Abstract 1847: An elevation in the mutation rate in progeria syndromes using the PIG-A gene as a sentinel
Authors: Araten David J. , Zamechek Leah 
Published: February 2011

Abstract
The mutation rate (µ) is a key parameter in oncogenesis but has historically been very difficult to measure. We have recently developed a method for measuring µ using the PIG-A gene, which is present in only one copy (in males) or one active copy (in females) because it is X-linked. PIG-A mutations confer a loss of GPI-linked proteins on the cell surface, such that rare mutants can be detected amongst a vastly larger normal population by flow cytometry. We have previously demonstrated elevations in µ in Fanconi Anemia, ataxia-telangiectasia, and in malignant cell lines. Here we investigated whether accelerated aging (progeria) syndromes might be associated with an elevation in µ. To measure µ, EBV transformed B cell lines are stained with an antibody specific for CD59 (a GPI-linked protein) and the upper 50th percentile of the distribution is collected, to eliminate pre-existing mutants. The collected GPI (+) cells are counted, returned to culture, and expanded over ∼ 3 weeks to allow new mutations to arise. The number of cell divisions (d) is then estimated from live cell counts, and the frequency (f) of spontaneously arising GPI (-) cells is determined by flow cytometry, after staining sequentially with a mixture of mouse antibodies specific for 3 GPI-linked proteins (CD48, CD55, and CD59), PE-conjugated rabbit anti-mouse antibody, and then FITC-conjugated anti- HLA-DR, a transmembrane protein not affected by PIG-A mutations. Dead cells are excluded by propidium iodide. Control GPI (-) cells from patients with PNH can serve as a negative control, and we have found it useful to define spontaneously arising GPI (-) cells as those having &lt; 4% of the number of PE molecules bound compared to the normal population, using QuantiBrite beads. The mutation rate is then calculated by the formula µ = f / d. We found that in a panel of 7 cell lines from different normal donors, the median µ value was 23.5 × 10−7 per cell division (range 6.5 to 29 × 10−7), which was similar to our previous data. In cell lines from 5 patients with Werner's syndrome, the median µ value was 134 × 10−7 per cell division (range 54 to 191 × 10−7, p = .006, 2-sided non-parametric test). This was as expected, based on previous reports by others suggesting a predisposition to deletions and an increased risk of malignancy in this syndrome. In cell lines from 7 patients with Hutchinson-Gilford Progeria Syndrome (HGPS), the median µ value was 44 × 10−7 (range 16 to 109 × 10−7, p = .04). Despite a lack of a known cancer predisposition in HGPS, we had suspected that µ might be elevated, due to known abnormalities in the nuclear membrane associated in this disorder. In 2 out of 3 cell lines from patients with Bloom's syndrome, µ was also clearly elevated (109 × 10−7 and 140 × 10−7). We conclude that an elevation in the mutation rate is a common feature of inherited accelerated aging syndromes.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1847.