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), determined by TaqMan COX-3 site technology. RNA extraction and RTPCR had been performed following the insert kit guidelines (Nanogen Inc., San Diego, CA, USA). The measurement in the cDNA of P210 was normalized towards the cDNA of ABL1 gene. Traditional cytogenetic evaluation on bone marrow showed on 22 metaphases a ACAT2 list reciprocal translocation involving the lengthy arm of chromosomes 12 and 22, t(12;22), with no 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 major chimeric transcript, having 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 of the fusion gene. The probe set can be a mixture of ASS-ABL1 probe labeled in red and of BCR probe together with the proximal BCR area labeled in blue and the distal 1 in green. FISH on 200 metaphases and nuclei showed the following: (i) one particular purple (bluered) fusion signal representing the fusion gene (BCRABL1) on der(22), (ii) 1 green signal of three 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 standard chromosome 9 (Figures 1(b) and 1(c)). The reciprocal fusion ABL1BCR signal was not detected. FISH evaluation on 200 nuclei and metaphases applying the subtelomeric 9qter probe was performed to additional investigate the involvement of chromosome 9 inside the complicated rearrangement: it showed a standard signal pattern.3. DiscussionWe describe a patient with CML related using a novel cryptic complicated variant t(9;22), involving chromosome 12 apart from chromosomes 9 and 22, which was unmasked and characterized by RT-PCR and FISH analyses. In agreement with ESMO clinical practice guidelines, this case report proves the function of these molecular approaches in detecting cryptic fusion gene in some sorts of variant translocations with masked Ph and der(9) chromosomes. As previously reported, the breakpoints location of complicated variant t(9;22) is nonrandom using a marked clustering to specific chromosome bands suggesting that some regions are much more prone to breakage. This finding may be explained by the presence of a distinct genomic structure mediating the recombination. Certainly a considerable clustering was described for higher CG content 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 complicated Philadelphia translocation and in some situations of three-way translocation t(9;22) [11]. Furthermore, this region is involved each in other chromosomal translocations, originating chimeric genes associated to diverse subtypes of leukemia as reported in Mitelman et al. [14] and in Atlas of chromosome in cancer databases [15], and within the fragile web page, FRA12A, that is caused by an expanded CGG repeat in the 5-prime untranslated area on the DIP2B gene (OMIM 611379) [16]. Combining all these information we are able to speculate that the presence of certain genomic motif in 12q13, for instance CGG repeats, could ha.