case_images: PMC4071805_01
case_images: 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).
case_images: 1755-8166-7-42-1.jpg
case_images: PMC4071805_01_1755-8166-7-42-1.jpg
case_images: F1
case_images: 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).
case_images: 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.
case_images: 1755-8166-7-42-2.jpg
case_images: PMC4071805_01_1755-8166-7-42-2.jpg
case_images: F2
case_images: 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).
case_images: 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).
case_images: 1755-8166-7-42-3.jpg
case_images: PMC4071805_01_1755-8166-7-42-3.jpg
case_images: F3
case_images: 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).