Background: No rapid reliable method exists for identifying ataxia-telangiectasia (A-T) homozygotes or heterozygotes. Heterozygotes are at an increased risk of cancer and are more sensitive to the effects of ionizing radiation (IR) than the general population. We report a rapid flow cytometry (FC)-based ataxia-telangiectasia mutated (ATM) kinase assay that measures ATM- dependent phosphorylation of structural maintenance of chromosomes 1 (SMC1) following DNA damage (FC-pSMC1 assay).

Methods: After optimizing conditions with lymphoblastoid cell lines (LCLs), we studied peripheral blood mononuclear cells (PBMCs) isolated from 16 healthy donors (unknowns), 10 obligate A-T heterozygotes, and 6 unrelated A-T patients. One hour after DNA damage (by either IR or bleomycin), the cells were fixed and incubated with a primary antibody to SMC1pSer966. We analyzed the stained cells by FC to determine the difference in geometric mean fluorescence intensity (ΔGMFI) of untreated and treated cells; this difference was expressed as a percentage of daily experimental controls.

Results: The FC-pSMC1 assay reliably distinguished ATM heterozygotes and homozygotes from controls. Average ΔGMFI percentages (SD) of daily controls were, for unknowns, 106.1 (37.6); for A-T heterozygotes, 37.0 (18.7); and for A-T homozygotes; −8.73 (16.2). Values for heterozygotes and homozygotes were significantly different from those of controls (P < 0.0001).

Conclusions: The FC-pSMC1 assay shortens the turnaround time for diagnosing A-T homozygotes from approximately 3 months to approximately 3 h. It also identifies A-T heterozygotes and can be used for prenatal counseling or for screening individuals in large study cohorts for potential ATM heterozygosity, which can then be confirmed by sequencing.