PMC4348613_01
Conventional cytogenetics (bone marrow) analysis revealing: (a) 46, XY, t(9;22)(p24;q11) in 60% of the metaphases: arrows indicate the balanced translocation between the short arm of the chr9 and the long arm of the chr22. (b) 47, XY, t(9;22)(p24;q11), +der(22) t(9;22)(q24;q11) in 10% of the metaphases: arrows indicate the chr9 and chr22 involved in the t(9;22)(p24;q11) and dotted arrow indicates the duplication of derived.
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fig1
Chromosome analysis (Figure 1) showed t(9;22)(p24;q11) as the sole abnormality in 60% of the analyzed metaphases (12/20).
FISH analysis. (a) Nucleus with two red signals (red arrow, ABL probe) on chr9 and two green signals (green arrow, BCR probe) on chr22, indicating that BCR-ABL1 FISH for the Ph-chromosome did not reveal any fusion signals, revealing a normal hybridization pattern negative for t(9;22)(q34;q11.2) BCR-ABL1 fusion. (b) Two red signals (red arrows, ABL probe) were present on the long arm of both the normal and the derivative chr9. Three green signals were present, indicating an extra signal of the BCR probe, suggestive of an extra chromosome 22 or additional chromosome material containing the 22q11.2 region: two intense green signals were on the normal chr22 (green arrows, BCR probe) and one reduced intensity green signal was localized on the derivative chr22 and on the short arm of derivative chr9 (dotted green arrow, telomeric part of the BCR probe). Notice that one of the derivative chromosome signals was too feint to be seen in some nuclei.
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FISH analysis (Figure 2) using dual fusion probes for BCR (22q11.2) and ABL1 (9q34) regions (LSI BCR/ABL ES Dual Color Translocation Probe, Abbott
Mechanism of BCR-JAK2 fusion and breakpoints direct sequencing. Sanger sequencing alignment of the RT-PCR product revealed a break at nucleotide 3458 of exon 13 of BCR (nucleotides highlighted in red) and at nucleotide 1 of exon 17 of JAK2 (nucleotides highlighted in blue). Amino acids of the respective fusion gene BCR-JAK2 reveal a new valine residue (V) that has been created at the fusion junction.
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Technologies, Carlsbad, CA, USA). cDNA synthesis was performed using Moloney Murine Leukemia Virus reverse transcriptase (Invitrogen, Life Technologies, Carlsbad, CA, USA). The breakpoint Sanger sequencing using ABI 3130 (Life Technologies, Carlsbad, CA, USA) identified an in-frame fusion of the last nucleotide of BCR exon 13 with the first nucleotide of JAK2 exon 17 (Figure 3). There was no loss or insertion of a base at these breakpoints.
Quantitative real time PCR expression levels of BCR-JAK2 follow-up. BCR-JAK2 follow-up carried out relative to the expression of the housekeeping gene ABL1, in the bone marrow and the peripheral blood, after allogeneic stem-cell transplantation (black arrow). It revealed one of the longest sustained complete hematologic and cytogenetic remissions at 35 months of follow-up in a BCR-JAK2 fusion MPN-U.
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With 35-month follow-up, the patient remains alive with undetectable BCR-JAK2 transcript levels in the blood and no transplant-related complications (Figure 4).