Richard A. Gatti

NIBRIN, A NOVEL DNA DOUBLE-STRAND BREAK REPAIR PROTEIN, IS MUTATED IN NIJMEGEN BREAKAGE SYNDROME

Nijmegen breakage syndrome (NBS) is an autosomal recessive chromosomal instability syndrome characterized by microcephaly, growth retardation, immunodeficiency, and cancer predisposition. Cells from NBS patients are hypersensitive to ionizing radiation with cytogenetic features indistinguishable from ataxia telangiectasia. We describe the positional cloning of a gene encoding a novel protein, nibrin. It contains two modules found in cell cycle checkpoint proteins, a forkhead-associated domain adjacent to a breast cancer carboxy-terminal domain. A truncating 5 bp deletion was identified in the majority of NBS patients, carrying a conserved marker haplotype. Five further truncating mutations were identified in patients with other distinct haplotypes. The domains found in nibrin and the NBS phenotype suggest that this disorder is caused by defective responses to DNA double-strand breaks.

Occurrence of malignancy in immunodeficiency diseases: A literature review†

The incidence of malignancy in patients with primary immunodeficiencies is roughly 10,000 times that of the general age‐matched population. It is apparent from this review of the literature that each type of immunodeficiency has a distinctive constellation of malignancies associated with it. In light of recent studies demonstrating both immunologically aggressive lymphocytes and the presence of blocking antibodies in the blood of neuroblastoma patients, a major role for immunity in ontogenesis seems almost certain. The role of such antibodies in the formation of lymphoid malignancies, such as occur so frequently in patients with immunologic deficiencies who often do not produce antibodies of any type well, remains unclear.

ATM-dependent phosphorylation of nibrin in response to radiation exposure

Mutations in the gene ATM are responsible for the genetic disorder ataxia-telangiectasia (A-T), which is characterized by cerebellar dysfunction, radiosensitivity, chromosomal instability and cancer predisposition. Both the A-T phenotype and the similarity of the ATM protein to other DNA-damage sensors suggests a role for ATM in biochemical pathways involved in the recognition, signalling and repair of DNA double-strand breaks (DSBs). There are strong parallels between the pattern of radiosensitivity, chromosomal instability and cancer predisposition in A-T patients and that in patients with Nijmegen breakage syndrome (NBS). The protein defective in NBS, nibrin (encoded by NBS1), forms a complex with MRE11 and RAD50 (refs 1,2). This complex localizes to DSBs within 30 minutes after cellular exposure to ionizing radiation (IR) and is observed in brightly staining nuclear foci after a longer period of time. The overlap between clinical and cellular phenotypes in A-T and NBS suggests that ATM and nibrin may function in the same biochemical pathway. Here we demonstrate that nibrin is phosphorylated within one hour of treatment of cells with IR. This response is abrogated in A-T cells that either do not express ATM protein or express near full-length mutant protein. We also show that ATM physically interacts with and phosphorylates nibrin on serine 343 both in vivo and in vitro. Phosphorylation of this site appears to be functionally important because mutated nibrin (S343A) does not completely complement radiosensitivity in NBS cells. ATM phosphorylation of nibrin does not affect nibrin-MRE11-RAD50 association as revealed by radiation-induced foci formation. Our data provide a biochemical explanation for the similarity in phenotype between A-T and NBS.

Functional link between ataxia-telangiectasia and Nijmegen breakage syndrome gene products.

