Figure 1.
Microsatellite instability in pediatric HGG.
Representative electropherogram traces for three MSI-positive pediatric HGG, presenting sequence alterations in more than one quasimonomorphic marker. Case RMH2452 (MSI-H) presented alterations in NR27, NR21, BAT25 and BAT26; RMH2458 (MSI-L) in NR21 and BAT25; and RMH4816 (MSI-L) in NR21 and NR24. Alterations in relation to a control trace are indicated with arrows.
Table 1.
Clinical characterization and MMR status of MSI cases.
Figure 2.
Genomic stability in MSI pediatric HGG.
Array CGH genome plots are shown for two MSI-positive pediatric HGG, the MSI-H RMH2452 (A) and the MSI-L RMH2458 (B), demonstrating lack of large scale alterations detectable on the 32K BAC platform. Log2 ratios for each clone (y axis) are plotted according to chromosomal location (x axis). The centromeres are represented by vertical lines.
Table 2.
Genomic alterations and Candidate MSI target genes frameshift mutations in MSI samples.
Figure 3.
Target gene screening in MSI pediatric HGG.
Electropherogram traces, direct sequencing, and immunohistochemistry for MSI target genes presenting frameshift mutations in pediatric HGG (DNA alterations indicated with arrows). (A) Case RMH2452 (MSI-H) presenting a homozygous insertion of one bp in the MSH6 poly(C)8 tract and loss of protein expression. (B) RMH3969 (MSI-L) presenting a heterozygous deletion of one bp in the DNAPKcs poly(A)10 tract, and retention of protein expression. (C) RMH2444 (MSI-L) presenting a heterozygous insertion of one bp in the MRE11 poly(T)11 tract and retention of protein expression.
Figure 4.
MRE11 poly(T)11/12 sequence variant has no apparent functional consequence on DNA damage detection and repair.
(A) Electropherograms and sequencing traces of the MRE11 poly(T)11 tract in leukemia/lymphoma cells: Daudi (heterozygous insertion of one bp, poly(T)11/12); Jurkat (heterozygous deletion of one bp, poly(T)10/11); and Raji (wild-type, poly(T)11). (B) MRE11 expression in Daudi, Jurkat and Raji cells. qRT-PCR was used to calculate the ratio of MRE11 expression (amplification of exons 2 to 4) relative to the GAPDH control, independently of the presence of the aberrant transcript observed in Jurkat. PCR products were loaded on the gel at saturation of the reaction, being not quantitative. MRE11 protein levels were assessed by Western blot, and were considerably lower in Jurkat. (C) ATM-pS1981 levels were assessed by Western blot in Daudi, Jurkat and Raji after 1.5 Gy irradiation of cells and 1 h, 4 h or 24 h recovery. There was an increase of ATM phosphorylation 1 h after irradiation in Daudi and Raji cells that decreased at the later time points. Jurkat showed no ATM phosphorylation after exposure to irradiation. (D) DNA repair foci was observed by immunofluorescence after 1.5 Gy IR followed by 1 h and 4 h recovery. Irradiation resulted in the formation of both MRE11 and 53BP1 foci in Daudi and Raji cells, whereas Jurkat failed to form MRE11 foci. Nuclei were counterstained with DAPI. Merged figure presents co-localization of MRE11 and 53BP1 foci visualized in yellow.