Genotype-phenotype relationships in ataxia-telangiectasia and variants.

Ataxia-telangiectasia (A-T) is an autosomal recessive disorder characterized by cerebellar degeneration, immunodeficiency, chromosomal instability, radiosensitivity, and cancer predisposition. A-T cells are sensitive to ionizing radiation and radiomimetic chemicals and fail to activate cell-cycle checkpoints after treatment with these agents. The responsible gene, ATM, encodes a large protein kinase with a phosphatidylinositol 3-kinase-like domain. The typical A-T phenotype is caused, in most cases, by null ATM alleles that truncate or severely destabilize the ATM protein. Rare patients with milder manifestations of the clinical or cellular characteristics of the disease have been reported and have been designated "A-T variants." A special variant form of A-T is A-TFresno, which combines a typical A-T phenotype with microcephaly and mental retardation. The possible association of these syndromes with ATM is both important for understanding their molecular basis and essential for counseling and diagnostic purposes. We quantified ATM-protein levels in six A-T variants, and we searched their ATM genes for mutations. Cell lines from these patients exhibited considerable variability in radiosensitivity while showing the typical radioresistant DNA synthesis of A-T cells. Unlike classical A-T patients, these patients exhibited 1%-17% of the normal level of ATM. The underlying ATM genotypes were either homozygous for mutations expected to produce mild phenotypes or compound heterozygotes for a mild and a severe mutation. An A-TFresno cell line was found devoid of the ATM protein and homozygous for a severe ATM mutation. We conclude that certain "A-T variant" phenotypes represent ATM mutations, including some of those without telangiectasia. Our findings extend the range of phenotypes associated with ATM mutations.     

Nuclear ataxia-telangiectasia mutated (ATM) mediates the cellular response to DNA double strand breaks in human neuron-like cells

The protein kinase ATM (ataxia-telangiectasia mutated) activates the cellular response to double strand breaks (DSBs), a highly cytotoxic DNA lesion. ATM is activated by DSBs and in turn phosphorylates key players in numerous damage response pathways. ATM is missing or inactivated in the autosomal recessive disorder ataxia-telangiectasia (A-T), which is characterized by neuronal degeneration, immunodeficiency, genomic instability, radiation sensitivity, and cancer predisposition. The predominant symptom of A-T is a progressive loss of movement coordination due to ongoing degeneration of the cerebellar cortex and peripheral neuropathy. A major deficiency in understanding A-T is the lack of information on the role of ATM in neurons. It is unclear whether the ATM-mediated DSB response operates in these cells similarly to proliferating cells. Furthermore, ATM was reported to be cytoplasmic in neurons and suggested to function in these cells in capacities other than the DNA damage response. Recently we obtained genetic molecular evidence that the neuronal degeneration in A-T does result from defective DNA damage response. We therefore undertook to investigate this response in a model system of human neuron-like cells (NLCs) obtained by neuronal differentiation in culture. ATM was largely nuclear in NLCs, and their ATM-mediated responses to DSBs were similar to those of proliferating cells. Knocking down ATM did not interfere with neuronal differentiation but abolished ATM-mediated damage responses in NLCs. We concluded that nuclear ATM mediates the DSB response in NLCs similarly to in proliferating cells. Attempts to understand the neurodegeneration in A-T should be directed to investigating the DSB response in the nervous system.     

ATM controls proper mitotic spindle structure

The recessive ataxia-telangiectasia (A-T) syndrome is characterized by cerebellar degeneration, immunodeficiency, cancer susceptibility, premature aging, and insulin-resistant diabetes and is caused by loss of function of the ATM kinase, a member of the phosphoinositide 3-kinase–like protein kinases (PIKKs) family. ATM plays a crucial role in the DNA damage response (DDR); however, the complexity of A-T features suggests that ATM may regulate other cellular functions. Here we show that ATM affects proper bipolar mitotic spindle structure independently of DNA damage. In addition, we find that in mitosis ATM forms a complex with the poly(ADP)ribose (PAR) polymerase Tankyrase (TNKS) 1, the spindle pole protein NuMA1, and breast cancer susceptibility protein BRCA1, another crucial DDR player. Our evidence indicates that the complex is required for efficient poly(ADP)ribosylation of NuMA1. We find further that a mutant NuMA1 version, non-phosphorylatable at potential ATM-dependent phosphorylation sites, is poorly PARylated and induces loss of spindle bipolarity. Our findings may help to explain crucial A-T features and provide further mechanistic rationale for TNKS inhibition in cancer therapy.     

