title: A rapid flow cytometry test based on histone H2AX phosphorylation for the sensitive and specific diagnosis of ataxia telangiectasia.
abstract: Ataxia telangiectasia (A-T) is a progressive neurodegenerative disease with onset in early childhood, caused by mutations in the ATM (ataxia-telangiectasia mutated) gene. Diagnosis relies on laboratory tests showing high levels of serum alphafetoprotein, cell sensitivity to ionizing radiation (IR) and absence or reduced levels of ATM protein. Many tests, however, are not sufficiently sensitive or specific for A-T, have long turnaround times, or require large blood samples. This prompted us to develop a new flow cytometry method for the diagnosis of A-T based on the measurement of histone H2AX phosphorylation. We established normal ranges of histone H2AX phosphorylation after 2 Gy IR by testing T-cell lines, lymphoblastoid cell lines (LCLs) and/or peripheral blood mononuclear cells (PBMCs) or both from 20 genetically proven A-T and 46 control donors. To further evaluate the specificity and sensitivity of the test, we analyzed cells from 19 patients suspected of having A-T, and from one Friedreich Ataxia, one Ataxia with Oculomotor Apraxia type 2, and one Nijmegen Breakage Syndrome patients. Phosphorylated histone H2AX mean fluorescence intensity of irradiated A-T cells was significantly lower than that of healthy donors. The intrastaining, intraassay, and interassay imprecisions were <or=13.22%. Sensitivity and specificity were virtually 100% when the test was performed on PBMCs. Screening of 19 consecutive new patients with suspected A-T classified 15 patients as non-A-T and four as A-T; diagnosis of the latter four was subsequently confirmed by DNA sequencing to identify ATM mutations. The Friedreich Ataxia patient, the Ataxia with Oculomotor Apraxia type 2 patient and the Nijmegen Breakage Syndrome patient were classified as non-A-T. This flow cytometry test is very sensitive, specific and rapid, and requires only 2 ml of blood. It may thus be proposed for the early differential diagnosis of A-T as an alternative to methods requiring the production of LCLs.
authors.affiliation: Department of Clinical and Biological Sciences, University of Turin, Turin, Italy. paola.porcedda@unito.it
authors.forename: Paola
authors.initials: P
authors.lastname: Porcedda
authors.forename: Valentina
authors.initials: V
authors.lastname: Turinetto
authors.forename: Alfredo
authors.initials: A
authors.lastname: Brusco
authors.forename: Simona
authors.initials: S
authors.lastname: Cavalieri
authors.forename: Erica
authors.initials: E
authors.lastname: Lantelme
authors.forename: Luca
authors.initials: L
authors.lastname: Orlando
authors.forename: Umberto
authors.initials: U
authors.lastname: Ricardi
authors.forename: Antonio
authors.initials: A
authors.lastname: Amoroso
authors.forename: Dario
authors.initials: D
authors.lastname: Gregori
authors.forename: Claudia
authors.initials: C
authors.lastname: Giachino
mesh_terms: D001260:Ataxia Telangiectasia / Q000175:diagnosis*; D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics; D003937:Diagnosis, Differential; D005434:Flow Cytometry / Q000379:methods*; D006657:Histones / Q000378:metabolism* / Q000528:radiation effects; D006801:Humans; D015151:Immunoblotting; D007963:Leukocytes, Mononuclear / Q000378:metabolism / Q000528:radiation effects; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D025521:Tumor Suppressor Proteins / Q000235:genetics
chemical_list: D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C468783:H2AX protein, human; D006657:Histones; D025521:Tumor Suppressor Proteins; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases