Chromosome 9 and 22 Breakpoints Cluster Regions Definition of Deleted Sequences on der(9) in Chronic Myeloid Leukemia.

Abstract Chronic myeloid leukemia (CML) is characterized by a reciprocal translocation t(9;22)(q34;q11) that generates a BCR-ABL fusion gene on the Philadelphia (Ph) chromosome. Large deletions adjacent to the t(9;22) breakpoint on the derivative 9 chromosome have now been found, which result in genomic loss at both sides of the translocation breakpoint. We report a FISH study in CML cases bearing deletions on the der(9) chromosome. FISH analysis with specific BAC/PAC clones for the ABL1 and BCR genes was performed on bone marrow cells of 305 CML patients at diagnosis. A set of BAC/PAC probes, belonging to chromosomes 9, 22 was selected according to the University of Santa Cruz (UCSC) database and employed in FISH experiments. We detected der(9) deletions in 56 (18.4%) CML cases. Deletions of chromosome 9 sequences on the der(9) were found in 47 (84%) cases; they ranged from 350 Kb to 41.6 Mb. Chromosome 22 deletions on der(9) were found in 49 (87.5%) of the analysed cases; the deleted chromosome 22 sequences were shorter than the deleted chromosome 9 sequences (ranging from 400 Kb to 12.7 Mb). Although deletions size appeared to be heterogeneous, 2 main breakpoints cluster regions were identified proximally (at about 1.9 Mb) to the ABL and distally (at about 0.7 Mb) to the BCR genes, respectively. In fact, a cluster region of 1.1Mb, delimited by RP11-203J24 and RP11-247A12, was defined on chromosome 9 whereas a 0.8 Mb region, included between CTA-322B1 and RP5-930L11, was identified on chromosome 22. Bioinformatic analysis of breakpoints cluster regions was performed to identify sequence motifs that could mediate the chromosomal rearrangement. The presence of Matrix Association Regions (MARs) and the topoisomerase II consensus was investigated by MAR-Wiz software (http://www.futuresoft.org/modules/MarFinder/); the RepeatMasker program (http://www.repeatmasker.org/) was employed to detect interspersed repeats and low complexity DNA sequences (such as SINE, LINE, and LTR). Potential sequence similarities between breakpoints cluster regions were searched for using Genalyzer software. Sequence analysis revealed that the frequency distribution of MARs, topoisomerase II sites, and repeated elements was no different from the frequency observed in other genomic regions. Genalyzer software did not detect sequence similarities between the 2 clustering regions; however, a 76 Kb duplicon, previously reported in literature, was located at a distance of 500 kb and 550 Kb from the chromosomes 9 and 22 cluster regions, respectively. In conclusion, our analysis revealed that sequence motifs do not seem to be involved in the occurrence of der(9) deletions. We hypothesize that the presence of a duplicon could mediate the juxtaposition of ABL and BCR genes and that a probable open chromatin status of chromosomes 9 and 22 sequences could confer to DNA more vulnerability to double strand breaks, mediating the t(9;22) rearrangement and the occurrence of der(9) deletions.