The ataxia telangiectasia-mutated (ATM) protein is a major coordinator of the DNA damage response pathway. ATM loss-of-function variants are associated with 2-fold increased breast cancer risk. We... Show moreThe ataxia telangiectasia-mutated (ATM) protein is a major coordinator of the DNA damage response pathway. ATM loss-of-function variants are associated with 2-fold increased breast cancer risk. We aimed at identifying and classifying spliceogenic ATM variants detected in subjects of the large-scale sequencing project BRIDGES. A total of 381 variants at the intron-exon boundaries were identified, 128 of which were predicted to be spliceogenic. After further filtering, we ended up selecting 56 variants for splicing analysis. Four functional minigenes (mgATM) spanning exons 4-9, 11-17, 25-29, and 49-52 were constructed in the splicing plasmid pSAD. Selected variants were genetically engineered into the four constructs and assayed in MCF-7/HeLa cells. Forty-eight variants (85.7%) impaired splicing, 32 of which did not show any trace of the full-length (FL) transcript. A total of 43 transcripts were identified where the most prevalent event was exon/multi-exon skipping. Twenty-seven transcripts were predicted to truncate the ATM protein. A tentative ACMG/AMP (American College of Medical Genetics and Genomics/Association for Molecular Pathology)-based classification scheme that integrates mgATM data allowed us to classify 29 ATM variants as pathogenic/likely pathogenic and seven variants as likely benign. Interestingly, the likely pathogenic variant c.1898+2T>G generated 13% of the minigene FL-transcript due to the use of a noncanonical GG-5'-splice-site (0.014% of human donor sites). Circumstantial evidence in three ATM variants (leakiness uncovered by our mgATM analysis together with clinical data) provides some support for a dosage-sensitive expression model in which variants producing >= 30% of FL-transcripts would be predicted benign, while variants producing <= 13% of FL-transcripts might be pathogenic. (c) 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland. Show less
Sampadi, B.; Mullenders, L.H.F.; Vrieling, H. 2022
Background: Although cancer risk is assumed to be linear with ionizing radiation (IR) dose, it is unclear to what extent low doses (LD) of IR from medical and occupational exposures pose a cancer... Show moreBackground: Although cancer risk is assumed to be linear with ionizing radiation (IR) dose, it is unclear to what extent low doses (LD) of IR from medical and occupational exposures pose a cancer risk for humans. Improved mechanistic understanding of the signaling responses to LD may help to clarify this uncertainty. Here, we per -formed quantitative mass spectrometry-based proteomics and phosphoproteomics experiments, using mouse embryonic stem cells, at 0.5 h and 4 h after exposure to LD (0.1 Gy) and high doses (HD; 1 Gy) of IR. Results: The proteome remained relatively stable (29; 0.5% proteins responded), whereas the phosphoproteome changed dynamically (819; 7% phosphosites changed) upon irradiation. Dose-dependent alterations of 25 IR-responsive proteins were identified, with only four in common between LD and HD. Mitochondrial metabolic proteins and pathways responded to LD, whereas transporter proteins and mitochondrial uncoupling pathways responded to HD. Congruently, mitochondrial respiration increased after LD exposure but decreased after HD exposure. While the bulk of the phosphoproteome response to LD (76%) occurred already at 0.5 h, an equivalent proportion of the phosphosites responded to HD at both time points. Motif, kinome/phosphatome, kinase-substrate, and pathway analyses revealed a robust DNA damage response (DDR) activation after HD exposure but not after LD exposure. Instead, LD-irradiation induced (de)phosphorylation of kinases, kinase-substrates and phosphatases that predominantly respond to reactive oxygen species (ROS) production. Conclusion: Our analyses identify discrete global proteome and phosphoproteome responses after LD and HD, uncovering novel proteins and protein (de)phosphorylation events involved in the dose-dependent ionizing ra-diation responses. Show less
