title: Rapid flow cytometry-based structural maintenance of chromosomes 1 (SMC1) phosphorylation assay for identification of ataxia-telangiectasia homozygotes and heterozygotes.
abstract: 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 (DeltaGMFI) 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 DeltaGMFI 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.
authors.affiliation: Molecular Toxicology Interdepartmental Doctoral Program, UCLA School of Medicine and School of Public Health, Los Angeles, CA 90095, USA.
authors.forename: Shareef A
authors.initials: SA
authors.lastname: Nahas
authors.forename: Anthony W
authors.initials: AW
authors.lastname: Butch
authors.forename: Liutao
authors.initials: L
authors.lastname: Du
authors.forename: Richard A
authors.initials: RA
authors.lastname: Gatti
mesh_terms: D001260:Ataxia Telangiectasia / Q000175:diagnosis* / Q000235:genetics / Q000378:metabolism*; D064007:Ataxia Telangiectasia Mutated Proteins; D001761:Bleomycin / Q000494:pharmacology; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002868:Chromosomal Proteins, Non-Histone / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005189:False Positive Reactions; D005434:Flow Cytometry / Q000379:methods*; D042822:Genomic Instability / Q000235:genetics; D006579:Heterozygote; D006720:Homozygote; D006801:Humans; D010766:Phosphorylation / Q000187:drug effects / Q000528:radiation effects; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D013997:Time Factors; D025521:Tumor Suppressor Proteins / Q000378:metabolism; D000098550:Structural Maintenance of Chromosome Protein 1
chemical_list: D018797:Cell Cycle Proteins; D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; D025521:Tumor Suppressor Proteins; D000098550:Structural Maintenance of Chromosome Protein 1; D001761:Bleomycin; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases