 Patient Artemis-2 showed a homozygous G>T mutation at position 47 in exon 5 of the Artemis gene. This resulted in mutation of a glycine to valine residue at position 111 in the SNM1 homology domain of the Artemis protein. We did not identify this point mutation in 18 healthy controls. Furthermore, glycine at position 111 is highly conserved between yeast PSO2, mouse SNM1, human SNM1A, and human SNM1B proteins and therefore is likely to represent an essential amino acid (aa).27 Patients Artemis-3.1 and -3.2 showed a homozygous G>A mutation at
position 42 in exon 6 of the Artemis gene. This resulted in
mutation of a glycine to glutamic acid residue at position 128 in the
SNM1 homology domain of the Artemis protein. We did not identify this
point mutation in 14 healthy controls. Furthermore, glycine at position
128 is highly conserved between yeast PSO2, mouse SNM1, human SNM1A,
and human SNM1B proteins and therefore likely to represent an essential
aa.27 Because the parents of patients Artemis-3.1 and -3.2 had a nonconsanguineous relationship, we investigated whether the point
mutation was really homozygous. We excluded a large deletion
(containing exon 6) of one allele by Southern blot analysis of the
Artemis gene using a PCR-based exon 6 probe (data not
shown). We did not find a lower level of hybridization (normalized to
Ig In addition to the full-length transcripts, one of the Artemis-3 patients showed alternatively spliced transcripts in which exon 4 was spliced to exon 6. Sequencing analysis of the alternatively spliced transcripts showed the presence of the disease-causing G>A mutation in exon 6, fully consistent with the homozygous mutation. Such alternatively spliced Artemis transcripts were not found in patient Artemis-2. No mutations were present in the donor and acceptor splice sites of any Artemis exon in the 4 studied patients. Additional nondisease-causing mutations in the Artemis gene The entire Artemis gene was sequenced in all 4 RS-SCID patients with Artemis mutations to exclude the presence of additional mutations. We identified a total of 4 additional alterations compared with the published sequence. Two base changes were present in all 4 RS-SCID patients analyzed and were therefore likely to represent polymorphisms or sequencing errors in the published sequence. The first alteration concerned a G>T change at position 522 in exon 14 of the Artemis gene. This alteration resulted in a change from a valine to leucine residue at position 553 outside the SNM1 homology domain of the Artemis protein. The second alteration concerned a C>T change at position 653 in exon 14 of the Artemis gene, which did not result in an aa substitution.We identified another silent alteration at position 106 in exon 8 of the Artemis gene (a T>C change) in patients Artemis-3.1 and -3.2. Finally, patient Artemis-1 with the large deletion carried a homozygous A>G change at position 50 in exon 9 of the Artemis gene. This alteration resulted in a change from the basic aa histidine to the basic aa arginine at position 236 in the SNM1 homology domain. We did not identify this point mutation in 12 healthy controls. Dsb repair Mutation of Ku80, DNA-PKcs, XRCC4, or DNA ligase IV leads to a shift from precise joining to microhomology-directed joining in mammalian cells.19 Therefore, we transfected linearized DNA constructs into fibroblasts of all 4 patients with Artemis gene mutations to analyze the use of direct joining and microhomology pathways. The Artemis-mutated cells showed equal usage of the direct joining and microhomology pathways (Figure 2), suggesting that Artemis is not involved in the ligation process itself.
V(D)J recombination also involves dsb repair by NHEJ. We analyzed
coding and signal joint formation in fibroblasts of all 4 patients with
Artemis gene mutations. In all cases we found normal levels
of precise signal joint formation but absence of coding joint formation
(Figure 3).
