PMC4071805_01
Molecular and cytogenetic studies. a) Metaphase FISH showed a rearrangement between NUP98 gene at 11p15 and the HOXA cluster at 7p15. b) Breakpoint falls between NUP98 exon 16 and HOXA13 exon 2. Red triangles indicate in-frame splicing variants; yellow triangle an out-of-frame variant. Sequence analysis confirmed the transcript.c) The fusion protein with NUP98 GLFG repeats and the HOXA13 homeodomain (HD).
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F1
Metaphase FISH (Figure 1a) showed a NUP98/HOXA gene rearrangement and Interphase FISH (data not shown) identified the fusion in 57% of nuclei. Three splicing variants had the same breakpoint but were lacking respectively NUP98 exon 11, 12 or 14 (which was out of frame) (Figure 1b and c).
Expression analysis. a)MSI2 and b)HOXA9 are over-expressed in the present patient with NUP98/HOXA13. BC1 and BC2: two other cases of Ph + blast crisis CML with additional karyotypic aberrations over-expressing MSI2 but not HOXA9. Expression values were referred to the average of two references. Fluorescence data were analyzed with the Second Derivative Maximum method; gene expression was expressed as Cp (Crossing point) values. c) Significance for MSI2 expression was tested by Mann-Whitney test (*p < 0.05/3); values are expressed as means +/- SD; AA. Additional aberrations.
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F2
In the present case both MSI2 and HOXA9 were over-expressed (Figure 2a, b and c). MSI2, but not HOXA9, was significantly over-expressed also in two other cases of Ph + blast crisis CML both harbouring additional cytogenetic aberrations (BC1 and BC2 in Figure 2a and b), with the following karyotypes: 46,XY,t(9;22)(q34;q11) /49-52,XY,idem,+8,+t(9;22)(q34;q11),+13,+15,+21[cp6] (patient BC1), and 46XY,t(3;7)(p21;q32),t(9;22)(q34;q11)[15/15]) (patient BC2).
Chromatin Immunoprecipitation. NUP98/HOXA13 binds both MSI2 and HOXA9 promoters. ChIP was performed on both NUP98/HOXA13 sample and a non-malignant disease sample (wt). 1,5 mug of rat IgG (Millipore Normal Rat IgG Polyclonal Antibody) and No Antibody (not shown) were used as negative controls. a) Semi-quantitative PCR showed an enrichment in NUP98/HOXA13 sample compared to controls. b) qPCR confirmed this result; data are presented as fold increase relative to the control sample (wt) based on the formula 2-DeltaDeltaCp. One out of three (for MSI2) or two (for HOXA9) ChIP experiments is shown. The results shown are the mean +- S.E.M. (error bars) of two independent qPCR experiments. c) NUP98/HOXA13 binds both MSI2 and HOXA9 promoters. HOXA9 binds MSI2 promoter. Protein structure: homeodomain (HD). Gene structure: exons (numbered boxes), transcription start site (TSS; +1), direction of transcription (flag), putative HOX binding element 1 kb upstream of TSS (oval).
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F3
ChIP confirmed the binding of NUP98/HOXA13, but not of wild-type NUP98, to both MSI2 and HOXA9 promoters in the present patient (Figure 3a and b).