Identification of ataxia telangiectasia heterozygotes by flow cytometric analysis of X-ray damage.

Flow cytometry was used to identify heterozygotes for the autosomal recessive DNA-repair deficiency disease ataxia telangiectasia (AT). Confluent G0/G1 fibroblasts from 4 homozygotes (at/at), 5 obligate heterozygotes (at/+) and 7 presumed normal controls (+/+) were X-irradiated with 200 Rad and subcultured immediately in medium containing 5-bromodeoxyuridine (BrdU). Cells were harvested 72 h later and stained with fluoresceinated anti-BrdU antibody to identify cells that had entered S phase. They were counterstained with propidium iodide to measure total DNA content. On the basis of relative release from G0/G1, the at/+ strains as a group (33 +/- 3% release) were distinguished from both the presumed +/+ strains (60 +/- 3%) and at/at strains (85 +/- 3%), although the individual values for some strains did show overlap between genotypes. When 10 cell strains were coded and analyzed in 'blind' experiments, all 4 heterozygotes were correctly assigned, although one poorly growing presumed normal line was incorrectly assigned as a heterozygote. By a similar assay in which exponentially growing cultures were pulsed briefly with BrdU 8 h after irradiation with 400 Rad and then harvested immediately, presumed +/+ cells as a group could be distinguished from at/at cells but not from at/- cells. This combination of assays assists in the identification of all 3 AT genotypes. This should be of both basic and diagnostic use, particularly in families known to segregate AT.