Complementation of the coding joint formation defect by cotransfection of wt Artemis constructs The genomic deletion in patient Artemis-1, resulting in a premature stop codon, was most probably the disease-causing defect. However, we considered the possibility that the missense mutations in patients Artemis-2, -3.1, and -3.2 could represent polymorphisms. Therefore, we cotransfected the wt Artemis expression vector together with the RAG1 and RAG2 expression vectors and the recombination substrates into fibroblasts of the RS-SCID patients and analyzed the signal and coding joint formation. As shown in Figure 3, this resulted in complementation of the coding joint formation defect, thereby proving the disease-causing effect of the missense mutations in the Artemis gene. Furthermore, transfection of an Artemis expression vector containing the mutation of Artemis-3 could not complement coding joint formation in Artemis-2 fibroblasts and vice versa (data not shown).Ig gene rearrangements in BMMCs To study the capacity of mutated Artemis proteins to perform DNA dsb repair during human precursor B-cell differentiation in vivo, we isolated DNA from BMMCs and investigated the occurrence of incomplete DH-JH and complete VH-JH gene rearrangements. BM samples were available from patients Artemis-1 and Artemis-2. In both patients, incomplete DH-JH gene rearrangements could be amplified by PCR, but no complete VH-JH gene rearrangements could be detected.Flow cytometric analysis of precursor B cells in BM of RS-SCID patients with Artemis gene mutations BM samples from 2 RS-SCID patients with Artemis gene mutations (Artemis-1 and -2) were available and analyzed as described before.22,23 The percentage of B cells within the lymphocyte gate differed between the 2 RS-SCID patients with Artemis gene mutations (Table 3). In BM from healthy children, approximately 15% of the precursor B-cell compartment consisted of CyIgµ precursor B cells, distributed over pro-B cells
and pre-B-I cells with a pro-B/pre-B-I ratio of 1.3 ± 0.8, whereas
in BM from both RS-SCID patients with Artemis gene
mutations, 100% of the precursor B-cell compartment was located in the
pro-B and pre-B-I cell stages (Figures 4
and 5). Therefore, our
results indicate that the differentiation arrest in the 2 RS-SCID
patients with Artemis gene mutations resulted in a more than
6-fold relative accumulation of precursor B-cell subpopulations located
before the transition from CyIgµ pre-B-I cells to
CyIgµ+ pre-B-II cells, with an inverted pro-B/pre-B-I
ratio of 0.6 ± 0.7 in the 2 RS-SCID patients with Artemis
gene mutations.
From 13 T Increased microhomology use in a plasmid recircularization assay proved to be highly diagnostic of mutations in the DNA-PK or ligase IV-XRCC4 complex.19 However, our 4 RS-SCID patients with mutations in the Artemis gene showed normal direct joining (Figure 2). This suggests that the Artemis protein functions in a different step of the end joining process. These results are consistent with the recent biochemical evidence that Artemis may be required for hairpin opening and DNA end processing.11 We previously described detection of IGH gene rearrangements in human BMMCs as a parameter for in vivo RAG activity.13 Now we have used detection of IGH gene rearrangements in BMMCs of Artemis-1 and Artemis-2 as a parameter for residual activity of the Artemis protein. The complete absence of normal coding joints in patient Artemis-1 is in line with the large genomic deletion, which results in a truncated Artemis protein with absence of part of the SNM1 homology domain. Patient Artemis-2 contained relatively high frequencies of precursor B cells (23% of BM lymphocytes, Table 3), but we could detect only incomplete DH-JH rearrangements and no VH-JH gene rearrangements. Flow cytometric evaluation of the BM precursor B-cell compartment in
patients Artemis-1 and -2 showed a complete arrest at the transition
from CyIgµ In conclusion, both deletions and missense mutations in the Artemis gene can cause RS-SCID with defective coding joint formation and lead to an early and complete B-cell differentiation block.
The authors thank Penny Jeggo for 180BR, Jean-Pierre de Villartay for communication of results before publication, Michael Lieber for the pGG49 and pGG51 constructs, Mauro Modesti for XRCC4 antisera, Steve Jackson for anti-ATM antiserum, R. E. E. van Lange for technical assistance, and H. Karasuyama for making available the MAb HSL96 directed against the human VpreB protein.
Submitted June 6, 2002; accepted September 22, 2002.
Prepublished online as Blood First Edition Paper, October 24, 2002; DOI 10.1182/blood-2002-01-0187.
Supported by the Revolving Fund 2000 of the University Hospital Rotterdam, the Dutch Cancer Society/Koningin Wilhelmina Fonds (grants EUR 98-1775 and EMCR 2002-2734), and the Netherlands Scientific Organization (NWO grant AGIKO 920-03-089).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: J. J. M. van Dongen, Department of Immunology, Erasmus MC/University Medical Center Rotterdam, Dr Molewaterplein 50, 3015 GE Rotterdam, The Netherlands; e-mail: vandongen{at}immu.fgg.eur.nl.
1.
Buckley RH.
Primary immunodeficiency diseases due to defects in lymphocytes.
N Engl J Med.
2000;343:1313-1324
2.
Cavazzana-Calvo M, Hacein-Bey S, de Saint Basile G, et al.
Gene therapy of human severe combined immunodeficiency (SCID)-X1 disease.
Science.
2000;288:669-672
3.
Schwarz K, Gauss GH, Ludwig L, et al.
RAG mutations in human B cell-negative SCID.
Science.
1996;274:97-99
4.
Nicolas N, Moshous D, Cavazzana-Calvo M, et al.
A human severe combined immunodeficiency (SCID) condition with increased sensitivity to ionizing radiations and impaired V(D)J rearrangements defines a new DNA recombination/repair deficiency.
J Exp Med.
1998;188:627-634 5. Van Gent DC, Hoeijmakers JH, Kanaar R. Chromosomal stability and the DNA double-stranded break connection. Nat Rev Genet. 2001;2:196-206[CrossRef][Medline] [Order article via Infotrieve].
