Figure 1
Genetic and molecular analysis of NOTCH1 aberrations. (A) Graphic representation of dic(9;14)(q34;q32) with indicated probes applied for FISH mapping of both breakpoints and their hybridization pattern. (B-D) Examples of FISH analysis performed on a diagnostic LN sample with (B) LSI IGH dual color break apart probe and CEP12 (green), (C) RP11-707O3 (green) and RT11-769N4 (red), (D) WI2-569D3 (G248P80019F4, green) and WI2-1851N4 (G248P8957B2, red). BAC and fosmid clones were selected using the UCSC Genome Browser on Human May 2004 (NCBI35/hg17) Assembly. Note in panel B the presence of the 3′IGH/red signal on dic(9;14) and loss of the distal IGVH/green signal in cell with trisomy 12, in panel C hybridization of RP11-707O3 (green) with dic(9;14) and loss of the distal RT11-769N4 (red) sequences, and in panel D hybridization of both fosmids covering NOTCH1 with dic.(9;14). (E) qRT-PCR analysis of NOTCH1 mRNA expression levels. The patient was analyzed at 2 different timepoints, at diagnosis (05/2003; PBL; 57% of cells with dic(9;14)) and during disease evolution (06/2011; PBL; dic(9;14)-negative), and relative NOTCH1 expression levels were compared with 2 control CLL samples (CLL1 and CLL2) with unmutated VH and trisomy12. (F) Western blot analysis of protein extract of diagnostic LN cells of the index case (15% of cells with dic(9;14)) and 2 control CLLs (unmutated VH, trisomy12); CLL3 with unmutated NOTCH1 and CLL4 positive for ΔCT7544-7545/P2515fs. The top panel shows detection of active, cleaved NOTCH1 (Val 1744 antibody; Cell Signaling Technology) in CLL4 and the index case, but not in CLL3 cells. The bottom panel shows expression of NOTCH1 with a general anti-NOTCH1 antibody (c-20 Santa Cruz Biotechnology). Because of a low content of cells with dic(9;14) in the only available LN sample, overexpression of an activated form of NOTCH1 could not be evidenced. (G) Sanger chromatogram illustrating a heterozygous ΔCT7544-7545/P2515fs of the NOTCH1 cDNA in the diagnostic BM sample. FISH images acquired with a 63×/1.4 oil immersion objective in an Axioplan 2 fluorescence microscope equiped with an Axiophot 2 camera (Carl Zeiss) and an Isis imaging system (MetaSystems). Images were imported directly into PowerPoint (Microsoft).

Genetic and molecular analysis of NOTCH1 aberrations. (A) Graphic representation of dic(9;14)(q34;q32) with indicated probes applied for FISH mapping of both breakpoints and their hybridization pattern. (B-D) Examples of FISH analysis performed on a diagnostic LN sample with (B) LSI IGH dual color break apart probe and CEP12 (green), (C) RP11-707O3 (green) and RT11-769N4 (red), (D) WI2-569D3 (G248P80019F4, green) and WI2-1851N4 (G248P8957B2, red). BAC and fosmid clones were selected using the UCSC Genome Browser on Human May 2004 (NCBI35/hg17) Assembly. Note in panel B the presence of the 3′IGH/red signal on dic(9;14) and loss of the distal IGVH/green signal in cell with trisomy 12, in panel C hybridization of RP11-707O3 (green) with dic(9;14) and loss of the distal RT11-769N4 (red) sequences, and in panel D hybridization of both fosmids covering NOTCH1 with dic.(9;14). (E) qRT-PCR analysis of NOTCH1 mRNA expression levels. The patient was analyzed at 2 different timepoints, at diagnosis (05/2003; PBL; 57% of cells with dic(9;14)) and during disease evolution (06/2011; PBL; dic(9;14)-negative), and relative NOTCH1 expression levels were compared with 2 control CLL samples (CLL1 and CLL2) with unmutated VH and trisomy12. (F) Western blot analysis of protein extract of diagnostic LN cells of the index case (15% of cells with dic(9;14)) and 2 control CLLs (unmutated VH, trisomy12); CLL3 with unmutated NOTCH1 and CLL4 positive for ΔCT7544-7545/P2515fs. The top panel shows detection of active, cleaved NOTCH1 (Val 1744 antibody; Cell Signaling Technology) in CLL4 and the index case, but not in CLL3 cells. The bottom panel shows expression of NOTCH1 with a general anti-NOTCH1 antibody (c-20 Santa Cruz Biotechnology). Because of a low content of cells with dic(9;14) in the only available LN sample, overexpression of an activated form of NOTCH1 could not be evidenced. (G) Sanger chromatogram illustrating a heterozygous ΔCT7544-7545/P2515fs of the NOTCH1 cDNA in the diagnostic BM sample. FISH images acquired with a 63×/1.4 oil immersion objective in an Axioplan 2 fluorescence microscope equiped with an Axiophot 2 camera (Carl Zeiss) and an Isis imaging system (MetaSystems). Images were imported directly into PowerPoint (Microsoft).

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