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P53 Centrosomal Localization Diagnoses Ataxia-Telangiectasia

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P53 Centrosomal Localization Diagnoses Ataxia-Telangiectasia Technical advance p53 centrosomal localization diagnoses ataxia-telangiectasia homozygotes and heterozygotes Andrea Prodosmo,1 Andrea De Amicis,2 Cecilia Nisticò,3 Mario Gabriele,4 Giuliana Di Rocco,1 Laura Monteonofrio,1 Maria Piane,2 Enrico Cundari,4 Luciana Chessa,2 and Silvia Soddu1 1Department of Experimental Oncology, Regina Elena National Cancer Institute, Rome, Italy. 2Department of Clinical and Molecular Medicine, Sapienza University, Rome, Italy. 3Department of Clinical Oncology, Regina Elena National Cancer Institute, Rome, Italy. 4Institute of Molecular Biology and Pathology, National Research Council, Rome, Italy. Ataxia-telangiectasia (A-T) is an autosomal recessive neurodegenerative disorder characterized by radiosensi- tivity, genomic instability, and predisposition to cancer. A-T is caused by biallelic mutations in the ataxia-te- langiectasia 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 prog- ress 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. Introduction general population, though slight variations can be encountered Ataxia-telangiectasia (A-T) is a rare autosomal recessive multi- in different countries (2, 11, 12). A-T heterozygotes are usually systemic syndrome characterized by progressive cerebellar asymptomatic and largely considered healthy carriers, but com- degeneration, telangiectasias, immunodeficiency, recurrent pared with the general population, they have been reported to be infections, insulin-resistant diabetes, premature aging, radio- more vulnerable to ionizing radiation (IR) and to have a higher sensitivity, and high risk for malignancy, particularly leukemia risk of ischemic heart disease (2) and breast, stomach, and lung and lymphoma in children and epithelial cancers in surviving cancers (4). Though definitive information on these susceptibili- adults (1, 2). No effective disease-modifying therapy is presently ties is not yet available, regular surveillance of A-T carriers is con- available, though early supportive symptomatic medications sidered part of A-T management in carrier families. and aggressive physical stimulation can minimize infections and A-T diagnosis is based on the combination of clinical features with delay neurological symptoms (3). laboratory tests showing high levels of serum alpha-fetoprotein, cell In A-T, mutations in the ATM (ataxia-telangiectasia mutated) sensitivity to IR, and reduced or absent levels of ATM protein. None gene result in a lack or inactivation of the ATM protein, a ubiqui- of these tests, alone or in combination, is sufficiently sensitive and tous serine/threonine kinase of 3056 amino acids mainly involved specific for early differential diagnosis, genetic counseling, and car- in the maintenance of genomic stability (4–7). The ATM gene con- rier prediction (3). Because of the complex genomic organization of tains 66 exons spanning approximately 160 Kb of genomic DNA the ATM gene, its direct sequencing is not yet cost effective, particu- (8), and a large variety (>600) of mutations occurs across the full- larly for large-scale surveys of A-T carriers. A further level of complex- length transcript without hotspots. In addition, like other large ity is established by the fact that a majority of the ATM mutations genes, ATM possesses many polymorphisms that must be distin- in A-T patients are protein truncations or splice junction variants, guished from mutations (9, 10). while missense mutations in evolutionarily conserved residues are A-T is present throughout the world, with an incidence of 1 in more common in breast cancer (BC) patients than in controls (13). 10,000 to 100,000 newborns (11). However, because of the diffi- Functional assays that are able to distinguish between deleterious culties in diagnosis, particularly in those children who die at a and neutral alterations in A-T carriers are thus necessary. In the past young age, A-T might actually be more frequent than currently 3 decades, an intense effort has therefore been dedicated to devel- estimated. The theoretical frequency of A-T mutant allele hete- oping rapid and reliable methods for identifying A-T homozygotes rozygosity (A-T carriers) has been calculated as 1.4%–2% of the and heterozygotes. However, with the exception of gene sequencing, none of the assays presently available is unambiguously diagnostic Conflict of interest: The authors have declared that no conflict of interest exists. without the support of clinical symptoms or can identify A-T carriers Citation for this article: J Clin Invest. 