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

Abstract

Ataxia telangiectasia (A-T) is a rare autosomal recessive disorder characterized by progressive cerebellar ataxia, oculocutaneous telangiectasia, immune defects and predisposition to malignancies. A-T is caused by biallelic inactivation of the ATM gene, in most cases by frameshift or nonsense mutations. More rarely, ATM missense mutations with unknown consequences on ATM function are found, making definitive diagnosis more challenging. In this study, a series of 15 missense mutations, including 11 not previously reported, were identified in 16 patients with clinical diagnosis of A-T belonging to 14 families and 1 patient with atypical clinical features. ATM function was evaluated in patient lymphoblastoid cell lines by measuring H2AX and KAP1 phosphorylation in response to ionizing radiation, confirming the A-T diagnosis for 16 cases. In accordance with previous studies, we showed that missense mutations associated with A-T often lead to ATM protein underexpression (15 out of 16 cases). In addition, we demonstrated that most missense mutations lead to an abnormal cytoplasmic localization of ATM, correlated with its decreased expression. This new finding highlights ATM mislocalization as a new mechanism of ATM dysfunction, which may lead to therapeutic strategies for missense mutation associated A-T.

Figure 1

Response to IR. (a) H2AX phosphorylation in response to IR, 1.5 h after 5 Gy irradiation. H2AX fluorescence intensity was analyzed as an FL1-FITC histogram. Filled histogram: irradiated cells; empty histogram: untreated cells. (b) H2AX phosphorylation was measured by FACS and expressed as the difference with basal level (0 Gy). The box plot represents the mean and SD of at least three experiments. WT represents the mean and SD of nine normal cell lines. The significance of each sample versus WT is indicated above each column; NS, not significant; ^*P<0.001. (c) Analysis of KAP1 phosphorylation 1 h after 5 Gy irradiation in cell lines derived from 17 patients bearing ATM missense mutations. β-actin is used as a loading control. WT cell line; A-T: A-T cell line carrying two truncating mutations in the ATM gene.

Figure 2

Western blot analysis of ATM. (a) Analysis of ATM protein level in cell lines derived from 17 patients bearing ATM missense mutations. α-Adaptin is used as a loading control. WT1-3 are WT cell lines, WT4 carries ATM SNP rs1800058 at the heterozygote state and an A-T cell line carries two truncating mutations in the ATM gene. This experiment is representative of at least three assays per sample. (b) Quantification was performed using ImageJ software. The histogram and error bars represent the mean and SD of ATM levels related to α-adaptin calculated from three independent western blots. The significance of each sample versus the control is indicated above each column; NS, not significant; ^*P<0.05 and ^***P<0.001.

Figure 3

Analysis of ATM subcellular localization. (a) The histograms represent the mean fraction of nuclear (light gray) versus cytoplasmic (dark gray) ATM calculated from three independent experiments. WT1-3 are WT cell lines, WT4 carries ATM SNP rs1800058 at the heterozygote state. Samples showing a significantly lower nuclear fraction as compared with WT controls are indicated by; NS, not significant; ^*P<0.05, ^**P<0.01 and ^***P<0.001. (b) Localization of ATM by immunofluorescence. Representative images of four ATM missense mutation bearing cell lines are shown, together with WT and A-T cell lines as positive and negative controls. DNA is stained with DAPI (magnification × 1000). (c) Graph illustrating the correlation between ATM protein expression (expressed as percentage of normal ATM level on the x-axis) and nuclear fraction (expressed as percentage of total ATM level on the y-axis).