ATM-heterozygous germline mutations contribute to breast cancer-susceptibility

Approximately 0.5%-1% of the general population has been estimated to be heterozygous for a germline mutation in the ATM gene. Mutations in the ATM gene are responsible for the autosomal recessive disorder ataxia-telangiectasia (A-T) (MIM 208900). The finding that ATM-heterozygotes have an increased relative risk for breast cancer was supported by some studies but not confirmed by others. In view of this discrepancy, we examined the frequency of ATM germline mutations in a selected group of Dutch patients with breast cancer. We have analyzed ATM germline mutations in normal blood lymphocytes, using the protein-truncation test followed by genomic-sequence analysis. A high percentage of ATM germline mutations was demonstrated among patients with sporadic breast cancer. The 82 patients included in this study had developed breast cancer at age <45 and had survived >/=5 years (mean 15 years), and in 33 (40%) of the patients a contralateral breast tumor had been diagnosed. Among these patients we identified seven (8.5%) ATM germline mutations, of which five are distinct. One splice-site mutation (IVS10-6T-->G) was detected three times in our series. Four heterozygous carriers were patients with bilateral breast cancer. Our results indicate that the mutations identified in this study are "A-T disease-causing" mutations that might be associated with an increased risk of breast cancer in heterozygotes. We conclude that ATM heterozygotes have an approximately ninefold-increased risk of developing a type of breast cancer characterized by frequent bilateral occurrence, early age at onset, and long-term survival. The specific characteristics of our population of patients may explain why such a high frequency was not found in other series.     

Functional consequences of sequence alterations in the ATM gene

The product of the gene (ATM) mutated in the human genetic disorder ataxia-telangiectasia (A-T) is a high molecular weight, protein ( approximately 350kDa) containing a C-terminal protein kinase domain and a number of other putative domains not yet functionally defined. The majority of ATM gene mutations in A-T patients are truncating, resulting in prematurely terminated products that are highly unstable. Missense mutations within the kinase domain and elsewhere in the molecule alter the stability of the protein and lead to loss of protein kinase activity. Only rarely are patients observed with two missense mutations and this gives rise to a milder disease phenotype. Evidence for a dominant interfering effect on normal ATM kinase activity has been reported in cell lines transfected with missense mutant ATM and in cell lines from some A-T heterozygotes. The dominant negative effect of mutant ATM is manifested by an enhancement of cellular radiosensitivity and may be responsible for the cancer predisposition observed in carriers of ATM missense mutations. In this review, we explore the domain structure of the ATM molecule, sites of interaction with other proteins and the consequences of specific amino acid changes on function.     

ATM activates the pentose phosphate pathway promoting anti-oxidant defence and DNA repair

Ataxia telangiectasia (A-T) is a human disease caused by ATM deficiency characterized among other symptoms by radiosensitivity, cancer, sterility, immunodeficiency and neurological defects. ATM controls several aspects of cell cycle and promotes repair of double strand breaks (DSBs). This probably accounts for most of A-T clinical manifestations. However, an impaired response to reactive oxygen species (ROS) might also contribute to A-T pathogenesis. Here, we show that ATM promotes an anti-oxidant response by regulating the pentose phosphate pathway (PPP). ATM activation induces glucose-6-phosphate dehydrogenase (G6PD) activity, the limiting enzyme of the PPP responsible for the production of NADPH, an essential anti-oxidant cofactor. ATM promotes Hsp27 phosphorylation and binding to G6PD, stimulating its activity. We also show that ATM-dependent PPP stimulation increases nucleotide production and that G6PD-deficient cells are impaired for DSB repair. These data suggest that ATM protects cells from ROS accumulation by stimulating NADPH production and promoting the synthesis of nucleotides required for the repair of DSBs.     

Requirement of the MRN complex for ATM activation by DNA damage

The ATM protein kinase is a primary activator of the cellular response to DNA double-strand breaks (DSBs). In response to DSBs, ATM is activated and phosphorylates key players in various branches of the DNA damage response network. ATM deficiency causes the genetic disorder ataxia-telangiectasia (A-T), characterized by cerebellar degeneration, immunodeficiency, radiation sensitivity, chromosomal instability and cancer predisposition. The MRN complex, whose core contains the Mre11, Rad50 and Nbs1 proteins, is involved in the initial processing of DSBs. Hypomorphic mutations in the NBS1 and MRE11 genes lead to two other genomic instability disorders: the Nijmegen breakage syndrome (NBS) and A-T like disease (A-TLD), respectively. The order in which ATM and MRN act in the early phase of the DSB response is unclear. Here we show that functional MRN is required for ATM activation, and consequently for timely activation of ATM-mediated pathways. Collectively, these and previous results assign to components of the MRN complex roles upstream and downstream of ATM in the DNA damage response pathway and explain the clinical resemblance between A-T and A-TLD.     

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.     

Iron chelators reduce chromosomal breaks in ataxia-telangiectasia cells

Ataxia-telangiectasia (A-T) is characterized by ataxia, genomic instability, and increased cancer incidence. Previously, iron chelator concentrations which suppressed normal cell colony formation increased A-T cell colony formation. Similarly, iron chelators preferentially increased A-T cell colony formation following peroxide exposure compared to normal cells. Last, A-T cells exhibited increased short-term sensitivity to labile iron exposure compared to normal cells, an event corrected by recombinant ATM (rATM) expression. Since chromosomal damage is important in A-T pathology and iron chelators exert beneficial effects on A-T cells, we hypothesized that iron chelators would reduce A-T cell chromosomal breaks. We treated A-T, normal, and A-T cells expressing rATM with labile iron, iron chelators, antioxidants, and t-butyl hydroperoxide, and examined chromosomal breaks and ATM activation. Additionally, the effect of ATM-deficiency on transferrin receptor (TfR) expression and TfR activity blockage in A-T and syngeneic A-T cells expressing rATM was examined. We report that (1) iron chelators and iron-free media reduce spontaneous and t-butyl hydroperoxide-induced chromosomal breaks in A-T, but not normal, or A-T cells expressing rATM; (2) labile iron exposure induces A-T cell chromosomal breaks, an event lessened with rATM expression; (3) desferal, labile iron, and copper activate ATM; (4) A-T cell TfR expression is lowered with rATM expression and (5) blocking TfR activity with anti-TfR antibodies increases A-T cell colony formation, while lowering chromosomal breaks. ATM therefore functions in iron responses and the maintenance of genomic stability following labile iron exposure.     

ATM mutations and phenotypes in ataxia-telangiectasia families in the British Isles: expression of mutant ATM and the risk of leukemia, lymphoma, and breast cancer.

We report the spectrum of 59 ATM mutations observed in ataxia-telangiectasia (A-T) patients in the British Isles. Of 51 ATM mutations identified in families native to the British Isles, 11 were founder mutations, and 2 of these 11 conferred a milder clinical phenotype with respect to both cerebellar degeneration and cellular features. We report, in two A-T families, an ATM mutation (7271T-->G) that may be associated with an increased risk of breast cancer in both homozygotes and heterozygotes (relative risk 12.7; P=. 0025), although there is a less severe A-T phenotype in terms of the degree of cerebellar degeneration. This mutation (7271T-->G) also allows expression of full-length ATM protein at a level comparable with that in unaffected individuals. In addition, we have studied 18 A-T patients, in 15 families, who developed leukemia, lymphoma, preleukemic T-cell proliferation, or Hodgkin lymphoma, mostly in childhood. A wide variety of ATM mutation types, including missense mutations and in-frame deletions, were seen in these patients. We also show that 25% of all A-T patients carried in-frame deletions or missense mutations, many of which were also associated with expression of mutant ATM protein.     

Elevated Cu/Zn-SOD exacerbates radiation sensitivity and hematopoietic abnormalities of Atm-deficient mice

Patients with the genetic disorder ataxia-telangiectasia (A-T) display a pleiotropic phenotype that includes neurodegeneration, immunodeficiency, cancer predisposition and hypersensitivity to ionizing radiation. The gene responsible is ATM, and ATM:-knockout mice recapitulate most features of A-T. In order to study the involvement of oxidative stress in the A-T phenotype, we examined mice deficient for Atm and overexpressing human Cu/Zn superoxide dismutase (SOD1). We report that elevated levels of SOD1 exacerbate specific features of the murine Atm- deficient phenotype, including abnormalities in hematopoiesis and radiosensitivity. The data are consistent with the possibility that oxidative stress contributes to some of the clinical features associated with the A-T phenotype.     

ATM deficiency and oxidative stress: a new dimension of defective response to DNA damage

ATM is one of the sentries at the gate of genome stability. This multifunctional protein kinase orchestrates the intricate array of cellular responses to DNA double-strand breaks. Absence or inactivation of ATM leads to the pleiotropic genetic disorder ataxia-telangiectasia (A-T), whose hallmarks are neuronal degeneration, immunodeficiency, genomic instability, premature aging and cancer predisposition. Several features of the complex clinical and cellular phenotype of A-T are reminiscent of other syndromes involving neurodegeneration, premature aging or genomic instability. A common denominator of many of these conditions is the perturbation of the cellular balance of reactive oxygen species, which leads to constant oxidative stress. Of these disorders, ATM deficiency is one of the most extensively studied with regard to the genome instability-oxidative stress connection. This connection may provide new insights into the phenotypes associated with genetic deficiencies of DNA damage responses, and point to new strategies to alleviate some of their clinical symptoms.     

ATM protein physically and functionally interacts with proliferating cell nuclear antigen to regulate DNA synthesis

Ataxia telangiectasia (A-T) is a pleiotropic disease, with a characteristic hypersensitivity to ionizing radiation that is caused by biallelic mutations in A-T mutated (ATM), a gene encoding a protein kinase critical for the induction of cellular responses to DNA damage, particularly to DNA double strand breaks. A long known characteristic of A-T cells is their ability to synthesize DNA even in the presence of ionizing radiation-induced DNA damage, a phenomenon termed radioresistant DNA synthesis. We previously reported that ATM kinase inhibition, but not ATM protein disruption, blocks sister chromatid exchange following DNA damage. We now show that ATM kinase inhibition, but not ATM protein disruption, also inhibits DNA synthesis. Investigating a potential physical interaction of ATM with the DNA replication machinery, we found that ATM co-precipitates with proliferating cell nuclear antigen (PCNA) from cellular extracts. Using bacterially purified ATM truncation mutants and in vitro translated PCNA, we showed that the interaction is direct and mediated by the C terminus of ATM. Indeed, a 20-amino acid region close to the kinase domain is sufficient for strong binding to PCNA. This binding is specific to ATM, because the homologous regions of other PIKK members, including the closely related kinase A-T and Rad3-related (ATR), did not bind PCNA. ATM was found to bind two regions in PCNA. To examine the functional significance of the interaction between ATM and PCNA, we tested the ability of ATM to stimulate DNA synthesis by DNA polymerase δ, which is implicated in both DNA replication and DNA repair processes. ATM was observed to stimulate DNA polymerase activity in a PCNA-dependent manner.     

Ataxia-telangiectasia, an evolving phenotype

Ataxia-telangiectasia (A-T) is a progressive neurodegenerative disorder, with onset in early childhood and a frequency of approximately 1 in 40,000 births in the United States. A-T is seen among all races and is most prominent among ethnic groups with a high frequency of consanguinity. The syndrome includes: progressive cerebellar ataxia, dysarthric speech, oculomotor apraxia, choreoathetosis and, later, oculocutaneous telangiectasia. Immunodeficiency with sinopulmonary infections, cancer susceptibility (usually lymphoid), and sensitivity to ionizing radiation are also characteristic. Laboratory findings include: (1) elevated alphafetoprotein (AFP), (2) cerebellar atrophy on magnetic resonance imaging, (3) reciprocal translocations between chromosomes 7 and 14 in lymphocytes, (4) absence or dysfunction of the ATM protein, (5) radiosensitivity, as demonstrated by colony survival assay (CSA), and (6) mutations in the ATM gene. The latter are usually truncating or splicing mutations; approximately 10% are missense mutations. Mutations are found across the entire gene. Almost all recurring mutations are found on unique haplotypes that represent founder effects and ancestral relationships between patients. In addition to radiosensitivity and sensitivity to radiomimetic chemicals, the phenotype of A-T cells includes defective damage-induced activation of the cell cycle checkpoints at G1, S and G2/M. With the aid of molecular testing, A-T can now be distinguished from other autosomal recessive cerebellar ataxias (ARCAs) such as Friedreich ataxia, Mre11 deficiency (AT-like disease), and the oculomotor apraxias 1 (aprataxin deficiency) and 2 (senataxin deficiency). Other "A-T variants" include: (1) Nijmegen breakage syndrome (NBS) or nibrin/Nbs1 deficiency, with microcephaly and mental retardation but without ataxia, apraxia, or telangiectasia, and 2) A-T(Fresno), a phenotype that combines features of both NBS and A-T, with mutations in the ATM gene. The term "A-T variant" has a diminishing usefulness.     

