No mutations have been noticed in JAK2 exons 12 14 by Sanger sequencing. Molecular Evaluation RT PCR and Sequencing of BCR JAK2 Fusion Transcript A possible BCR JAK2 fusion was suspected based on the chromosome evaluation revealing a translocation t and clinical diagnosis of MPD. Total RNA was isolated from individuals EDTA plasma sample by EasyMagW extraction kit following manu facturers instructions. A total of six person RT PCR reactions were developed to determine the possible break points inside BCR and JAK2 resulting inside a fusion transcript. The RT PCR was performed using SuperScript III a single step RT PCR systems with PlatinumW Taq DNA polymer ase. The PCR situations were as follows, initial annealing step at 55 C for 30 min and 94 C for two min, followed by 40 cycles of 94 C for 15 second, 60 C for 30 second and 68 C for 1 min and a final exten sion step of 68 C for 7 min.
Distinct PCR solutions, had been purified by MinElute gel extraction. The PCR products were then sequenced in each forward and reverse direc tions utilizing ABI PRISMW 3730XL genetic analyzer. Sequencing description data are base referred to as by Sequencing Analysis computer software and NCBI blast web page. RT PCR was performed employing forward primers mapping for the cod ing sequences of exons 1 of your minor, significant, and micro breakpoint regions on the BCR locus, respectively Final results A presumptive diagnosis of MPD and probable BCR JAK2 fusion was suspected from chromosome and FISH analysis revealing a translocation t. Confirmation and delineation of your fusion was pursued by further molecular evaluation. A specific amplification item of about 340 bp was obtained in the RT PCR reaction. Direct sequencing on the RT PCR product and sequence alignment revealed a fusion at nucleotide 1279 of BCR and at nucleotide 2435 of JAK2 coding transcripts, respectively, the fusion item integrated the whole exon 1 of BCR fused to nt 1 of exon 19 of JAK2, in frame.
There was no loss or insertion of a base at the breakpoints. This would predict a break upstream of exon 1 at the BCR genomic selelck kinase inhibitor locus and within intron 18 of JAK2 locus. The breakpoint within the BCR gene corresponds for the minor breakpoint cluster area that benefits in the p190 BCR ABL fusion protein in CML. The in frame fusion item is predicted to create a 747 amino acid protein. The predicted protein product probably includes the coiled coil oligomerization domain of BCR plus the segment immedi ately distal towards the JH2 pseudokinase domain of JAK2, thus preserving its active protein tyrosine kinase domain. Conclusions Though reasonably rare and likely below diagnosed, the BCR JAK2 fusion occasion in this case with CML MPD adds towards the spectrum of rare but recurrent translocation partners for every of your genes, respectively. The BCR gene harbors two popular breakpoints involved within the formation of the two alternative forms from the Philadelphia chromosome translocation observed in chronic myeloid leukemia and acute lymphoblastic leukemia.