Mechanisms of cellular radiosensitivity and human molecular genetic disorders

The importance of determining the responses to DNA damage by ionizing radiation (IR) is reflected in the radiosensitivity, and cancer susceptibility of chromosomal instability syndromes. We investigated underlying causes of radiosensitivity in radiation sensitive lymphoblastoid cell lines, (RS-LCLs) with unknown molecular genetic defects.;In addition, heterozygotes for the chromosomal instability syndrome, Ataxia-Telangiectasia (A-T), are at a greater risk for cancer. Prompted by this, we developed a rapid flow cytometry-based assay for distinguishing A-T heterozygotes from the normal population.;To establish a foundation for studying the causes of radiosensitivity in RS-LCLs we analyzed IR-induced cellular responses in cells from patients with known chromosomal instability syndromes, such as A-T, NBS, AOA1, and MRE11. We expanded these studies to examine uncharacterized cells from Fanconi Anemia (FA), Ataxia Ocular Motor Apraxia 2, (AOA2) and Immunodeficiency, Chromosomal instability, Facial anomaly syndrome, (ICF) patients. Cells from FA patients were radiosensitive, whereas AOA2 and ICF patient cells were not. These findings allow us to use radiosensitivity testing to exclude a diagnosis of AOA2 and ICF.;We analyzed the mechanisms of RS using functional assays, (1) colony survival assay (CSA), (2) microarray expression profiling; (3) radioresistant DNA synthesis (RDS) (4) in-vitro DNA end joining, (DNA-EJ) and (5) mitochondrial resazurin reduction. In addition, we used cytogenetic techniques to investigate the integrity of the cells. Overall fourteen RS-LCLs had an S-phase delay defect, 5 were defective in DNA-EJ, 18 exhibited chromosomal aberrations, and 12 had reduced mitochondrial respiration. After interrogating proteins known to regulate each pathway, we focused on two RS-LCLs: a patient with a Chk2 kinase defect, and another with an aprataxin deficiency who was unexpectedly RS.;A clinical diagnosis of A-T takes, >3 months, and is confirmed by RS and absence of ATM protein. To expedite the time of diagnosis, we first developed a rapid immunoassay to measure ATM protein, ATM-ELISA. This assay diagnoses A-T within 2 days directly from whole blood, but requires a standard source of purified ATM protein and cannot identify A-T heterozygotes. As an adjunctive test, we developed a flow cytometry (FC)-based ATM kinase assay that measures ATM-dependent phosphorylation of Structural Maintenance Chromosomes-1 (SMC1) protein following DNA damage, allowing rapid identification of A-T homozygotes and heterozygotes.