Rase chain reaction (RT-PCR) for chimeric BCR-ABL1 transcript on peripheral blood
Rase chain reaction (RT-PCR) for chimeric BCR-ABL1 transcript on peripheral blood was performed with Philadelphia p210 Q-PCR Alert kit (Nanogen Inc., San Diego, CA, USA), depending on TaqMan technologies. RNA extraction and RTPCR were performed following the insert kit instructions (Nanogen Inc., San Diego, CA, USA). The measurement with the cDNA of P210 was normalized for the cDNA of ABL1 gene. Conventional cytogenetic evaluation on bone marrow showed on 22 metaphases a reciprocal IL-1 MedChemExpress translocation involving the long arm of chromosomes 12 and 22, t(12;22), devoid of the involvement of chromosome 9 (Figure 1(a)). The presence of a cryptic BCRABL1 fusion transcript was detected by RT-PCR and subsequently by interphase FISH analyses on bone marrow. Quantitative RT-PCR analysis for BCRABL1 on peripheral blood revealed the key chimeric transcript, with a BCR-ABL1(P210)ABL1 ratio of 14.95 (International Scale). FISH evaluation with BCRABL1 t(9;22) Triple-Color and Dual-Fusion probe was performed to characterize the t(12;22) translocation and to detect the localization with the fusion gene. The probe set can be a mixture of ASS-ABL1 probe labeled in red and of BCR probe with the proximal BCR area labeled in blue and the distal one particular in green. FISH on 200 metaphases and nuclei showed the following: (i) one purple (bluered) fusion CCR1 medchemexpress signal representing the fusion gene (BCRABL1) on der(22), (ii) one green signal of 3 BCR sequences on chromosome 12 involved in translocation t(12;22), (iii) a greenblue signal on regular chromosome 22, and (iv) a red signal on typical chromosome 9 (Figures 1(b) and 1(c)). The reciprocal fusion ABL1BCR signal was not detected. FISH evaluation on 200 nuclei and metaphases using the subtelomeric 9qter probe was performed to further investigate the involvement of chromosome 9 in the complicated rearrangement: it showed a typical signal pattern.3. DiscussionWe describe a patient with CML associated using a novel cryptic complicated variant t(9;22), involving chromosome 12 besides chromosomes 9 and 22, which was unmasked and characterized by RT-PCR and FISH analyses. In agreement with ESMO clinical practice recommendations, this case report proves the role of those molecular approaches in detecting cryptic fusion gene in some forms of variant translocations with masked Ph and der(9) chromosomes. As previously reported, the breakpoints place of complicated variant t(9;22) is nonrandom using a marked clustering to certain chromosome bands suggesting that some regions are additional prone to breakage. This getting could be explained by the presence of a distinct genomic structure mediating the recombination. Certainly a important clustering was described for higher CG content material regions, Alu repeats, LINE, genes, and miRNA explaining the presence of recombination hotspots [11, 12]. The 12q13 chromosome area, involved in our case, was described by Costa et al. [13] in association with complex Philadelphia translocation and in some circumstances of three-way translocation t(9;22) [11]. Additionally, this region is involved each in other chromosomal translocations, originating chimeric genes connected to different subtypes of leukemia as reported in Mitelman et al. [14] and in Atlas of chromosome in cancer databases [15], and inside the fragile web-site, FRA12A, which can be caused by an expanded CGG repeat inside the 5-prime untranslated area on the DIP2B gene (OMIM 611379) [16]. Combining all these information we can speculate that the presence of particular genomic motif in 12q13, for example CGG repeats, could ha.