One of the most prevalent abnormalities in leukemia patients, the Philadelphia chromosome, a translocation of the ABL1 oncogene on chromosome 9 to the BCR gene on chromosome 22, occurs in adult chronic myelogenous leukemia and childhood acute chronic leukemia. In addition to the translocation, the commercial ES-probe used in clinical cytogenetics laboratories sometimes detects deletions of DNA sequences upstream of the ABL1 gene on the derivative 9 chromosome (in 1/10 to 1/3 of patients). These deletions appear to be prognostic for early blast crisis. We hypothesize that these deletions result in hemizygosity of additional genes adjacent to ABLI. These deletion breakpoints can be refined with high- resolution fluorescence in situ studies (scFISH), which uses short single copy (sc) DNA probes designed from the draft genome sequence. Chromosomal preparations of patient specimens diagnosed with Philadelphia chromosomes in clinical cytogenetic laboratories will be analyzed with these probes. ScFISH, which has been developed in our laboratories, provides an unprecedented level of resolution for delineating sequences associated with inherited chromosomal rearrangements by FISH (Rogan, Cazcarro, Knoll, 2001; Knoll, Cazcarro, Rogan, 2000). We have developed and validated single copy DNA probes (quickly and without cloning) for FISH analysis of more than 20 distinct chromosomal regions.
We aim (1) to develop scFISH probes for ABL1 oncogene, BCR and their adjacent regions from the draft genome sequence and verify their locations by FISH; (2) to compare results with scFISH probes to those obtained using the commercially available ES-probes for t(9;22) on the same patient specimens; and (3a) to categorize cytogenetically positive t(9;22) into molecular subgroups based on the sizes of deletion centromeric to ABL1 and the breakpoints within the BCR gene, (3b) to determine if there are preferential sites of breakage on chromosome 9 adjacent to ABL1, and to localize these sites. In contrast with scFISH probes, commercial reagents detecting this translocation routinely used in clinical laboratories comprise large multigenic segments. We anticipate that both the commercial and scFISH probes will detect the chromosomal translocation in some patients, however, only the scFISH probes are expected to delineate deletions in sequences immediately adjacent to ABL 1, and to distinguish the major and minor sites of breakage in the BCR gene that correspond to chronic myelogenous leukemia and acute lymphocytic leukemia, respectively. The proposed study will delineate the chromosomal regions that undergo breakage in these types of leukemia and will establish whether there are common deletion breakage intervals on the translocated chromosome 9. Our long range goal is to classify patients based on the sites of translocation and deletion size, and then to determine if the disease complications can be attributed to the loss of specific genes adjacent to ABL1.
| Knoll, Joan H M (2007) Human metaphase chromosome FISH using quantum dot conjugates. Methods Mol Biol 374:55-66 |
| Newkirk, Heather L; Rogan, Peter K; Miralles, Mauricio et al. (2006) Determination of genomic copy number with quantitative microsphere hybridization. Hum Mutat 27:376-86 |
| Newkirk, Heather L; Knoll, Joan H M; Rogan, Peter K (2005) Distortion of quantitative genomic and expression hybridization by Cot-1 DNA: mitigation of this effect. Nucleic Acids Res 33:e191 |
| Knoll, Joan H M; Rogan, Peter K (2003) Sequence-based, in situ detection of chromosomal abnormalities at high resolution. Am J Med Genet A 121A:245-57 |