6.
Smith GC, Jackson SP.
The DNA-dependent protein kinase.
Genes Dev.
1999;13:916-934 7. Critchlow SE, Bowater RP, Jackson SP. Mammalian DNA double-strand break repair protein XRCC4 interacts with DNA ligase IV. Curr Biol. 1997;7:588-598[CrossRef][Medline] [Order article via Infotrieve]. 8. Grawunder U, Wilm M, Wu X, et al. Activity of DNA ligase IV stimulated by complex formation with XRCC4 protein in mammalian cells. Nature. 1997;388:492-495[CrossRef][Medline] [Order article via Infotrieve].
9.
Moshous D, Li L, Chasseval R, et al.
A new gene involved in DNA double-strand break repair and V(D)J recombination is located on human chromosome 10p.
Hum Mol Genet.
2000;9:583-588 10. Moshous D, Callebaut I, de Chasseval R, et al. Artemis, a novel DNA double-strand break repair/V(D)J recombination protein, is mutated in human severe combined immune deficiency. Cell. 2001;105:177-186[CrossRef][Medline] [Order article via Infotrieve]. 11. Ma Y, Pannicke U, Schwarz K, Lieber MR. Hairpin opening and overhang processing by an Artemis/DNA-dependent protein kinase complex in nonhomologous end joining and V(D)J recombination. Cell. 2002;108:781-794[CrossRef][Medline] [Order article via Infotrieve].
12.
Li L, Moshous D, Zhou Y, et al.
A founder mutation in Artemis, an SNM1-like protein, causes SCID in Athabascan-speaking Native Americans.
J Immunol.
2002;168:6323-6329
13.
Noordzij JG, Verkaik NS, Hartwig NG, De Groot R, Van Gent DC, Van Dongen JJM.
N-terminal truncated human RAG1 proteins can direct T-cell receptor but not immunoglobulin gene rearrangements.
Blood.
2000;96:203-209 14. Verhagen OJHM, Wijkhuis AJM, Van der Sluijs-Gelling AJ, et al. Suitable DNA isolation method for the detection of minimal residual disease by PCR techniques. Leukemia. 1999;13:1298-1299[CrossRef][Medline] [Order article via Infotrieve]. 15. Van Dongen JJM, Macintyre EA, Gabert JA, et al. Standardized RT-PCR analysis of fusion gene transcripts from chromosome aberrations in acute leukemia for detection of minimal residual disease. Report of the BIOMED-1 Concerted Action: investigation of minimal residual disease in acute leukemia. Leukemia. 1999;13:1901-1928[CrossRef][Medline] [Order article via Infotrieve].
16.
Szczepanski T, Pongers-Willemse MJ, Langerak AW, et al.
Ig heavy chain gene rearrangements in T-cell acute lymphoblastic leukemia exhibit predominant DH6-19 and DH7-27 gene usage, can result in complete V-D-J rearrangements, and are rare in T-cell receptor 17. Pongers-Willemse MJ, Seriu T, Stolz F, et al. Primers and protocols for standardized detection of minimal residual disease in acute lymphoblastic leukemia using immunoglobulin and T cell receptor gene rearrangements and TAL1 deletions as PCR targets. Report of the BIOMED-1 Concerted Action: investigation of minimal residual disease in acute leukemia. Leukemia. 1999;13:110-118[CrossRef][Medline] [Order article via Infotrieve].
18.
de Jager M, Dronkert ML, Modesti M, Beerens CE, Kanaar R, van Gent DC.
DNA-binding and strand-annealing activities of human Mre11: implications for its roles in DNA double-strand break repair pathways.
Nucleic Acids Res.
2001;29:1317-1325 19. Verkaik NS, Esveldt-van Lange RE, van Heemst D, et al. Different types of V(D)J recombination and end-joining defects in DNA double-strand break repair mutant mammalian cells. Eur J Immunol. 2002;32:701-709[CrossRef][Medline] [Order article via Infotrieve]. 20. Gauss GH, Lieber MR. Mechanistic constraints on diversity in human V(D)J recombination. Mol Cell Biol. 1996;16:258-269[Abstract].
21.
Sadofsky MJ, Hesse JE, McBlane JF, Gellert M.
Expression and V(D)J recombination activity of mutated RAG-1 proteins.
Nucleic Acids Res.
1993;21:5644-5650
22.
Noordzij JG, De Bruin-Versteeg S, Verkaik NS, et al.
The immunophenotypic and immunogenotypic B-cell differentiation arrest in bone marrow of RAG deficient SCID patients corresponds to residual recombination activities of mutated RAG proteins.
Blood.
