NGS Detects Extensive Genomic Alterations in Survivors of Irradiated Normal Human Fibroblast Cells

It is thought that cells surviving ionizing radiation exposure repair DNA double-strand breaks (DSBs) and restore their genomes. However, the recent biochemical and genetic characterization of DSB repair pathways reveals that only homologous recombination (HR) can function in an error-free manner and that the non-homologous end joining (NHEJ) pathways canonical NHEJ (c-NHEJ), alternative end joining (alt-EJ), and single-strand annealing (SSA) are error-prone, and potentially leave behind genomic scars and altered genomes. The strong cell cycle restriction of HR to S/G2 phases and the unparalleled efficiency of c-NHEJ throughout the cell cycle, raise the intriguing question as to how far a surviving cell "reaches" after repairing the genome back to its pre-irradiation state. Indeed, there is evidence that the genomes of cells surviving radiation treatment harbor extensive genomic alterations. To directly investigate this possibility, we adopted next-generation sequencing (NGS) technologies and tested a normal human fibroblast cell line, 82-6 hTert, after exposure up to 6 Gy. Cells were irradiated and surviving colonies expanded and the cells froze. Sequencing analysis using the Illumina sequencing platform and comparison with the unirradiated genome detected frequent genomic alterations in the six investigated radiation survivor clones, including translocations and large deletions. Translocations detected by this analysis and predicted to generate visible cytogenetic alterations were frequently (three out of five) confirmed using mFISH cytogenetic analysis. PCR analysis of selected deletions also confirmed seven of the ten examined. We conclude that cells surviving radiation exposure tolerate and pass to their progeny a wide spectrum of genomic alterations. This recognition needs to be integrated into the interpretation of biological results at all endpoints, as well as in the formulation of mathematical models of radiation action. NGS analysis of irradiated genomes promises to enhance molecular cytogenetics by increasing the spectrum of detectable genomic alterations and advance our understanding of key molecular radiobiological effects and the logic underpinning DSB repair. However, further developments in the technology will be required to harness its full potential.