We provide here a preliminary setup and validation of a new functional assay for evaluating mutations in the human ATM gene by measuring the percentage of mitotic cells with p53 localization at the centrosome. At variance with existing tests, p53-MCL unambiguously distinguishes between A-T homozygotes and heterozygotes in a very highly sensitive and specific manner. Indeed, the results obtained by analyzing all the wtATM-carrying donors and ATM mutation carriers were stable enough within each genotype to allow for significant discrimination, without the need for reference samples. In addition, none of the LCLs derived from patients with genetic diseases causing ataxia (i.e., A-T–like disorder), or showing radiosensitivity (i.e., Nijmegen breakage syndrome, Werner syndrome, or Fanconi anemia group A) have reduced p53-MCL. Similar data were also obtained by analyzing ATR-defective and p53-mutated LCLs (Seckel syndrome and Li-Fraumeni syndrome) or other disorders (Cornelia de Lange syndrome and cylindromatosis). Although the definitive validation of this assay requires larger numbers, our data indicate that the p53-MCL test has a sensitivity and specificity not attained by other existing A-T functional assays.
From clinical and epidemiological points of view, screening for mutations by full gene sequencing is still too expensive and difficult in most countries (34
). Thus, young children developing ataxia experience a long diagnostic process before their PBMCs are immortalized or their ATM
gene is sequenced. The issue is even more challenging for the identification of heterozygous carriers. Since the discovery of IR hypersensitivity in A-T cells in vitro in 1975 (35
), and the development of the colony survival assay (15
), which remains one of the most important laboratory tests for A-T diagnosis, several assays have been developed in an attempt to improve A-T identification. Unfortunately, none of these tests, with the exception of ATM
sequencing, has yet obtained unambiguous results. In our opinion, this is due to two inherent reasons. The first is linked to ATM’s role in the DNA damage response, the best-characterized ATM function, on which most A-T tests are based. ATM works within a network of several sensor and effector proteins whose mutations can cause cell phenotypes to partially overlap those of the A-T cells, thereby reducing the test’s specificity. The second reason is linked to the quantitative differences induced by the diverse amounts of wild-type 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
). These problems have a limited impact on the identification of A-T homozygotes, but constitute a formidable obstacle to carrier prediction. To our surprise, at variance with these assays, the p53-MCL assay does not measure a continuous quantitative variation (e.g., radiosensitivity), but rather a “binary” outcome. At the single-cell level, p53 does or does not localize at the centrosome, and we did not find any detectable quantitative difference, such as lower p53 quantities, at the centrosome. At the cell-population level, the number of cells showing one or the other phenotype allowed us to clearly discriminate the genotype.
From a biological point of view, it is possible that more precise and quantitative assays than double indirect IF will allow us to discover more subtle differences in the amount of p53 at the centrosomes. However, in anaphase and telophase, the last steps of mitosis, p53 was at the centrosome in all of the cells we analyzed, independently of the genotype (data not shown). This suggests the existence of compensatory mechanisms for the final phase of cell division and supports the concept that p53-MCL is an ATM-dependent qualitative characteristic.
We serendipitously discovered this ATM-dependent behavior of p53, though we still do not understand why half of the A-T carriers’ cells behave like wild-type cells and the other half like A-T cells. We found no differences in gender, type of ATM mutations, amounts of residual ATM protein, or p53 polymorphisms at codon 72. Limiting dilution cloning of heterozygous LCLs yielded only clones with a percentage of mitotic cells with p53-MCL, comparable to that of healthy donors. This indicates that the 50% distribution is not linked to each cell division, and that among the A-T heterozygous cells, those able to localize p53 at their centrosomes have a cloning advantage (data not shown). It will be interesting to compare the single-cell clones with the parental-cell populations in order to understand the molecular cause of the peculiar distribution found in A-T carriers.
We took advantage of these observations and set up a p53-MCL assay on PBMCs. We found that as little as 1 ml of whole blood is sufficient to calculate the percentage of mitotic cells with p53-MCL with a high degree of precision. In addition, when we applied our test to search for unknown A-T carriers among BC patients, in whom A-T–carrier frequency should be 2- to 4-fold higher than in the general population, we identified 7 putative A-T heterozygotes (i.e., the expected increment) and 2 cancer-predisposition ATM
variants. The role of monoallelic ATM
mutations in BC susceptibility has been a subject of debate. Some studies have indicated that ATM
mutations causing A-T in biallelic carriers (i.e., protein truncations or splice junction variants) confer a risk for BC, others have reported little evidence of an associated risk, and still other studies have shown that a subset of rare, evolutionarily unlikely missense substitutions are important (13
). These studies indicate that useful screenings for ATM
mutations linked to BC susceptibility require full gene sequencing to identify the different classes of ATM variants and to further discriminate between deleterious missense mutations and neutral ATM
polymorphisms. Of interest, our initial evaluation of 80 BC patients showed that p53-MCL can functionally recognize at least some of the ATM cancer predisposition variants. Future studies will show whether and to what extent p53-MCL can discriminate between ATM
-neutral polymorphisms and deleterious missense mutations.
In conclusion, we have established a simple, fast, minimally invasive, reliable, and inexpensive test to determine mutant ATM zygosity, opening the possibility of performing large-scale screening in the general population for different clinical aspects, such as early diagnosis, genetic counseling, cancer predisposition, susceptibility to IR, and selection for specific targeted therapies.