Increased Oxidative Stress and Genomic Instability in Peripheral Blood and Bone Marrow of Individuals with Fanconi Anemia,

Abstract 3425

Impaired anti-oxidant defenses and overproduction of reactive oxygen species (ROS) in individuals with Fanconi anemia (FA) are associated with a pro-oxidant state in this population. Cells derived from individuals with FA demonstrate increased sensitivity to DNA damage by ambient oxygen and increased frequency of chromosomal aberrations. Studies in animals and human subjects indicate that higher levels of and increased sensitivity to both ROS and tumor necrosis factor-alpha in individuals with FA play key roles in the pathogenesis of bone marrow failure (BMF) and neoplastic transformation. Accumulation of somatic DNA damage can be assessed using the Glycophorin A assay (GPA), which is designed to detect potentially inactivating mutations caused by chromosome loss, large deletions, or recombination events at the erythrocyte GPA locus. Previous cross-sectional studies in individuals with FA have shown a markedly increased frequency of GPA variant cells, demonstrating increased DNA damage. Knowledge regarding the timecourse of the accumulation of this somatic DNA damage is lacking. The goal of the current study is to evaluate serum ROS levels and accumulation of somatic DNA damage in individuals with FA to better understand the contribution of oxidative stress to disease phenotype and progression to marrow failure or malignant transformation. ROS levels were assessed ex vivo from peripheral blood and bone marrow of individuals with FA and healthy controls. We used a flow cytometric method to quantify ROS level by incubation of samples with CM-H2DCFDA, a cell-permeable fluorescence dye that reacts to a broad spectrum of ROS. Peripheral blood was also serologically typed on first submission using anti-M and anti-N sera. As only heterozygous (MN) individuals are informative, only these individuals were studied further. We used the GPA assay to quantify the accumulation of somatic cell damage in peripheral blood samples as measured by GPA variant cell frequencies. Peripheral blood and/or bone marrow samples from 19 individuals with FA and normal controls were evaluated for ROS on 23 occasions. ROS levels were variable, with 13 of the FA patients demonstrating high levels of ROS as measured by MFI signal strength. Overall ROS levels in individuals with FA were higher than those of normal control individuals (132% of control ROS levels, p<0.09, see Table). When stratified by age, older individuals with FA (age ≥10 years) had significantly higher ROS than normal controls (159% of control levels, p<0.05). Evaluation of 32 children at our center with FA by MN genotyping has identified 13 who are MN heterozygotes eligible for GPA testing. The frequency of NO and NN variant cell frequencies were markedly increased in children with FA (range of 1.2 to 187.5 fold and 0.46 to 201.8 fold over controls respectively). Additionally, longitudinal samples over a 5 year period revealed stable elevation of NO variant frequency with increasing NN variant cell frequency. NN variant frequency was closer to normal in younger individuals with FA and progressively increased with age and development of bone marrow failure.

We demonstrate that individuals with FA have increased levels of ROS, and that ROS seems to increase with age. This correlates with accumulation of somatic DNA damage over time in this patient population. We hypothesize that ROS levels are low in younger FA individuals and progressively increase with age, resulting in accumulation of DNA damage. Additional longitudinal studies evaluating timecourse, intra-individual variability, and effects of complementation as well as environmental triggers are underway. We expect these studies to identify biomarkers that allow for earlier diagnosis leading to more timely therapy and to elucidate additional targets for novel therapeutic interventions to prevent BMF and myelodysplastic/leukemic transformation.