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 13.22%. Sensitivity and specificity were virtually 100% when the test was performed on PBMCs.

p53 centrosomal localization diagnoses ataxia-telangiectasia homozygotes and heterozygotes

“…The second reason is linked to the quantitative differences induced by the diverse amounts of wildtype protein present in A-T carriers compared with that found in wild-type homozygotes and A-T homozygotes. Because of the huge number of mutations and the complexity of the pathways analyzed in the different assays, these quantitative differences consist of a continuum of partially overlapping readouts that are unable to yield unambiguous results (13,(15)(16)(17)(18)(19)(20)(21). These problems have a limited impact on the identification of A-T homozygotes, but constitute a formidable obstacle to carrier prediction.…”

Underexpression and abnormal localization of ATM products in ataxia telangiectasia patients bearing ATM missense mutations

“…FACS analysis of H2AX phosphorylation (gH2AX) after IR was shown to correctly discriminate A-T cell lines from cell lines derived from normal individuals as well as from related syndromes such as Nijmegen Breakage Syndrome, A-T-like disease, Friedreich ataxia and ataxia with oculomotor apraxia type 2 (AOA2). 24 LCLs were irradiated at a dose of 5 Gy and the H2AX phosphorylation status was measured by FACS at the peak of phosphorylation (1.5 h). As shown in Figures 1a and b, the intensity of the gH2AX peak was markedly decreased in an A-T control cell line as compared with nine WT cell lines.…”

Measurement of H2AX Phosphorylation as a Marker of Ionizing Radiation Induced Cell Damage

“…This finding has been confirmed in other studies using similar methods (Bourton, Plowman et al). Flow cytometry measurement of gamma-H2AX has been used to diagnose Ataxia telangiectasia, a disease where patients are radiosensitive and show a delayed clearance of H2AX foci after in vitro irradiation of lymphocytes (Porcedda, Turinetto et al 2008). Similar methods have been used to find patients with severe normal tissue toxicity following radiotherapy (Bourton, Plowman et al) and to study the gamma-H2AX response in several subsets of nucleated blood cells (Andrievski and Wilkins 2009).…”