Cytogenetic investigations in a family with ataxia telangiectasia

Summary Cytogenetic findings on a family with ataxia telangiectasia (A-T) in which three of four sibs were affected are described. The affected individuals had approximately twice the level of spontaneous chromosome breakage of a normla control, while the parents and the normal sib had no significant increase. Lymphocytes from all three A-T homozygotes showed specific stable chromosomal rearrangements involving chromosomes 7 and 14. All of these abnormalities involved breakage at the usual four sites associated with A-T (7p14, 7q35, 14q12, and 14q32). Two rearrangements detected in the eldest and most severely affected patient were clones, one of which [t(14;14)(p11;q12)] is not commonly found in A-T cells. No chromosomal rearrangements were encountered in lymphocytes from the control, the parents, or the normal sib. Lymphocytes from the A-T patients also were found to be 7–11 times more sensitive to the induction of chromatid aberrations by X-irradiation than control cells. Lymphocytes from the parents and normal sib showed a moderately increased frequency of X-ray induced aberrations compared with that of the control.     

A double missense mutation in the ATM gene of a Dutch family with ataxia telangiectasia

Ataxia telangiectasia (AT) is an autosomal recessive disorder characterized by cerebellar ataxia, telangiectasia, immunodeficiency, elevated α-fetoprotein levels, chromosomal instability, predisposition to cancer, and radiation sensitivity. We report the identification of a new, double missense mutation in the ataxia telangiectasia gene (ATM) of a Dutch family. This homozygous mutation consists of two consecutive base substitutions in exon 55: a T→G transversion at position 7875 of the ATM cDNA and a G→C transversion at position 7876. These transversions were confirmed by polymerase chain reaction/primer-induced restriction analysis with CelII. The double base substitution results in an amino acid change of an aspartic acid to a glutamic acid at codon 2625 and of an alanine to a proline at codon 2626 of the ATM protein. Both amino acids are conserved between the ATM protein and its functional homolog, the Atm gene product in the mouse. Furthermore, the Chou-Fasman and Robson predictions both demonstrate a change in the secondary structure of the ATM protein carrying the D2625E/A2626P mutation. These findings suggest that the double base substitution in the ATM gene is a disease-causing mutation. Received: 6 October 1997 / Accepted: 5 November 1997     

Molecular characterization of ataxia telangiectasia T cell clones

Summary To delimit the 14q32.1 recurrent breakpoint of ataxia telangiectasia clones, we performed an in situ hybridization study with various probes located on the 14q32 band. We thus mapped this breakpoint between the D14S1 and Pi loci. Furthermore, an interstitial duplication including D14S1 and a part of the IgH locus was demonstrated on a t(14;14) clone.     

Molecular characterization of ataxia telangiectasia T cell clones

Summary We compared inversions of chromosome 14 in an ataxia telangiectasia clone and in a malignant T cell line (SUPT1). The R-banding chromosome analysis showed a clear difference between the distal breakpoint of the two inversions. Fine mapping of the distal breakpoint in the ataxia telangiectasia inv(14) was performed by in situ hybridization. We conclude that this breakpoint is centromeric to the immunoglobulin heavy chain locus and to the D14S1 anonymous locus. Our results favor the existence of an unknown oncogene in band 14q32.1.     

Oxidative stress in ataxia telangiectasia

Ataxia telangiectasia is one of a group of recessive hereditary genomic instability disorders and is characterized by progressive neurodegeneration, immunodeficiency and cancer susceptibility. Heterozygotes for the mutated gene are more susceptible to cancer and to ischaemic heart disease. The affected gene, ATM (ataxia telangiectasia mutated), has been cloned and codes for a protein kinase (ATM), which orchestrates the cellular response to DNA double-strand breaks after ionising radiation. An underlying feature of ataxia telangiectasia is oxidative stress and there is chronic activation of stress response pathways in tissues showing pathology such as the cerebellum, but not in the cerebrum or liver. ATM has also been shown to be activated by insulin and to have a wider role in signal transduction and cell growth. Many, but not all, aspects of the phenotype can be attributed to a defective DNA damage response. The oxidative stress may result directly from accumulated DNA damage in affected tissues or ATM may have an additional role in sensing/modulating redox homeostasis. The basis for the observed tissue specificity of the oxidative damage in ataxia telangiectasia is not clear.     

