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.
Rudolph
N S
NS
Division of Genetics and Mental Retardation Center, Children's Hospital, Boston, MA 02115.
Nagasawa
H
H
Little
J B
JB
Latt
S A
SA
D000293:Adolescent; D000328:Adult; D001260:Ataxia Telangiectasia / Q000235:genetics* / Q000473:pathology; D002478:Cells, Cultured; D002648:Child; D002675:Child, Preschool; D004260:DNA Repair / Q000528:radiation effects*; D005260:Female; D005347:Fibroblasts / Q000528:radiation effects; D005434:Flow Cytometry; D006580:Genetic Carrier Screening; D005838:Genotype / Q000528:radiation effects; D006801:Humans; D007231:Infant, Newborn; D007399:Interphase / Q000528:radiation effects; D008297:Male; D008875:Middle Aged