Ataxia-telangiectasia (A-T) and Nijmegen breakage syndrome (NBS) are recessive genetic disorders with susceptibility to cancer and similar cellular phenotypes. The protein product of the gene responsible for A-T, designated ATM, is a member of a family of kinases characterized by a carboxy-terminal phosphatidylinositol 3-kinase-like domain. The NBS1 protein is specifically mutated in patients with Nijmegen breakage syndrome and forms a complex with the DNA repair proteins Rad50 and Mre11. Here we show that phosphorylation of NBS1, induced by ionizing radiation, requires catalytically active ATM. Complexes containing ATM and NBS1 exist in vivo in both untreated cells and cells treated with ionizing radiation. We have identified two residues of NBS1, Ser 278 and Ser 343 that are phosphorylated in vitro by ATM and whose modification in vivo is essential for the cellular response to DNA damage. This response includes S-phase checkpoint activation, formation of the NBS1/Mre11/Rad50 nuclear foci and rescue of hypersensitivity to ionizing radiation. Together, these results demonstrate a biochemical link between cell-cycle checkpoints activated by DNA damage and DNA repair in two genetic diseases with overlapping phenotypes.

DNA Ligase IV Mutations Identified in Patients Exhibiting Developmental Delay and Immunodeficiency

DNA ligase IV functions in DNA nonhomologous end-joining and V(D)J recombination. Four patients with features including immunodeficiency and developmental and growth delay were found to have mutations in the gene encoding DNA ligase IV (LIG4). Their clinical phenotype closely resembles the DNA damage response disorder, Nijmegen breakage syndrome (NBS). Some of the mutations identified in the patients directly disrupt the ligase domain while others impair the interaction between DNA ligase IV and Xrcc-4. Cell lines from the patients show pronounced radiosensitivity. Unlike NBS cell lines, they show normal cell cycle checkpoint responses but impaired DNA double-strand break rejoining. An unexpected V(D)J recombination phenotype is observed involving a small decrease in rejoining frequency coupled with elevated imprecision at signal junctions.

Splicing Defects in the Ataxia-Telangiectasia Gene, ATM: Underlying Mutations and Consequences

Mutations resulting in defective splicing constitute a significant proportion (30/62 [48%]) of a new series of mutations in the ATM gene in patients with ataxia-telangiectasia (AT) that were detected by the protein-truncation assay followed by sequence analysis of genomic DNA. Fewer than half of the splicing mutations involved the canonical AG splice-acceptor site or GT splice-donor site. A higher percentage of mutations occurred at less stringently conserved sites, including silent mutations of the last nucleotide of exons, mutations in nucleotides other than the conserved AG and GT in the consensus splice sites, and creation of splice-acceptor or splice-donor sites in either introns or exons. These splicing mutations led to a variety of consequences, including exon skipping and, to a lesser degree, intron retention, activation of cryptic splice sites, or creation of new splice sites. In addition, 5 of 12 nonsense mutations and 1 missense mutation were associated with deletion in the cDNA of the exons in which the mutations occurred. No ATM protein was detected by western blotting in any AT cell line in which splicing mutations were identified. Several cases of exon skipping in both normal controls and patients for whom no underlying defect could be found in genomic DNA were also observed, suggesting caution in the interpretation of exon deletions observed in ATM cDNA when there is no accompanying identification of genomic mutations.

ATAXIA-TELANGIECTASIA: AN INTERDISCIPLINARY APPROACH TO PATHOGENESIS

Ataxia-telangiectasia is a syndrome with many facets, involving a progressive cerebellar ataxia, immunodeficiency, cancer susceptibility, radiosensitivity, defects in DNA repair/processing, chromosomal breakage and rearrangements, elevated serum alphafetoprotein, and premature aging. Ataxia-telangiectasia is an autosomal recessive disorder, rare in outbred populations; carriers of the ataxia-telangiectasia gene may be as common as 1 in 60 and have subclinical radiosensitivity and cancer susceptibility. One estimate suggests that 8.8% of patients with breast cancer could be carriers of ataxia-telangiectasia. These carriers may be responsible for underestimating normal tolerance doses for radiation therapy by 15% to 20%; thus by preselecting and excluding carriers of ataxia-telangiectasia from cohorts of patients with cancer, conventional radiation doses might be increased so as to improve greatly the efficacy of radiotherapy. The genes for the 3 most common ataxia-telangiectasia complementation groups, which include 97% of tested families, have recently been localized to the long arm of chromosome 11.

