Homozygous mutation of MTPAP causes cellular radiosensitivity and persistent DNA double-strand breaks.

The study of rare human syndromes characterized by radiosensitivity has been instrumental in identifying novel proteins and pathways involved in DNA damage responses to ionizing radiation. In the present study, a mutation in mitochondrial poly-A-polymerase (MTPAP), not previously recognized for its role in the DNA damage response, was identified by exome sequencing and subsequently associated with cellular radiosensitivity. Cell lines derived from two patients with the homozygous MTPAP missense mutation were radiosensitive, and this radiosensitivity could be abrogated by transfection of wild-type mtPAP cDNA into mtPAP-deficient cell lines. Further analysis of the cellular phenotype revealed delayed DNA repair, increased levels of DNA double-strand breaks, increased reactive oxygen species (ROS), and increased cell death after irradiation (IR). Pre-IR treatment of cells with the potent anti-oxidants, α-lipoic acid and n-acetylcysteine, was sufficient to abrogate the DNA repair and clonogenic survival defects. Our results firmly establish that mutation of the MTPAP gene results in a cellular phenotype of increased DNA damage, reduced repair kinetics, increased cell death by apoptosis, and reduced clonogenic survival after exposure to ionizing radiation, suggesting a pathogenesis that involves the disruption of ROS homeostasis.

Increased frequency of ATM mutations in breast carcinoma patients with early onset disease and positive family history.

BACKGROUND: An increased incidence of breast carcinoma has been reported among relatives of individuals who are affected with the rare recessive disorder, ataxia-telangiectasia (A-T), and who are heterozygous for mutations in the ataxia-telangiectasia mutated (ATM) gene. However, most studies of breast carcinoma cases from the general population have failed to find a higher incidence of ATM mutations in cases when compared with controls. METHODS: Genomic DNA samples from 258 individuals were screened for mutations of all types in each of the 62 coding exons of the ATM gene; 142 of these were from breast carcinoma cases with a first-degree family history or early age at diagnosis, 35 were from cases selected for the presence of either known disease-related mutations (n = 25) or missense alterations of unknown consequences (n = 10) in BRCA1 or BRCA2, and 81 were from matched controls. RESULTS: A total of 12 individuals with ATM mutations were identified, 11 among 142 breast carcinoma cases (7.7%; 95% CI, 3.9-13.4%) and 1 among 81 controls (1.2%; 95% CI, 0.0-6.7%) (P = 0.06). All mutations detected were of the missense type; none were predicted to truncate the ATM protein. Among cases, mutations were found exclusively in patients with a family history of breast carcinoma (12.1%; 95% CI, 6.2-20.6%) (P = 0.02). Similar frequencies of ATM mutations were found in 35 additional cases selected for the presence of BRCA1 or BRCA2 mutations when compared with cases overall. CONCLUSIONS: ATM mutations, specifically missense mutations, are more common in breast carcinoma cases selected for first-degree family history and early age at diagnosis.

Splicing defects in the ataxia-telangiectasia gene, ATM: underlying mutations and consequences.

Mutations resulting in defective splicing constitute a significant proportion (30/62 [48%]) of a new series of mutations in the ATM gene in patients with ataxia-telangiectasia (AT) that were detected by the protein-truncation assay followed by sequence analysis of genomic DNA. Fewer than half of the splicing mutations involved the canonical AG splice-acceptor site or GT splice-donor site. A higher percentage of mutations occurred at less stringently conserved sites, including silent mutations of the last nucleotide of exons, mutations in nucleotides other than the conserved AG and GT in the consensus splice sites, and creation of splice-acceptor or splice-donor sites in either introns or exons. These splicing mutations led to a variety of consequences, including exon skipping and, to a lesser degree, intron retention, activation of cryptic splice sites, or creation of new splice sites. In addition, 5 of 12 nonsense mutations and 1 missense mutation were associated with deletion in the cDNA of the exons in which the mutations occurred. No ATM protein was detected by western blotting in any AT cell line in which splicing mutations were identified. Several cases of exon skipping in both normal controls and patients for whom no underlying defect could be found in genomic DNA were also observed, suggesting caution in the interpretation of exon deletions observed in ATM cDNA when there is no accompanying identification of genomic mutations.