2002;100:2145-2152 23. Noordzij JG, De Bruin-Versteeg S, Comans-Bitter WM, et al. Composition of the precursor B-cell compartment in bone marrow from patients with X-linked agammaglobulinemia compared to healthy children. Pediatr Res. 2002;51:159-168[Medline] [Order article via Infotrieve].
24.
Tsuganezawa K, Kiyokawa N, Matsuo Y, et al.
Flow cytometric diagnosis of the cell lineage and developmental stage of acute lymphoblastic leukemia by novel monoclonal antibodies specific to human pre-B-cell receptor.
Blood.
1998;92:4317-4324 25. Groeneveld K, Te Marvelde JG, Van den Beemd MW, Hooijkaas H, Van Dongen JJM. Flow cytometric detection of intracellular antigens for immunophenotyping of normal and malignant leukocytes. Leukemia. 1996;10:1383-1389[Medline] [Order article via Infotrieve]. 26. Van Lochem EG, Groeneveld K, Te Marvelde JG, Van den Beemd MW, Hooijkaas H, Van Dongen JJM. Flow cytometric detection of intracellular antigens for immunophenotyping of normal and malignant leukocytes: testing of a new fixation-permeabilization solution. Leukemia. 1997;11:2208-2210[CrossRef][Medline] [Order article via Infotrieve].
27.
Dronkert ML, de Wit J, Boeve M, et al.
Disruption of mouse SNM1 causes increased sensitivity to the DNA interstrand cross-linking agent mitomycin C.
Mol Cell Biol.
2000;20:4553-4561
© 2003 by The American Society of Hematology.
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  L. Du, M. van der Burg, S. W. Popov, A. Kotnis, J. J.M. van Dongen, A. R. Gennery, and Q. Pan-Hammarstrom Involvement of Artemis in nonhomologous end-joining during immunoglobulin class switch recombination J. Exp. Med., December 22, 2008; 205(13): 3031 - 3040. [Abstract] [Full Text] [PDF]
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 J. Hejna, S. Philip, J. Ott, C. Faulkner, and R. Moses The hSNM1 protein is a DNA 5'-exonuclease Nucleic Acids Res., September 25, 2007; 35(18): 6115 - 6123. [Abstract] [Full Text] [PDF]
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 |
|
 V. J. McAlister and R. A. Owens Preferential Integration of Adeno-Associated Virus Type 2 into a Polypyrimidine/Polypurine-Rich Region within AAVS1 J. Virol., September 15, 2007; 81(18): 9718 - 9726. [Abstract] [Full Text] [PDF]
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 |
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 D. Niewolik, U. Pannicke, H. Lu, Y. Ma, L.-C. V. Wang, P. Kulesza, E. Zandi, M. R. Lieber, and K. Schwarz DNA-PKcs Dependence of Artemis Endonucleolytic Activity, Differences between Hairpins and 5' or 3' Overhangs J. Biol. Chem., November 10, 2006; 281(45): 33900 - 33909. [Abstract] [Full Text] [PDF]
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|
 A. Liu, C. A. J. Vosshenrich, C. Lagresle-Peyrou, M. Malassis-Seris, C. Hue, A. Fischer, J. P. Di Santo, and M. Cavazzana-Calvo Competition within the early B-cell compartment conditions B-cell reconstitution after hematopoietic stem cell transplantation in nonirradiated recipients Blood, August 15, 2006; 108(4): 1123 - 1128. [Abstract] [Full Text] [PDF]
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M. Ege, Y. Ma, B. Manfras, K. Kalwak, H. Lu, M. R. Lieber, K. Schwarz, and U. Pannicke Omenn syndrome due to ARTEMIS mutations Blood, June 1, 2005; 105(11): 4179 - 4186. [Abstract] [Full Text] [PDF]
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 S. Rooney, J. Sekiguchi, S. Whitlow, M. Eckersdorff, J. P. Manis, C. Lee, D. O. Ferguson, and F. W. Alt Artemis and p53 cooperate to suppress oncogenic N-myc amplification in progenitor B cells PNAS, February 24, 2004; 101(8): 2410 - 2415. [Abstract] [Full Text] [PDF]
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K. N. Mahajan and B. S. Mitchell Role of human Pso4 in mammalian DNA repair and association with terminal deoxynucleotidyl transferase PNAS, September 16, 2003; 100(19): 10746 - 10751. [Abstract] [Full Text] [PDF]
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J. Mansilla-Soto and P. Cortes VDJ Recombination: Artemis and Its In Vivo Role in Hairpin Opening J. Exp. Med., March 3, 2003; 197(5): 543 - 547. [Full Text] [PDF]
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Top
Abstract
Introduction
that is, at the transition from CyIgµ
J5) in the patients relative to a healthy control, indicating
that these patients are homozygous for the point mutation.
lineage.
Blood.
1999;93:4079-4085