This research proposal is focused on cross-validating three complementary genome integrity assays in multiple types of primary human cells from different individuals. In addition to a repeated measures study with multiple blood draws from the same individuals over time, we will compare genome integrity assays in multiple types of blood cells, fibroblasts, and keratinocytes from the same individuals. These samples are from participants in IRB approved studies supported by the parent projects, and will be shared without identifying information. Each of the three parent projects involved in this highly synergistic collaborative supplement will benefit from cross- testing of assays to reveal possible sources of error that may impact a single assay, but are unlikely to impact all three. Multiple complementary data streams will also enable a more comprehensive understanding the biological implications of the assay outputs. In particular, the project lead by Dr. Nagel will benefit by having a second approach, namely a fluorescence based unscheduled DNA synthesis assay that measures repair in the context of chromatin, and can be directly compared to fluorescence multiplex host cell reactivation (FM-HCR) assays, particularly those for nucleotide excision repair. The project lead by Dr. Niedernhofer will benefit in a reciprocal way. The project lead by Dr. Vijg will benefit from the establishment of a platform for using functional data to distinguish mutations arising from elevated exposure to DNA damaging agents from those arising from inefficient DNA repair. The projects led by Niedernhofer and Nagel will also both benefit from determining the extent to which DNA repair deficiencies as detected using functional assays in ex vivo biological samples are reflected in the number of mutations acquired in tissues from the same individuals in vivo. By determining the robustness and reproducibility of our assays, and by benchmarking them against other approaches in primary samples from human subjects with defined defects in the NER pathway, this supplement also advances the overall objective of the consortium, namely to facilitate the wider use of genome integrity assays in human populations.
Projective Narrative Several important questions regarding the application of genome integrity assays in human populations require cross-testing of multiple assays in different types of human tissues. These include whether apparent inter- individual variation is consistent across tissues, over time, and by different assays. We will address these questions and advance the use of genome integrity assays in human populations by comparing two functional assays with mutation assays in a multiple human cell types from different individuals.
