Table 1.
Primers used to amplify TP53 coding exons (NC_000017.10).
Table 2.
FISH: NPM1 status over time (median 9 months; range 2–156) in 10 adult patients with MDS/AML.
Figure 1.
FISH, cytogenetics and mutational analysis.
A) Results of FISH in 38 patients with MDS/AML and NPM1 monoallelic deletion (NPM1+/−): schema of 5q breakpoints. Upper: ideogram of the long arm of chromosome 5 in G banding. Lower: genomic clones at proximal (q11.2– q14.1 sub-bands) and distal (q35.1–q35.3 sub-bands) breakpoints with percentages of cases. B) G-banded karyotype of a representative case (n.103, Supporting Table S1) showed a complex karyotype including monosomies, unbalanced translocations, five markers, and a very small deleted chromosome 5 corresponding to the largest 5q deletion including NPM1 with breakpoints at q11.2 and q35.3 (Right ideogram). C) FISH of case n.103: concomitant deletion of RP11-117L6/NPM1 (green) and RP11-266N13 (red) indicate centromeric breakpoint (left) and RP1-240G13/subtelomeric sequences (red) indicate telomeric breakpoint (right). Only one copy of each clone is present. D) Gene sequencing of case n.103 shows no NPM1 exon 12 mutation in the non-deleted chromosome 5. The last 12 amino acids encoded by exon 12 of wild type NPM1 (NM_002520) are annotated on top of the sequence.
Figure 2.
RT-qPCR and NPM1 gene expression.
NPM1 expression in 9 patients with MDS/AML NPM1+/− (median: 0.5443; range: 0.18÷0.70), in 15 with MDS/AML NPM1+/+ (median: 0.96; range: 0.44÷3.20) and in 11 healthy controls (median 1.07; range 0.61÷1.50). Horizontal lines indicate median NPM1 expression in each group. Y axis = 2∧−ΔΔCt value (NPM1 gene expression level normalized to endogenous ABL1 gene) ΔCt = Ct NPM1 −Ct ABL1, ΔΔCt = ΔCt sample−ΔCtcalibrator. (P values according to the Mann-Whitney U test and Bonferroni correction); n.s. = non significant.
Table 3.
TP53 monoallelic deletion and/or mutations in 49/57 patients with MDS/AML and non-isolated 5q-.
Figure 3.
FISH investigations on clones encompassing 14 genes that are putatively involved in genomic stability.
Gains (upper) and losses (lower) in NPM1+/+ patients (white columns) and NPM1+/− (grey columns).
Figure 4.
Mutually exclusive NPM1 abnormalities indicate different leukemogenic pathways.