Genetic and biochemical studies with ataxia telangiectasia

Summary This article summarizes the genetics and clinical features of ataxia telangiectasia (AT) and then reviews recent cytogenetic, cellular, and biochemical studies which support the hypothesis that a defect in DNA repair is responsible for the various manifestations of the disease. The biochemical evidence further indicates that the defect specifically reduces the cellular capacity to remove bases and nucleotides damaged by ionizing radiation, without affecting the cells' ability to scavenge free radicals or to rejoin breaks in the sugar-phosphate backbone of DNA. Suggestions for additional research to more precisely identify the repair defect will also be presented.     

Gamma-ray-sensitive mutants of Neurospora crassa with characteristics analogous to ataxia telangiectasia cell lines

Well characterized gamma-ray sensitive mutants of the fungus Neurospora crassa have been screened for characteristics analogous to those of cell lines derived from humans with the genetic disease, ataxia telangiectasia (AT). Two Neurospora mutants, uvs-6 and mus-9, show the AT cell line characteristics of gamma-ray and bleomycin sensitivity, and little or no repression of DNA synthesis following treatment with these agents. Normal human or Neurospora cells show an extensive biphasic DNA synthesis repression (to 50% of control) and when DNA synthesis is analyzed by alkaline sucrose gradient centrifugation, repression of DNA synthesis by low doses of gamma-radiation occurs primarily in low molecular weight (MW) DNA pieces in both organisms. In AT cells and the uvs-6 mutant, no repression in synthesis of low or higher MW DNA is seen at low doses, while the mus-9 mutant shows little repression of high MW DNA, but an intermediate level of low MW DNA synthesis. Both mutants have been shown previously to have an increased level of spontaneous chromosome instability as do AT lines. The uvs-6 and mus-9 mutations are known to be due to two different genes in two different epistatic groups. These results demonstrate that AT-like cellular characteristics can arise from defects in at least two and probably any of several genes, and that lower eukaryotes such as Neurospora can provide an inexpensive and useful model for AT while avoiding the problems inherent in using transformed cell lines.     

The effects of ionizing radiation on cell cycle progression in ataxia telangiectasia

Although ataxia telangiectasia (AT) cells are more sensitive than normal cells to killing by ionizing radiation, their DNA synthesis is more resistant to inhibition by radiation. It was thought that this anomaly in DNA synthesis was likely to perturb cell cycle progression. Flow cytometry and the fraction of labelled mitoses (FLM) were used to investigate effects of irradiation in normal and AT cell lines. The FLM indicated that radiation apparently induced a longer G2 delay in normal cells than in AT cells. However, flow cytometry showed that radiation induced much larger and more prolonged increases in the proportion of G2 cells in AT than in normals. AT populations also showed much larger postirradiation decreases in viable cell numbers. These data suggest that a large proportion of the radiosensitive AT cells are not reversibly blocked in G2 but die there, and never proceed through mitosis. The less radiosensitive normal cells are delayed in G2 and then proceed through mitosis. We suggest that the apparently shorter radiation-induced mitotic delay seen in AT cells by FLM is not real but is an artifact arising from perturbation of steady state conditions by selective elimination of a particular cohort of AT cells. Accumulation of AT cells in G2 is compatible with radiosensitivity of these cells and may arise from a defect in DNA repair or an anomaly in DNA replication.     

Measurement of DNA polymerase beta in skin fibroblast cell lines from patients with ataxia telangiectasia

DNA polymerase beta levels were measured in 4 cell lines of normal human skin fibroblasts and in 5 cell lines of skin fibroblasts from patients with ataxia telangiectasia, an autosomal recessive disease exhibiting marked X-ray sensitivity. The enzyme specific activities for the normal lines were similar and the mean value was 2-fold lower than the mean value for the ataxia lines. With both kinds of cells, the enzyme level did not change as the cultures progressed from logarithmic to stationary phase of growth. Thus, this putative DNA repair enzyme appears to be 'constitutive' in human skin fibroblast lines, and a modest elevation of beta-polymerase activity is associated with ataxia telangiectasia. These results are discussed in the context to current views about DNA-repair enzymes in X-ray-sensitive cultured mammalian cells.     

Chromosomal radiosensitivity of ataxia telangiectasia cells at different cell cycle stages

Summary X-ray induced chromosomal aberrations in peripheral blood lymphocytes as well as in skin fibroblasts from ataxia telangiectasia patients, and from normal individuals were studied. At all stages of cell cycles—namely G₀, G₁, and G₂, more aberrations were induced in AT cells than in normal cells. In addition, AT cells were sensitive to induction of chromosomal aberrations by tritium beta rays from incorporated radioactive thymidine. Possible reasons for the increased sensitivity of AT cells for induction of chromosomal aberrations by ionizing radiations are discussed.     

Impaired insulin secretion in a mouse model of ataxia telangiectasia

Ataxia telangiectasia (A-T) is an autosomal recessive disease caused by mutations in the A-T mutated (ATM) gene. The gene encodes a serine/threonine kinase with important roles in the cellular response to DNA damage, including the activation of cell cycle checkpoints and induction of apoptosis. Although these functions might explain the cancer predisposition of A-T patients, the molecular mechanisms leading to glucose intolerance and diabetes mellitus (DM) are unknown. We have investigated the pathogenesis of DM in a mouse model of A-T. Here we show that young Atm-deficient mice show normal fasting glucose levels and normal insulin sensitivity. However, oral glucose tolerance testing revealed delayed insulin secretion and resulting transient hyperglycemia. Aged Atm-/- mice show a pronounced increase in blood glucose levels and a decrease in insulin and C-peptide levels. Our findings support a role for ATM in metabolic function and point toward impaired insulin secretion as the primary cause of DM in A-T.     

Cytogenetic investigations in families with ataxia-telangiectasia.

Chromosomal studies were performed on peripheral blood lymphocytes and cultured skin fibroblasts from five Israeli-Moroccan families with ataxia-telangiectasia. A total of 24 individuals, including seven propositi, was investigated. Among the probands, significantly elevated rates of chromosome damage were observed in both blood and skin. Skin fibroblasts of affected individuals showed several orders of magnitude more chromosome breakage than lymphocytes. Increased rates of chromosome damage were also observed in the fibroblasts of some phenotypically normal family members (obligate heterozygotes and sibs) when compared to normal controls. An apparent abnormal clone of cells, possessing a large acrocentric marker chromosome (14q+), was observed in varying proportions among cells of all the propositi (2-5% of lymphocytes; 1-9% of fibroblasts).     

Overproduction of topoisomerase II in an ataxia telangiectasia fibroblast cell line: comparison with a topoisomerase II-overproducing hamster cell mutant.

Ataxia telangiectasia (AT) cell lines are characterised by their hypersensitivity to ionizing radiation and bleomycin, and their failure to inhibit DNA synthesis after DNA damage. A recent report [Singh et al. (1988) Nucl. Acids Res. 16, 3919-3929] indicated that a reduction in topoisomerase II (topo II) activity was a feature of AT lymphoblast cell lines. We have studied the possible role of DNA topoisomerases in determining the phenotype of an AT fibroblast cell line. AT5BIVA cells are sensitive to the topo II inhibitors etoposide (VP16) and amsacrine (m-AMSA), compared to normal human fibroblasts (MRC5-V1 and VA13). AT5BIVA cells express a 3-fold higher level of topo II protein than MRC5-V1 cells, and 6-fold higher than VA13. This is reflected in elevated topo II activity in AT5BIVA cells. Untransformed AT5BI cells also show elevated topo II activity compared to untransformed normal cells. The extent of overproduction of topo II in AT5BIVA cells is comparable with that seen in a mutant Chinese hamster cell line, ADR-1, which is similarly hypersensitive to both bleomycin and topo II inhibitors. However, ADR-1 cells show neither hypersensitivity to ionizing radiation nor abnormal inhibition of DNA synthesis following DNA damage. Topo II overproduction per se does not appear sufficient to generate an "AT-like" phenotype. AT5BIVA cells express a reduced level of topoisomerase I (topo I) and are hypersensitive to the topo I inhibitor, camptothecin. ADR-1 cells express a normal level of topo I, indicating that a reduction in the level of topo I is not the inevitable consequence of an elevation in topo II.     