ATM is down-regulated by N-Myc–regulated microRNA-421

Ataxia-telangiectasia mutated (ATM) is a high molecular weight protein serine/threonine kinase that plays a central role in the maintenance of genomic integrity by activating cell cycle checkpoints and promoting repair of DNA double-strand breaks. Little is known about the regulatory mechanisms for ATM expression itself. MicroRNAs are naturally existing regulators that modulate gene expression in a sequence-specific manner. Here, we show that a human microRNA, miR-421, suppresses ATM expression by targeting the 3′-untranslated region (3′UTR) of ATM transcripts. Ectopic expression of miR-421 resulted in S-phase cell cycle checkpoint changes and an increased sensitivity to ionizing radiation, creating a cellular phenotype similar to that of cells derived from ataxia-telangiectasia (A-T) patients. Blocking the interaction between miR-421 and ATM 3′UTR with an antisense morpholino oligonucleotide rescued the defective phenotype caused by miR-421 overexpression, indicating that ATM mediates the effect of miR-421 on cell cycle checkpoint and radiosensitivity. Overexpression of the N-Myc transcription factor, an oncogene frequently amplified in neuroblastoma, induced miR-421 expression, which, in turn, down-regulated ATM expression, establishing a linear signaling pathway that may contribute to N-Myc-induced tumorigenesis in neuroblastoma. Taken together, our findings implicate a previously undescribed regulatory mechanism for ATM expression and ATM-dependent DNA damage response and provide several potential targets for treating neuroblastoma and perhaps A-T.

Cellular localisation of the ataxia-telangiectasia (ATM) gene product and discrimination between mutated and normal forms.

The recently cloned gene (ATM) mutated in the human genetic disorder ataxia-telangiectasia (A-T) is involved in DNA damage response at different cell cycle checkpoints and also appears to have a wider role in signal transduction. Antibodies prepared against peptides from the predicted protein sequence detected a ∼ 350 kDa protein corresponding to the open reading frame, which was absent in 13/23 A-T homozygotes. Subcellular fractionation, immunoelectronmicroscopy and immunofluorescence showed that the ATM protein is present in the nucleus and cytoplasmic vesicles. This distribution did not change after irradiation. We also provide evidence that ATM protein binds to p53 and this association is defective in A-T cells compatible with the defective p53 response in these cells. These results provide further support for a role for the ATM protein as a sensor of DNA damage and in a more general role in cell signalling, compatible with the broader phenotype of the syndrome.

The Wiskott-Aldrich Syndrome: RESULTS OF TRANSFER FACTOR THERAPY

12 patients with Wiskott-Aldrich syndrome were treated with therapeutic doses of transfer factor in an attempt to induce cellular immunity. Clinical improvement was noted after transfer factor therapy in 7 of the 12 patients treated. Because this disease has a variable course and temporary spontaneous improvement can occur, the observed improvement cannot necessarily be attributed to the transfer factor. However, in two patients repeated remissions consistently followed transfer factor administration on repeated occasions. This included freedom from infections, regression of splenomegaly, and clearing of eczema. An unexpected finding was a decrease in bleeding in 3 of the 10 patients who had bleeding. Conversion of skin reactivity was obtained in all seven patients who clinically seemed to respond to transfer factor. In vitro studies performed after the administration of transfer factor demonstrated that the lymphocytes of the patients now produced migration inhibitory factor in response to appropriate test antigens, but did not undergo increased radioactive thymidine incorporation in response to the same antigens. A defect in the monocyte IgG receptors has been found in certain patients with the disease, and the current study shows that all patients with defective monocyte IgG receptors responded to transfer factor, whereas only one patient with normal receptors showed any response. This test may thus prove to be useful in predicting the results of transfer factor therapy in patients with Wiskott-Aldrich syndrome, although evaluation of a larger series of patients will be necessary to confirm this point. We conclude that cellular immunity can be induced, that there appears to be clinical benefit in certain patients with Wiskott-Aldrich syndrome by the use of transfer factor, and that this mode of therapy warrents trial in these patients and others with defects of cellular immunity.