Showing papers by "Yosef Shiloh published in 1998"

ATM: From Gene to Function

Abstract: The identification of ATM , the gene responsible for the pleiotropic recessive disease ataxia telangiectasia, has initiated extensive research to determine the functions of its multifaceted protein product. The ATM protein belongs to a family of protein kinases that share similarities at their C-terminal region with the catalytic domain of phosphatidylinositol 3-kinases. Studies with ataxia telangiectasia (A-T) cells and Atm-deficient mice have shown that ATM is a key regulator of multiple signaling cascades which respond to DNA strand breaks induced by damaging agents or by normal processes, such as meiotic or V(D)J recombination. These responses involve the activation of cell cycle checkpoints, DNA repair and apoptosis. Other roles outside the cell nucleus might be carried out by the cytoplasmic fraction of ATM. In addition, ATM appears to function as a 'caretaker', suppressing tumorigenesis in specific T cell lineages.

Genotype-Phenotype Relationships in Ataxia-Telangiectasia and Variants

Abstract: 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.

ATM binds to β-adaptin in cytoplasmic vesicles

Abstract: Inherited mutations in the ATM gene lead to a complex clinical phenotype characterized by neuronal degeneration, oculocutaneous telangiectasias, immune dysfunction, and cancer predisposition Using the yeast two-hybrid system, we demonstrate that ataxia telangiectasia mutated (ATM) binds to β-adaptin, one of the components of the AP-2 adaptor complex, which is involved in clathrin-mediated endocytosis of receptors The interaction between ATM and β-adaptin was confirmed in vitro, and coimmunoprecipitation and colocalization studies show that the proteins also associate in vivo ATM also interacts in vitro with β-NAP, a neuronal-specific β-adaptin homolog that was identified as an autoantigen in a patient with cerebellar degeneration Our data describing the association of ATM with β-adaptin in vesicles indicate that ATM may play a role in intracellular vesicle and/or protein transport mechanisms

Selective loss of dopaminergic nigro-striatal neurons in brains of Atm-deficient mice

Abstract: Ataxia-telangiectasia (AT) is a human disease caused by mutations in the ATM gene. The neural phenotype of AT includes progressive cerebellar neurodegeneration, which results in ataxia and eventual motor dysfunction. Surprisingly, mice in which the Atm gene has been inactivated lack distinct behavioral ataxia or pronounced cerebellar degeneration, the hallmarks of the human disease. To determine whether lack of the Atm protein can nonetheless lead to structural abnormalities in the brain, we compared brains from male Atm-deficient mice with male, age-matched controls. Atm-deficient mice exhibited severe degeneration of tyrosine hydroxylase-positive, dopaminergic nigro-striatal neurons, and their terminals in the striatum. This cell loss was accompanied by a large reduction in immunoreactivity for the dopamine transporter in the striatum. A reduction in dopaminergic neurons also was evident in the ventral tegmental area. This effect was selective in that the noradrenergic nucleus locus coeruleus was normal in these mice. Behaviorally, Atm-deficient mice expressed locomotor abnormalities manifested as stride-length asymmetry, which could be corrected by peripheral application of the dopaminergic precursor l-dopa. In addition, these mice were hypersensitive to the dopamine releasing drug d-amphetamine. These results indicate that ATM deficiency can severely affect dopaminergic neurons in the central nervous system and suggest possible strategies for treating this aspect of the disease.

Identification of atm mutations using extended rt-pcr and restriction endonuclease fingerprinting, and elucidation of the repertoire of a-t mutations in israel

Abstract: Ataxia-telangiectasia (A-T) is an autosomal recessive disorder characterized by neurodegeneration, immunodeficiency, cancer predisposition, and radiation sensitivity. The responsible gene, ATM, has an extensive genomic structure and encodes a large transcript with a 9.2 kb open reading frame (ORF). A-T mutations are extremely variable and most of them are private. We streamlined a high throughput protocol for the search for ATM mutations. The entire ATM ORF is amplified in a single RT-PCR step requiring a minimal amount of RNA. The product can serve for numerous nested PCRs in which overlapping portions of the ORF are further amplified and subjected to restriction endonuclease fingerprinting (REF) analysis. Splicing errors are readily detectable during the initial amplification of each portion. Using this protocol, we identified 5 novel A-T mutations and completed the elucidation of the molecular basis of A-T in the Israeli population. Hum Mutat 11:69–75, 1998. © 1998 Wiley-Liss, Inc.

Defective potassium currents in ataxia telangiectasia fibroblasts.

Abstract: Similarities exist between the progressive cerebellar ataxia in ataxia telangiectasia (AT) patients and a number of neurodegenerative diseases in both mouse and man involving specific mutations in ion channels and/or ion channel activity. These relationships led us to investigate the possibility of defective ion channel activity in AT cells. We examined changes in the membrane potential of AT fibroblasts in response to extracellular cation addition and found that the ability of AT fibroblasts to depolarize in response to increasing concentrations of extracellular K+ is significantly reduced when compared with control fibroblasts. Electrophysiological measurements performed with a number of AT cell lines, as well as two matched sets of primary AT fibroblast cultures, reveal that outward rectifier K+ currents are largely absent in AT fibroblasts in comparison with control cells. These K+ current defects can be corrected in AT fibroblasts transfected with the full-length ATM cDNA. These data implicate, for the first time, a role for ATM in the regulation of K+ channel activity and membrane potential.

Ataxia‐telangiectasia in the Japanese population: Identification of R1917X, W2491R, R2909G, IVS33+2T→A, and 7883del5, the latter two being relatively common mutations

Abstract: We analyzed the data regarding six Japanese ataxia-telangiectasia (A-T) patients from four unrelated families, at the DNA level, to search for possible common mutations in the Japanese population. Among eight mutant alleles in the four families, c. 4612del165 (exon 33 skipping) was identified in two alleles, and c. 5749A to T (R1917X), c. 7471T to C (W2491R), c.7883de15, and c. 8725A to G (R2909G) were identified in one allele each. We found no mutations in the other two alleles. The IVS33 + 2T-->A mutation was identified at the genomic level as the cause of exon 33 skipping. We also identified the IVS33 + 2T-->A mutation in a Japanese patient ATL105 who was previously found to be a homozygote of c. 4612del165. W2491R and R2909G mutations were not detected in more than 100 control Japanese alleles. The latter is located in a highly conserved PI-3 kinase domain and is a completely conserved residue among ATM-related proteins. Taken together with previously documented mutations in five other Japanese A-T patients, IVS33 + 2T-->A and 7883del5 were identified in four and five alleles, respectively, in a total of 18 mutant alleles of Japanese A-T patients. These results suggest that these two mutations are relatively common mutations in the Japanese population.

Cloning and Expression of Large Mammalian cDNAs: Lessons from ATM

Abstract: The ATM gene and its protein product have recently become a focus of interest for clinicians, biologists and cancer epidemiologists. The ATM protein is involved in a complex junction of signal transduction pathways linking cellular stress responses, cell cycle control and specific developmental processes (1). The ATM gene was identified by virtue of its responsibility for a human genetic disorder, ataxia- telangiectasia (A-T). A-T is an autosomal recessive disease characterized by cerebellar degeneration, immunodeficiency, premature aging, cancer predisposition and radiation sensitivity. A-T cells exhibit chromosomal instability, extreme sensitivity to ionizing radiation and radiomimetic chemicals, defective cell cycle checkpoints, and defects in several signal transduction pathways that respond to mitogenic stimuli and genotoxic stress (see refs. 1, 2 for recent reviews).