title: TaqMan PCR-based gene dosage assay for predictive testing in individuals from a cancer family with INK4 locus haploinsufficiency.
abstract: BACKGROUND
  A genetic syndrome of cutaneous malignant melanoma and nervous system tumors recently has been characterized and shown to be linked to the INK4 locus in the 9p21 region. Hemizygosity at adjacent physically mapped microsatellite markers indicated deletion of p16, p19, and p15 clustered tumor suppressors. Because individuals from this family could benefit from predictive testing in terms of cancer prevention, we developed a direct test without need to analyze parental DNAs to comply with the rules of individual consent and secrecy.
  
  
  METHODS
  We developed an assay using TaqManTM real-time quantitative PCR, with p15 as the test sequence and albumin (ALB) as the reference gene. The normalized ratio of p15/ALB is expected to yield a value of approximately 1 in individuals without the deletion, whereas a ratio of approximately 0.5, indicating p15 haploinsufficiency, is expected in predisposed individuals.
  
  
  RESULTS
  All patients harboring the previously defined at-risk haplotype were correctly identified using this approach. In six individuals with deletions, the p15/ALB ratios were 0.472-0.556 (SD, 0.013-0.078). In the five individuals without deletions, the ratios were 0.919-1.019 (SD, 0.006-0.075).
  
  
  CONCLUSIONS
  This is the first report of a high-throughput, automatable gene dosage assay successfully applied to the identification of a germ-line deletion. This approach, not limited by marker informativeness or the need for harvesting live cells, can be applied to any condition with haploinsufficiency and extended to the characterization of most abnormalities of the ploidy.
authors.affiliation: Laboratoire de Génétique Moléculaire, Faculté des Sciences Pharmaceutiques et Biologiques, Université Paris V, 4 Avenue de l'Observatoire, 75006 Paris, France.
authors.forename: I
authors.initials: I
authors.lastname: Laurendeau
authors.forename: M
authors.initials: M
authors.lastname: Bahuau
authors.forename: N
authors.initials: N
authors.lastname: Vodovar
authors.forename: C
authors.initials: C
authors.lastname: Larramendy
authors.forename: M
authors.initials: M
authors.lastname: Olivi
authors.forename: I
authors.initials: I
authors.lastname: Bieche
authors.forename: M
authors.initials: M
authors.lastname: Vidaud
authors.forename: D
authors.initials: D
authors.lastname: Vidaud
mesh_terms: D002352:Carrier Proteins / Q000235:genetics*; D018797:Cell Cycle Proteins; D050762:Cyclin-Dependent Kinase Inhibitor p15; D019941:Cyclin-Dependent Kinase Inhibitor p16; D018628:Gene Dosage; D016147:Genes, Tumor Suppressor; D006239:Haplotypes; D006801:Humans; D008545:Melanoma / Q000235:genetics*; D009423:Nervous System Neoplasms / Q000235:genetics*; D010375:Pedigree; D016133:Polymerase Chain Reaction; D013577:Syndrome; D019914:Taq Polymerase; D025521:Tumor Suppressor Proteins
chemical_list: C493966:CDKN2B protein, human; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D050762:Cyclin-Dependent Kinase Inhibitor p15; D019941:Cyclin-Dependent Kinase Inhibitor p16; D025521:Tumor Suppressor Proteins; D019914:Taq Polymerase