Figure 1
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).

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal