Journal
HUMAN MUTATION
Volume 30, Issue 1, Pages 12-21Publisher
WILEY
DOI: 10.1002/humu.20805
Keywords
missense mutations; mutagenesis; ATM; cancer risk
Categories
Funding
- Joseph Drown Foundation
- Ataxia-Telangiectasia Medical Research Foundation
- National Institutes of Health (NIH) [NS35322, NS052528, AI067769]
- NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES [U19AI067769] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE [R01NS035322, R01NS052528] Funding Source: NIH RePORTER
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The functional consequences of missense variants are often difficult to predict. This becomes especially relevant when DNA sequence changes are used to determine a diagnosis or prognosis. To analyze the consequences of 12 missense variants in patients with mild forms of ataxia-telangiectasia (A-T), we employed site-directed mutagenesis of ataxia-telangiectasia mutated (ATM) cDNA followed by stable transfections into a single A-T cell line to isolate the effects of each allele on the cellular phenotype. After induction of the transfected cells with CdCl2, we monitored for successful ATM transcription and subsequently assessed: 1) intracellular ATM protein levels; 2) ionizing radiation (IR)-induced ATM kinase activity; and 3) cellular radiosensitivity. We then calculated SIFT and PolyPhen scores for the missense changes. Nine variants produced little or no correction of the AT cellular phenotype and were interpreted to be ATM mutations; SIFT/PolyPhen scores supported this. Three variants corrected the cellular phenotype, suggesting that they represented benign variants or polymorphisms. SIFT and PolyPhen scores supported the functional analyses for one of these variants (c.1709T > C); the other two were predicted to be not tolerated (c.6188G > A and c.6325T > G) and were classified, as operationally neutral. Genotype/phenotype relationships were compared: three deleterious missense variants were associated with an increased risk of cancer (c.6679C > T, c.7271T > G, and c.8494C > T). In situ mutagenesis represents an effective experimental approach for distinguishing deleterious missense mutations from benign or operationally neutral missense variants.
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