PMC5765657_01
a Karyotype of the patient displaying the t(12;14)(q23.2;q32.3). Arrows mark the translocation breakpoint regions on the derivative chromosomes. Note that der(12) is duplicated, leading to a partial trisomy 12. b Karyotype evolution (about three years later). Additional aberrations include a del(3)(p21), monosomy 13 and add(17)(p11). For detailed information see also Table 1. c FISH with the Cytocell IGH Breakapart probe on metaphase and interphase nuclei. The normal chromosome 14 generates a red-green fusion fluorescence signal. Der(14) yields only a red fluorescence signal with the distal green-labeled probe being translocated on der(12). A second green fluorescence signal is present due to the der(12) duplication. On the upper right side, a normal interphase with two red-green fusion signals is shown, next to an interphase bearing the translocation (lower right). A white arrow marks the fusion signal from the normal chromosome 14
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Fig1
Consistent findings in all patient probes included the t(12;14)(q23.2;q32.3), a partial trisomy 12 due to duplication of der(12) chromosome (Fig. 1a) and a submicroscopic deletion of the 13q14 region. Signal splitting of the Cytocell IGH Breakapart probe confirmed the involvement of the IGH locus on chromosome 14 in the translocation (Fig. 1b). We found 176 significantly deregulated genes (FC >= 2, FDR p <= 0.01) (Additional file 3: Figure S1) and (Additional file 4: Table S2).
Translocation breakpoints and derivative chromosome composition. Horizontal gray arrows indicate the transcriptional direction of the depicted genes. Vertical black arrows indicate breakpoints (BP). a Breakpoint region on chromosome 12. The breakpoint took place 86.5 kb distal of the ASCL1 gene. b The IGH locus on chromosome 14. The breakpoint took place within the pentameric repeat region of Switch mu. Dots indicate the IGH enhancer elements. c Composition of der(12) and sequence around the breakpoint. The enhancer element is part of the translocated IGH sequence and is juxtaposed to ASCL1. d der(14) and breakpoint sequence. The C12orf42 gene is translocated to chromosome 14
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Fig2
The breakpoint on der(14) was localized within the pentameric repeat region of Smu. There were no deletions or insertions of sequences at the breakpoints of both chromosomes (Fig. 2).
Validation of the ASCL1 overexpression. a Comparison of the BM expression of ASCL1 between the CLL patient with the t(12;14) translocation and healthy controls as well as CLL with normal karyotype, mono- and biallelic del(13) and trisomy 12 respectively. Results are displayed as log2 fold change. HB2M was used as housekeeping control. Comparisons of the ASCL1 expression in the patient versus all other groups were highly significant (ANOVA p-value = 5.12E-10). Comparisons between normal BM and all other groups were not significant. b Immunohistochemistry for ASCL1 on a peripheral blood cytospin of the patient bearing the t(12;14). Note the strong nuclear reaction in the center. In contrast to that a sample from a B-CLL control (c) showed no antibody reaction. Nuclei are counterstained with hematoxylin
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Fig3
ASCL1 was highly overexpressed in the sample of the patient bearing the translocation as opposed to all other groups with average fold change (FC) values greater than 5600 in all samples (ANOVA p-value = 5.12E-10) (Fig. 3a). Immunohistochemistry with a monoclonal anti-ASCL1 antibody on peripheral blood cytospins of the patient and two CLL control samples confirmed the ASCL1 overexpression at the protein level (Fig. 3b and c).