Figure 1
Figure 1. Cytogenetic and molecular analysis of the t(4;9)(q21;p24) found in case 1. (A) Conventional (left) and M-FISH karyotype (right). Note 2 copies of the t(4;9)(q21;p24) in both karyotypes (red and white arrows) and 1 additional copy of the der(4) in the M-FISH karyotype (white arrows). (B) An ideogram of chromosome 9 with probes used for the FISH characterization of the t(4;9) (left) and examples of FISH experiments (right). BAC probes included in the dual color JAK2 break-apart (JAK2 BA) assay are shown in the middle figure, fosmid probes covering JAK2 are shown in the right figure. Split of JAK2 BA signals on der(4) and der(9) (metaphase cell) illustrates the t(4;9)-associated rearrangement of JAK2. Further analysis with fosmid probes covering JAK2 mapped the breakpoint in the region flanked by WI2-2850C2 and WI2-1990K3 (split of red and green fosmid signal in an interphase cell). (C) An ideogram of chromosome 4 with probes used for the FISH characterization of the 4q21 breakpoint in the t(4;9) (left) and examples of FISH experiments (right). The 4q21 breakpoint was mapped with a BAC/fosmid walking interphase FISH strategy. The telomeric (5′) JAK2 probes (labeled with SpectrumGreen-dUTP) marking the der(4) were combined with particular 4q21 probes (labeled with SpectrumOrange-dUTP). The colocalized red and green signals (metaphase cell) indicate that the analyzed 4q21 probe hybridized with the der(4), while separated signals indicate that the probe is translocated to the der(9). Using this strategy, we narrowed down the 4q21 breakpoint to the region flanked by RP11-164M11 and RP11-93E17 that harbors SEC31A. Fosmid probes flanking the SEC31A gene (WI2-2194A15 and WI2-2877K7) were used to prove rearrangement of this gene (interphase cell; separated red and green signals). (D) Schematic representation of the SEC31A, JAK2 and SEC31A-JAK2 protein structures (top panel). Sequencing of the fragment amplified by SEC31A-JAK2 nested RT-PCR identified an in-frame fusion between exon 22 of SEC31A and exon 17 of JAK2 as shown in the electropherogram (bottom panel). FISH probes were directly labeled with SpectrumOrange- and SpectrumGreen-dUTP (Abbott Molecular). FISH images were acquired with a 63×/1.40 oil-immersion objective in an Axioplan 2 fluorescence microscope equipped with an Axiophot 2 camera (Carl Zeiss Microscopy) and a MetaSystems Isis imaging system (MetaSystems).

Cytogenetic and molecular analysis of the t(4;9)(q21;p24) found in case 1. (A) Conventional (left) and M-FISH karyotype (right). Note 2 copies of the t(4;9)(q21;p24) in both karyotypes (red and white arrows) and 1 additional copy of the der(4) in the M-FISH karyotype (white arrows). (B) An ideogram of chromosome 9 with probes used for the FISH characterization of the t(4;9) (left) and examples of FISH experiments (right). BAC probes included in the dual color JAK2 break-apart (JAK2 BA) assay are shown in the middle figure, fosmid probes covering JAK2 are shown in the right figure. Split of JAK2 BA signals on der(4) and der(9) (metaphase cell) illustrates the t(4;9)-associated rearrangement of JAK2. Further analysis with fosmid probes covering JAK2 mapped the breakpoint in the region flanked by WI2-2850C2 and WI2-1990K3 (split of red and green fosmid signal in an interphase cell). (C) An ideogram of chromosome 4 with probes used for the FISH characterization of the 4q21 breakpoint in the t(4;9) (left) and examples of FISH experiments (right). The 4q21 breakpoint was mapped with a BAC/fosmid walking interphase FISH strategy. The telomeric (5′) JAK2 probes (labeled with SpectrumGreen-dUTP) marking the der(4) were combined with particular 4q21 probes (labeled with SpectrumOrange-dUTP). The colocalized red and green signals (metaphase cell) indicate that the analyzed 4q21 probe hybridized with the der(4), while separated signals indicate that the probe is translocated to the der(9). Using this strategy, we narrowed down the 4q21 breakpoint to the region flanked by RP11-164M11 and RP11-93E17 that harbors SEC31A. Fosmid probes flanking the SEC31A gene (WI2-2194A15 and WI2-2877K7) were used to prove rearrangement of this gene (interphase cell; separated red and green signals). (D) Schematic representation of the SEC31A, JAK2 and SEC31A-JAK2 protein structures (top panel). Sequencing of the fragment amplified by SEC31A-JAK2 nested RT-PCR identified an in-frame fusion between exon 22 of SEC31A and exon 17 of JAK2 as shown in the electropherogram (bottom panel). FISH probes were directly labeled with SpectrumOrange- and SpectrumGreen-dUTP (Abbott Molecular). FISH images were acquired with a 63×/1.40 oil-immersion objective in an Axioplan 2 fluorescence microscope equipped with an Axiophot 2 camera (Carl Zeiss Microscopy) and a MetaSystems Isis imaging system (MetaSystems).

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal