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A nonsense mutation of human XRCC4 is associated with adult-onset progressive encephalocardiomyopathy.

Bee L, Nasca A, Zanolini A, Cendron F, d'Adamo P, Costa R, Lamperti C, Celotti L, Ghezzi D, Zeviani M - EMBO Mol Med (2015)

Bottom Line: XRCC4 transcript levels were profoundly reduced, and the protein was undetectable in patients' skin fibroblasts.Gamma-irradiated mutant cells demonstrated reduction, but not abolition, of DSB repair.Surprisingly, neither immunodeficiency nor predisposition to malignancy was reported in these patients.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Padua, Padua, Italy.

No MeSH data available.


Related in: MedlinePlus

Analysis of γ-H2AX nuclear fociγ-H2AX nuclear foci in XRCC4wt and mutant fibroblasts (XRCC4m/m1 and XRCC4m/m2) were determined at different times after irradiation with 0.5 Gy of γ-rays. Values, subtracted of their non-irradiated control (1.3 foci/nucleus in XRCC4wt cells and 1.4 and 0.9 foci/nucleus in XRCC4m/m1 and XRCC4m/m2 cells, respectively), are the mean ± SD of three independent experiments.Percentages of γ-H2AX foci in XRCC4wt and XRCC4m/m fibroblasts (pooled values) remaining at the indicated time-points.Immunofluorescence micrographs of γ-H2AX foci in XRCC4wt and XRCC4m/m fibroblasts.Data information: ***P < 0.001; **P < 0.01; *P < 0.05 (XRCC4m/m vs. XRCC4wt cells), two-way ANOVA, Bonferroni post hoc test.
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fig02: Analysis of γ-H2AX nuclear fociγ-H2AX nuclear foci in XRCC4wt and mutant fibroblasts (XRCC4m/m1 and XRCC4m/m2) were determined at different times after irradiation with 0.5 Gy of γ-rays. Values, subtracted of their non-irradiated control (1.3 foci/nucleus in XRCC4wt cells and 1.4 and 0.9 foci/nucleus in XRCC4m/m1 and XRCC4m/m2 cells, respectively), are the mean ± SD of three independent experiments.Percentages of γ-H2AX foci in XRCC4wt and XRCC4m/m fibroblasts (pooled values) remaining at the indicated time-points.Immunofluorescence micrographs of γ-H2AX foci in XRCC4wt and XRCC4m/m fibroblasts.Data information: ***P < 0.001; **P < 0.01; *P < 0.05 (XRCC4m/m vs. XRCC4wt cells), two-way ANOVA, Bonferroni post hoc test.

Mentions: To test the consequence of the XRCC4 mutation on DNA repair efficiency, wt and mutant fibroblasts were first exposed to γ-rays (0.5 Gy), in order to induce DNA DSBs. After exposure to IR, the extensive phosphorylation at Ser139 of histone H2AX results in the formation of discrete γ-H2AX foci, which can be identified by immunostaining and constitute a useful tool to highlight the presence of DSBs (Fernandez-Capetillo et al, 2004; Sharma et al, 2012). Because this phosphorylation is abundant, fast, and correlates well with each DSB, it can be used to examine the DNA damage produced by irradiation and the subsequent repair of the DNA lesion (Svetlova et al, 2010). After irradiation, DSB rejoining was monitored for 24 h, by measuring the formation of γ-H2AX nuclear foci, as a DNA damage index, and their disappearance, as an index of DNA repair. During the 24-h monitoring, the decrease in foci number was significantly slower in XRCC4-mutant fibroblasts (XRCC4m/m #1, from individual II-1; XRCC4m/m #2, from individual II-2) compared to that in wt cells, whereas at the endpoint, the differences became statistically non-significant (Fig2A). Since the two XRCC4-mutant cell lines showed no significant differences in DSB rejoining, these data were pooled and expressed as a mean in subsequent analyses. The kinetics of γ-H2AX foci disappearance (Fig2B) confirmed that in XRCC4-mutant cells, the DNA lesions were repaired, although with lower efficiency than in wt cells, as the percentage of foci remaining at 24 h was 5 ± 8% in XRCC4wt cells and 19 ± 7% in XRCC4m/m. This result suggests that undetectable levels of XRCC4 protein do not completely abolish DNA repair capability.


A nonsense mutation of human XRCC4 is associated with adult-onset progressive encephalocardiomyopathy.

Bee L, Nasca A, Zanolini A, Cendron F, d'Adamo P, Costa R, Lamperti C, Celotti L, Ghezzi D, Zeviani M - EMBO Mol Med (2015)

Analysis of γ-H2AX nuclear fociγ-H2AX nuclear foci in XRCC4wt and mutant fibroblasts (XRCC4m/m1 and XRCC4m/m2) were determined at different times after irradiation with 0.5 Gy of γ-rays. Values, subtracted of their non-irradiated control (1.3 foci/nucleus in XRCC4wt cells and 1.4 and 0.9 foci/nucleus in XRCC4m/m1 and XRCC4m/m2 cells, respectively), are the mean ± SD of three independent experiments.Percentages of γ-H2AX foci in XRCC4wt and XRCC4m/m fibroblasts (pooled values) remaining at the indicated time-points.Immunofluorescence micrographs of γ-H2AX foci in XRCC4wt and XRCC4m/m fibroblasts.Data information: ***P < 0.001; **P < 0.01; *P < 0.05 (XRCC4m/m vs. XRCC4wt cells), two-way ANOVA, Bonferroni post hoc test.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4520657&req=5

fig02: Analysis of γ-H2AX nuclear fociγ-H2AX nuclear foci in XRCC4wt and mutant fibroblasts (XRCC4m/m1 and XRCC4m/m2) were determined at different times after irradiation with 0.5 Gy of γ-rays. Values, subtracted of their non-irradiated control (1.3 foci/nucleus in XRCC4wt cells and 1.4 and 0.9 foci/nucleus in XRCC4m/m1 and XRCC4m/m2 cells, respectively), are the mean ± SD of three independent experiments.Percentages of γ-H2AX foci in XRCC4wt and XRCC4m/m fibroblasts (pooled values) remaining at the indicated time-points.Immunofluorescence micrographs of γ-H2AX foci in XRCC4wt and XRCC4m/m fibroblasts.Data information: ***P < 0.001; **P < 0.01; *P < 0.05 (XRCC4m/m vs. XRCC4wt cells), two-way ANOVA, Bonferroni post hoc test.
Mentions: To test the consequence of the XRCC4 mutation on DNA repair efficiency, wt and mutant fibroblasts were first exposed to γ-rays (0.5 Gy), in order to induce DNA DSBs. After exposure to IR, the extensive phosphorylation at Ser139 of histone H2AX results in the formation of discrete γ-H2AX foci, which can be identified by immunostaining and constitute a useful tool to highlight the presence of DSBs (Fernandez-Capetillo et al, 2004; Sharma et al, 2012). Because this phosphorylation is abundant, fast, and correlates well with each DSB, it can be used to examine the DNA damage produced by irradiation and the subsequent repair of the DNA lesion (Svetlova et al, 2010). After irradiation, DSB rejoining was monitored for 24 h, by measuring the formation of γ-H2AX nuclear foci, as a DNA damage index, and their disappearance, as an index of DNA repair. During the 24-h monitoring, the decrease in foci number was significantly slower in XRCC4-mutant fibroblasts (XRCC4m/m #1, from individual II-1; XRCC4m/m #2, from individual II-2) compared to that in wt cells, whereas at the endpoint, the differences became statistically non-significant (Fig2A). Since the two XRCC4-mutant cell lines showed no significant differences in DSB rejoining, these data were pooled and expressed as a mean in subsequent analyses. The kinetics of γ-H2AX foci disappearance (Fig2B) confirmed that in XRCC4-mutant cells, the DNA lesions were repaired, although with lower efficiency than in wt cells, as the percentage of foci remaining at 24 h was 5 ± 8% in XRCC4wt cells and 19 ± 7% in XRCC4m/m. This result suggests that undetectable levels of XRCC4 protein do not completely abolish DNA repair capability.

Bottom Line: XRCC4 transcript levels were profoundly reduced, and the protein was undetectable in patients' skin fibroblasts.Gamma-irradiated mutant cells demonstrated reduction, but not abolition, of DSB repair.Surprisingly, neither immunodeficiency nor predisposition to malignancy was reported in these patients.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Padua, Padua, Italy.

No MeSH data available.


Related in: MedlinePlus