Genetic heterogeneity in patients having ataxia telangiectasia

UV-irradiated Chinese hamster cells on post-irradiation treatment with caffeine in growth medium for 24 h gave rise to biphasic UV-survival curves. At caffeine concentrations between 0.001 and 0.1 mM, control and caffeine-grown cells had similar survival curves initially from 0 to 30 J/m². At fluences greater than 30 J/m², there was effectively only little further killing of caffeine-grown cells beyond that observed at 30 J/m². At concentrations of caffeine greater than 0.5 mM, there was a gradual sensitization in the early part of the survival curve with increasing caffeine concentrations; but at fluences greater than 3 J/m², the slopes in the survival curves decreased very much.It has been proposed that the initial sensitization observed at low UV fluences is due to the suppression of post-replication repair by caffeine. After high fluences of UV exposures in these excision-deficient cells, in the presence of caffeine, the possibility of an induced ‘SOS’-like repair process has been suggested. This suggestion was supported by the observation that caffeine increased the yield of the UV-induced 8-azaguanine-resistant mutants only for the cell population exposed to UV fluences greater than 30 J/m².     

Premature aging syndrome in ataxia telangiectasia patients

Ataxia telangiectasia (AT) is a genetic disorder caused by the mutation of the atm gene. It is characterized by progressive neurological abnormalities in combination with oculocutaneous telangiectasias, immunodeficiency, and increased frequency of malignancy. Cells of AT patients display increased radiosensitivity and premature aging markers, including shortened telomer length beginning at birth and limited proliferation potential. We studied radiosensitivity (at a dose 2 Gy) and the manifestation of premature aging markers in cultured skin fibroblasts derived from two unrelated AT patients and their heterozygous parents. We have shown that all the markers studied, i.e., HP1-γ, histone H2AX phosphorylated for serine-139 (γ-H2AX) and foci of 53BP1 protein, indicate the premature aging of the cells of both patients and their blood relatives. However, cells of heterozygous carriers express premature aging to a lesser extent. A study of the repair process (the amount of γ-H2AX and the number of cells with 53BP1 foci in their nuclei) after X-ray irradiation showed that patients’ cells only halfway completed repairs, even 24 h after irradiation, while the healthy donor cells completed repairs in 24 h. In cells from atm heterozygous donors, DNA repair was also slower. Heterozygous cells also differ reliably from healthy donor cells. Only amounts of p21^Waf1/Cip1 protein, an inhibitor of cyclin-dependent kinases, in heterozygous cells do not differ from normal cells. However, the patients’ cells differ significantly. It was found that the mutation of the atm gene was related to the suppression of the reparation of DNA double-strand breaks (DSBs), which is in good agreement with increased radiosensitivity and premature aging in AT families at the cellular level.     

DNA-repair synthesis in ataxia telangiectasia lymphoblastoid cells

The ability of a number of Epstein-Barr virus-transformed lymphoblastoid cells from ataxia telangiectasia (AT) patients to repair γ-radiation damage to DNA was determined. All of these AT cells were previously shown to be hypersensitive to γ-irradiation. Two methods were used to determine DNA-repair synthesis: isopycnic gradient analysis and a method employing hydroxyurea to inhibit semiconservative DNA synthesis. Control, AT heterozygote and AT homozygote cells were demonstrateed to have similar capacities for repair of radiation damage to DNA. In addition at high radiation doses (10–40 krad) the extent of inhibition of DNA synthesis was similar in the different cell types.     

New mutations in the ataxia telangiectasia gene

We report the detection of four new mutations in the ataxia telangiectasia gene (ATM). Reverse-transcribed RNA extracted from cultured cells was analysed for mutations by polymerase chain reaction amplifications and restriction endonuclease fingerprinting. Three deletions and a base substitution are described. The deletions reported here would result in severe disruptions of the ATM gene product by leading either to a protein truncation (a 4-bp deletion) or the loss of stretches of 53 and 58 amino acids (a 159-bp deletion and a 174-bp deletion, respectively); whereas the base substitution would lead to an amino acid change from a highly conserved glycine to an arginine residue. Received: 15 April 1996 / Revised: 24 April 1996