p53 centrosomal localization diagnoses ataxia-telangiectasia homozygotes and heterozygotes
Andrea Prodosmo, … , Luciana Chessa, Silvia Soddu
Andrea Prodosmo, … , Luciana Chessa, Silvia Soddu
Published February 1, 2013
Citation Information: J Clin Invest. 2013;123(3):1335-1342. https://doi.org/10.1172/JCI67289.
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Technical Advance Genetics

p53 centrosomal localization diagnoses ataxia-telangiectasia homozygotes and heterozygotes

Abstract

Ataxia-telangiectasia (A-T) is an autosomal recessive neurodegenerative disorder characterized by radiosensitivity, genomic instability, and predisposition to cancer. A-T is caused by biallelic mutations in the ataxia-telangiectasia mutated (ATM) gene, but heterozygous carriers, though apparently healthy, are believed to be at increased risk for cancer and more sensitive to ionizing radiation than the general population. Despite progress in functional and sequencing-based assays, no straightforward, rapid, and inexpensive test is available for the identification of A-T homozygotes and heterozygotes, which is essential for diagnosis, genetic counseling, and carrier prediction. The oncosuppressor p53 prevents genomic instability and centrosomal amplification. During mitosis, p53 localizes at the centrosome in an ATM-dependent manner. We capitalized on the latter finding and established a simple, fast, minimally invasive, reliable, and inexpensive test to determine mutant ATM zygosity. The percentage of mitotic lymphoblasts or PBMCs bearing p53 centrosomal localization clearly discriminated among healthy donors (>75%), A-T heterozygotes (40%–56%), and A-T homozygotes (<30%). The test is specific for A-T, independent of the type of ATM mutations, and recognized tumor-associated ATM polymorphisms. In a preliminary study, our test confirmed that ATM is a breast cancer susceptibility gene. These data open the possibility of cost-effective, early diagnosis of A-T homozygotes and large-scale screenings for heterozygotes.

Authors

Andrea Prodosmo, Andrea De Amicis, Cecilia Nisticò, Mario Gabriele, Giuliana Di Rocco, Laura Monteonofrio, Maria Piane, Enrico Cundari, Luciana Chessa, Silvia Soddu

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Figure 3

Assessment of p53-MCL test precision.

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Assessment of p53-MCL test precision. (A) Summary data of p53-MCL percen...
(A) Summary data of p53-MCL percentages in PBMCs and LCLs derived from 1 A-T patient and 1 healthy donor. No significant difference was observed between the 2 groups. Two-tailed Student’s t test. (B) Summary data of p53-MCL percentages in fresh PBMCs and frozen and thawed PBMCs. No significant difference was observed after freezing and thawing PBMCs from 9 different A-T carriers. (C) Intraday assay variability testing was performed on a single blood sample from 1 healthy donor as follows: PBMCs were PHA-stimulated for 60 hours and used to make 8 different coverslip preparations, each of which was immunostained at the indicated times after fixation. No significant difference was observed between the preparations. (D) Interday assay variability was performed by collecting and assessing PBMCs from the same healthy donor on 5 different days. No significant difference was observed among the samples. (E) To verify the minimal amount of blood required to perform the test, we collected 15 ml of blood from a single healthy donor and made 5 aliquots with different amounts of blood (from 1 to 5 ml). The test performed on PBMCs purified from the 5 different samples showed no significant differences, demonstrating that the assay can be performed with as little as 1 ml of whole blood. A 2-tailed Student’s t test was performed. All samples were analyzed by examining 100 metaphases per sample in quadruplicate. Data are expressed as mean ± SD. NS, not significant.