Early diagnosis of ataxia-telangiectasia using radiosensitivity testing

Sun, Xia; Becker-Catania, Sara G.; Chun, Helen H.; Hwang, Mee Jeong; Huo, Yong; Wang, Zhijun; Mitui, Midori; Sanal, Özden; Chessa, Luciana; Crandall, Barbara F.; Gatti, Richard A.

doi:10.1067/mpd.2002.123879

Cited by 114 publications

(131 citation statements)

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“…The assessment of the gene expression phenotype has been proposed as a possible means of identifying ATM heterozygotes (Watts et al, 2002), and these results would lend support to this proposal. However, it should be noted that some variation between the different LCLs was noted and no direct relationship between the ATM protein and mRNA levels was seen in all cell lines (Jongmans et al, 1997;Becker-Catania et al, 2000;Sun et al, 2002). In this study, the three AT heterozygote cell lines carrying a missense mutation had higher levels of mRNA levels, as measured by a semiquantitative PCR-based technique compared to the lines carrying truncating mutations, reflecting the observations in ATM homozygote lines.…”

Section: Discussionmentioning

confidence: 46%

“…Many authors have reported a level of cell survival intermediate between that seen in normal and ATM homozygote cells, but, in other studies, some overlap or no statistically significant differences between normal and AT het cell types have been detected (Chen et al, 1978;Kinsella et al, 1982;Nagasawa et al, 1985;Weeks et al, 1991;West et al, 1995;Shigeta et al, 1999;Sun et al, 2002). Many of these studies have used a fibroblast-cloning assay, which requires the trypsinising and replating of treated cells, which may lead to additional cell death, particularly in radiationsensitive cell types.…”

Section: Discussionmentioning

confidence: 98%

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Cellular responses to ionising radiation of AT heterozygotes: differences between missense and truncating mutation carriers

et al. 2004

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It has been estimated that approximately 1% of the general population are ataxia telangiectasia (AT) mutated (ATM) heterozygotes. The ATM protein plays a central role in DNA-damage response pathways; however, the functional consequences of the presence of either heterozygous truncating or missense mutations on ATM expression and the ionising radiation (IR)-induced cellular phenotype remain to be fully determined. To investigate this relationship, the ATM mRNA and protein levels and several cellular end points were characterised in 14 AT heterozygote (AT het) lymphoblastoid cell lines, compared to normal and AT homozygote lines. The AT het cell lines displayed a wide range of IR-induced responses: despite lower average levels of ATM mRNA and protein expression compared to normal cells, 13 out of 14 were capable of phosphorylating the ATM substrates p53-ser15 and Chk2, leading to a normal cell cycle progression after irradiation. However, cell survival was lower than in the normal cell lines. The presence of a missense compared to a truncating mutation was associated with lower cell survival after exposure to 2 Gy irradiation (P ¼ 0.005), and a higher level of ATM mRNA expression (P ¼ 0.047). Our results underline the difficulty in establishing a reliable test for determining ATM heterozygosity.

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Section: Discussionmentioning

confidence: 46%

Section: Discussionmentioning

confidence: 98%

Cellular responses to ionising radiation of AT heterozygotes: differences between missense and truncating mutation carriers

et al. 2004

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“…Whereas RDS evaluates the S phase checkpoint, the CSA was used to assess the long-term effects of geneticin on A-T cell survival after radiation damage (1 Gy) (8). Fig.…”

Section: Resultsmentioning

confidence: 99%

“…CSA was performed as described (8). After 4 days of incubation with or without aminoglycosides, LCLs were plated, in duplicate, in 96-well plates at 50, 100, or 200 cells per well.…”

Section: Methodsmentioning

confidence: 99%

Correction of ATM gene function by aminoglycoside-induced read-through of premature termination codons

Lai

Chun

Nahas

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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Cells were irradiated with 10 Gy and were incubated for 45 min at 37°C before fixation on coverslips. ATM pS1981 IRIFs were not observed in untreated cells; maximum intensity of staining was seen in the nuclei of cells exposed to 75 g͞ml. Nonirradiated cells, untreated or exposed to comparable concentrations of geneticin, showed only occasional foci. A taxia-telangiectasia (A-T) is an autosomal recessive neurodegenerative disorder with onset in early childhood, resulting from mutations in the A-T mutated (ATM) gene (1). The ATM protein is a hierarchical serine-threonine kinase, phosphorylating many substrates involved in repair of doublestranded DNA breaks, control of cell cycle checkpoints, and responses to oxidative stress, as well as in radiosensitivity, cancer susceptibility, immune function, and neurological development (2). ATM protein levels are undetectable in the cells of most A-T patients by conventional testing. However a few patients, often with a milder phenotype, have some detectable ATM protein (3,4). This finding encouraged us to seek compounds, such as aminoglycosides, that have the potential to read through premature termination codons and restore ATM protein function (5, 6).To test these effects, we selected 13 lymphoblastoid cell lines (LCLs) with primary premature termination codon (PTC) mutations from a repository of Ͼ400 lines derived from A-T patients. Herein, we show representative data from 5 of the 13 LCLs. Using protein truncation testing (PTT) driven by plasmid templates containing PTC mutations in the ATM gene (7), we observed in vitro read-through effects of various magnitudes with three of four aminoglycosides tested. Full-length ATM protein was also documented ex vivo by immunoprecipitation. Correction of radioresistant DNA synthesis and radiosensitivity, as well as autophosphorylation of ATM, suggested that this readthrough produces functional ATM protein. Experimental ProceduresCell Lines. Lymphoblastoid cell lines were maintained in RPMI medium 1640 (Invitrogen) with 15% FBS (HyClone) and 1% penicillin͞streptomycin (Invitrogen) at 37°C and 5% CO 2 . In ex vivo experiments, aminoglycosides were added daily into culture media at the indicated doses and times. Because streptomycin is also an aminoglycoside that can perturb proofreading by binding to another ribosomal site, ex vivo experiments were performed with or without streptomycin; no differences were noted.Mutations. We selected only PTC mutations that resulted directly from the mutation, i.e., primary PTC mutations, and not those caused indirectly by upstream mutations or aberrant splicing, because the latter would have no potential therapeutic benefits. The LCLs carried the following mutations (www.benaroyaresearch.org͞bri investigators͞atm.htm): AT185LA, homozygous 3673C 3 T (a UAA G stop codon in PTT fragment 4); TAT51, homozygous 5623C 3 T (a UGA C stop codon in PTT fragment 5); AT187LA, homozygous 5908C 3 T (a UAA G stop codon in PTT fragment 6); AT153LA, homozygous 8977C 3 T (a UGA A stop codon in fragment 8); and AT...

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“…88 (4) 26 (2) 72 (5) 174 (10) 104-1448 745-3499 858-3774 CD3-CD16/56+ NK cells /mm3 (%) 638 (29) 680 (66) 1123 (73) 1169 (68) 60-434 (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14) 194-994 (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14) 182-1581 (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13) IgG (mg/dL) [ Hercules, CA) and incubated with antibodies to nibrin (Novus, NB100-143 at 1:5000, Littleton, CO) overnight at 4°C. The immunoblots were subsequently incubated with an HRP-conjugated anti-rabbit secondary antibody at room temperature for 40 min for detection by enhanced chemiluminescence (ECL) (Amersham Pharmacia, Piscataway, NJ).…”

Section: Immunoblottingmentioning

confidence: 99%

Nijmegen Breakage Syndrome Detected By Newborn Screening for T Cell Receptor Excision Circles (TRECs)

Patel

Puck

Kundu³

et al. 2015

Journal of Allergy and Clinical Immunology

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Early diagnosis of ataxia-telangiectasia using radiosensitivity testing

Cited by 114 publications

References 32 publications

Cellular responses to ionising radiation of AT heterozygotes: differences between missense and truncating mutation carriers

Cellular responses to ionising radiation of AT heterozygotes: differences between missense and truncating mutation carriers

Correction of ATM gene function by aminoglycoside-induced read-through of premature termination codons

Nijmegen Breakage Syndrome Detected By Newborn Screening for T Cell Receptor Excision Circles (TRECs)

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