2013;123(3):1335–1342. doi:10.1172/JCI67289. in the absence of a direct intratest comparison (14–21). The Journal of Clinical Investigation http://www.jci.org Volume 123 Number 3 March 2013 1335 technical advance Figure 1 p53 centrosomal localization in mitotic LCLs. (A) p53 centrosomal localization detected by confocal IF microscopy with anti-p53 (green) and anti–γ-tubulin (red) Abs. DNA was stained with DAPI (blue); representative image and its software analysis are shown in A. (B) Representative IF images of the indicated proteins in mitotic LCLs derived from the indicated donors. p53, centrosomes, and DNA are marked as in A. Scale bar = 5 μm. (C) Summary data of percentages of p53-MCL measured by 2 different operators who counted 100 metaphases per sample in quadruplicate. Data are expressed as mean ± SD. Four different families, each including 1 A-T patient (A-T), 1 parent (Htz), and 1 wtATM-carrying relative (WT) were evaluated. (D) Comparison of p53-MCL percentages, evaluated as in C, between LCLs derived from wtATM donors (n = 11), A-T carriers (n = 20), and A-T patients (n = 10). The 3 groups are significantly different. Box-and-whisker plot: horizontal lines within the box represent median values, and the bottom and top of the box show the lower and upper quartiles. ***P < 0.0001; 2-tailed Student’s t test. In the process of studying the role of the tumor suppressor Results p53 in response to mitotic inhibitory drugs, we discovered that, p53 mitotic centrosomal localization determines mutant ATM zygos- in mitosis, p53 localizes at the centrosomes in an ATM-depen- ity. Centrosomes duplicate at each cell cycle and are the major dent manner and monitors mitotic spindle integrity (Figure 1A) organizers of the bipolar mitotic spindle. A number of differ- (22–24). These findings led us to propose that both the ATM and ent proteins reside at the centrosome permanently, while some p53 proteins might contribute to the “centrosomal checkpoint,” a transiently localize to the centrosome during the cell cycle or in network that integrates cell cycle arrest and repair signals (25, 26). response to damage (27). Since we previously demonstrated that Although a clear mechanistic proof for this hypothesis remains p53 moves to centrosomes at each mitosis in an ATM-dependent to be established, we show here that p53 does not localize at manner (22–24), we blindly measured the percentage of mitotic the centrosomes in almost 100% of mitotic lymphoblastoid cell cells with p53 centrosomal localization (p53 mitotic centrosomal lines (LCLs) or PBMCs derived from A-T patients. Surprisingly, localization, or p53-MCL) in EBV-derived LCLs obtained from 4 we consistently observed that in A-T heterozygous carriers, p53 A-T patients, 1 of their parents (the obligate A-T carriers), and localizes at the centrosomes in approximately 50% of the mitotic from 4 wild-type ATM (wtATM) relatives (families 1–4). To this cells. Based on these findings, we have developed a straightfor- end, a double indirect immunofluorescence (IF) analysis was per- ward, rapid, and inexpensive test to unambiguously diagnose A-T formed with anti-p53 and anti–γ-tubulin Abs (see Methods for homozygotes and heterozygotes. details). We observed 3 completely different behaviors: wtATM 1336 The Journal of Clinical Investigation http://www.jci.org Volume 123 Number 3 March 2013 technical advance Table 1 ATM genomic mutations, consequences, and protein levels in LCLs from A-T patients and carriers LCLs p53-MCL (%) Status ATM genomic mutation Consequence ATM protein levelA AT44RM 0 A-T c.1607+1G>T p.(Cys536fs) 0 AT28RM 0 A-T c.7792C>T/c.8283_8284delTC p.(Arg2598X)/p.(Ser2762fs) 0 AT91RM 9 A-T c.7408T>G p.(Tyr2470Asp) 50 AT24RM 9 A-T c.755_756delGT p.(Cys252fs) 0 AT52RM 29 A-T c.7327C>T/c.8368_8368delA p.(Arg2443X)/p.(Arg2790fs) 0 AT65RM 24 A-T c.6573-9G>A/c.8814_8824del11 p.(Ser2192fs)/p.(Met2938fs) 0 AT50RM 30 A-T c.5319+2T>C/c.5692C>T p.(Cys1726fs)/p.(Arg1898X) 0 AT58RM 28 A-T c.2250G>A/c.8122G>A p.(Glu709_Lys750del42)/p.(Asp2708Gln) 0 AT62RM 28 A-T c.1524_1524delT/c.5979_5983delTAAAG p.(Leu508fs)/p.(Ser1993fs) 0 247RM 49 Htz c.1607+1G>T/N p.(Cys536fs) 45 374RM 40 Htz c.8283_8284delTC/N p.(Ser2762fs) n.d. 248RM 54 Htz c.1607+1G>T/N p.(Cys536fs) n.d. 458RM 52.5 Htz c.6573-9G>A/N p.(Ser2192fs) n.d.
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