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 c-Rel custom synthesis technologies. RNA extraction and RTPCR had been performed following the insert kit guidelines (Nanogen Inc., San Diego, CA, USA). The measurement of the cDNA of P210 was normalized to the cDNA of ABL1 gene. Standard cytogenetic evaluation on bone marrow showed on 22 metaphases a reciprocal mAChR4 list translocation involving the long arm of chromosomes 12 and 22, t(12;22), without the need 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 evaluation 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 is often a mixture of ASS-ABL1 probe labeled in red and of BCR probe with the proximal BCR area labeled in blue plus 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) one 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 regular 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 additional investigate the involvement of chromosome 9 within the complex rearrangement: it showed a typical signal pattern.three. DiscussionWe describe a patient with CML associated having a novel cryptic complex variant t(9;22), involving chromosome 12 in addition to 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 part of those molecular approaches in detecting cryptic fusion gene in some varieties of variant translocations with masked Ph and der(9) chromosomes. As previously reported, the breakpoints location of complicated variant t(9;22) is nonrandom having a marked clustering to precise chromosome bands suggesting that some regions are far more prone to breakage. This discovering might be explained by the presence of a certain genomic structure mediating the recombination. Indeed a substantial 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 region, involved in our case, was described by Costa et al. [13] in association with complicated Philadelphia translocation and in some cases of three-way translocation t(9;22) [11]. In addition, this region is involved both in other chromosomal translocations, originating chimeric genes associated to various subtypes of leukemia as reported in Mitelman et al. [14] and in Atlas of chromosome in cancer databases [15], and inside the fragile site, FRA12A, which can be brought on by an expanded CGG repeat in the 5-prime untranslated region with the DIP2B gene (OMIM 611379) [16]. Combining all these data we can speculate that the presence of certain genomic motif in 12q13, for example CGG repeats, could ha.