PMC

Mutant frequency at microsatellite repeats is significantly increased in rAML. (A) Spontaneous mutant frequency measured at six non-coding polyadenine microsatellites increases with age in spleen cells of unirradiated animals (Pearson’s, P < 0.0001). A linear fit is shown. (B) Non-clonal mutant frequency is increased greater than 2-fold in AML cells compared to control spleen cells from age-matched animals (*, t test P < 0.001). No persistent increase in mutant frequency was detected in normal spleen cells of irradiated animals compared with those from unirradiated age-matched controls. Error bars indicate standard error.

Both high- and low-LET radiation-induced rAML show hemizygous deletion of chromosome 2 including the PU.1 gene. (A) Deletion of chromosome 2 in rAML spleen cells was assessed by FISH with a PU.1-containing BAC probe (pink) and DNA was counterstained with DAPI (blue). Cells were scored as having a hemizygous deletion if signals were absent from both chromatids of one chromosome 2 homolog. Representative metaphases are shown with 10 um scale bars. (B) Array CGH was used to identify deletions on chromosome 2. The location of PU.1 is indicated with a vertical line. Chromosomal gains (blue) and losses (red) are depicted for individual AML samples, demonstrating single copy PU.1 loss in all cases except AML 2704.

Chromatid-type aberrations are significantly increased in rAML, indicative of ongoing instability. (A) Representative rAML mouse metaphase hybridised with peptide nucleic acid G-rich telomere probe (pink) and counterstained with DAPI (blue). Examples of chromatid breaks are shown with arrows. Original magnification x100. (B) A significant increase in chromatid-type aberrations was seen in rAML (*, P = 0.033). Total telomere aberration frequencies and total chromosome aberration frequencies were not significantly different between AML and control spleens. The control group includes age-matched animals from unirradiated (n = 4), iron irradiated (n = 10) and gamma irradiated (n = 5) cohorts that did not develop AML. Error bars indicate standard error.

Characterisation of mutations at the undeleted PU.1 allele. (A) High resolution melt analysis was capable of genotyping samples with mutations in PU.1 R235. A 73bp amplicon containing the R235 coding sequence was amplified for each sample and a difference curve was used to compare melt profiles to reference DNA with wildtype PU.1. (Red = reference DNA and rAML samples with wildtype R235 allele; Green = rAML samples with mutant R235 allele). (B) Sequencing validated high resolution melt results for all samples. All mutations were detected at the first cytosine (asterisk) of PU.1 codon R235 (nucleotide 855 of NM_011355.1). Representative wildtype (top) and mutant (bottom) chromatograms are shown. (C) Bisulfite sequencing of bulk bone marrow demonstrated methylation of the target cytosine (asterisk). The sequencing chromatogram is shown, where methylated cytosines (indicated by blue circles) are unaffected by bisulfite conversion while unmethylated cytosines (red squares) are converted and amplified as thymine.