ATM mediates repression of DNA end-degradation in an ATP-dependent manner

Ataxia telangiectasia mutated (ATM) is a PI3-kinase-like kinase (PIKK) associated with DNA double-strand break (DSB) repair and cell cycle control. We have previously reported comparable efficiencies of DSB repair in nuclear extracts from both ATM deficient (A-T) and control (ATM+) cells; however, the repair products from the A-T nuclear extracts contained deletions encompassing longer stretches of DNA compared to controls. These deletions appeared to result from end-joining at sites of microhomology. These data suggest that ATM hinders error-prone repair pathways that depend on degradation of DNA ends at a break. Such degradation may account for the longer deletions we formerly observed in A-T cell extracts. To address this possibility we assessed the degradation of DNA duplex substrates in A-T and control nuclear extracts under DSB repair conditions. We observed a marked shift in signal intensity from full-length products to shorter products in A-T nuclear extracts, and addition of purified ATM to A-T nuclear extracts restored full-length product detection. This repression of degradation by ATM was both ATP-dependent and inhibited by the PIKK inhibitors wortmannin and caffeine. Addition of pre-phosphorylated ATM to an A-T nuclear extract in the presence of PIKK inhibitors was insufficient in repressing degradation, indicating that kinase activities are required. These results demonstrate a role for ATM in preventing the degradation of DNA ends possibly through repressing nucleases implicated in microhomology-mediated end-joining.     

ATM gene expression is associated with differentiation and angiogenesis in infiltrating breast carcinomas

The product of the ATM gene, mutated in the human genetic disorder ataxia-telangiectasia (A-T) plays a key role in the detection and repair of DNA double-strand breaks. A-T is defined by progressive cerebellar ataxia, telangiectasia, sensitivity to ionising radiation and genomic instability with cancer predisposition. On the other hand, increased angiogenesis is essential for tumor growth and metastasis. The aim of this study was to investigate ATM expression in breast carcinomas and its relationship to neoangiogenesis. METHODS AND RESULTS: Fifty-two breast tumors from 51 patients, 38 of them with concomitant in situ component (CIS), were analyzed by immunohistochemistry for the expression of ATM. CD34 expression was used for the morphometric evaluation of vasculature. ATM was positive in 1 to 10% of normal epithelial cells. ATM expression was reduced in 55.8% of infiltrating carcinomas, non-reduced in 34.6%, and increased in 9.6%. Expression of ATM in CIS was similar to the infiltrating component in 71% of cases and reduced in 23.7% of them. High-grade ductal infiltrating carcinomas showed lower ATM expression than low-grade ones. Reduced ATM expression also correlated with increased microvascular area. CONCLUSIONS: Reduced ATM expression in breast carcinomas correlated with tumor differentiation and increased microvascular parameters, supporting its role in neoangiogenesis and tumor progression in breast carcinogenesis.     

Targeted disruption of Ataxia-telangiectasia mutated gene in miniature pigs by somatic cell nuclear transfer

Ataxia telangiectasia (A-T) is a recessive autosomal disorder associated with pleiotropic phenotypes, including progressive cerebellar degeneration, gonad atrophy, and growth retardation. Even though A-T is known to be caused by the mutations in the Ataxia telangiectasia mutated (ATM) gene, the correlation between abnormal cellular physiology caused by ATM mutations and the multiple symptoms of A-T disease has not been clearly determined. None of the existing ATM mouse models properly reflects the extent to which neurological degeneration occurs in human. In an attempt to provide a large animal model for A-T, we produced gene-targeted pigs with mutations in the ATM gene by somatic cell nuclear transfer. The disrupted allele in the ATM gene of cloned piglets was confirmed via PCR and Southern blot analysis. The ATM gene-targeted pigs generated in the present study may provide an alternative to the current mouse model for the study of mechanisms underlying A-T disorder and for the development of new therapies.