| Summary: | 碩士 === 高雄醫學大學 === 醫學研究所碩士班 === 92 === Title:
The Molecular Characterization of a Complex Rearrangement Involving BCR/ABL Gene
Abstract
Objective: Conventional chromosome analysis has not been successful in detecting unusual cases of leukemia in recent studies. These recent studies reported that mRNA-Bar/Abl as an only back up method to detect Bcr/Abl gene rearrangement in leukemia cell were inconclusive. To detect abnormality, these recent studies probed into the molecular characterization of a complex rearrangement involving Bcr/AbL gene in CML. The FISH and an extra long PCR, a method used by Lange was able to detect the bcr/abl gene rearrangement in the early stage of CML.
In this current study, we developed two hypotheses. 1) The mRNA-Bcr/Abl as a back up method to detect Bcr/Abl gene rearrangement in leukemia cell was insufficient. We tested the reliability and the accuracy of Lange and conventional chromosome theories using a high resolution chromosome analysis, a series of PCR method, and various FISH probes which were whole painting probe and Bcr/Abl fusion gene probe to detect CML cases in our collected population. 2) Due to a possibility of a complex rearrangement in rare leukemia cases, CML breakpoints might occur in a very large genomic region on bcr/abl fusion gene instead of appearing in the mRNA or protein region.
Method: Sixty patients (16 females, 44 males; including 5 normal samples as controls) were collected over a 2-year period. All of the retrospective material is submitted to Institutional Review Board of Kaohsiung Medical University. The samples of patients having onset leukemia-like symptoms were referred by doctors from Kaohsiung Veterans General Hospital and Kaohsiung Chang-Gung Memorial Hospital to the Cytogenetics Laboratory at Kaoshiung Medical University Hospital where the study took place. This study employed a three-phase experiment. At the first phase, high resolution chromosome study by G-banding was performed on each case. FISH studies also performed on these cases. At the second phase, first, we used an extra long-template DNA-PCR (LT-DNA-PCR) (23k) which covered whole genomic from 1b, 1a and a2 on ABL gene parts to b2 and b3 on bcr gene parts. Then, we performed a series of long DNA PCR primer (5k) on chromosome 22 between centromere and Bcr gene. We used whole painting probe of FISH to identify abnormality among patients and to verify if their long PCR were positive.
Results: Among these total 60 cases, all 5 samples from normal control group and 14 of 55 samples from patient group were found with normal karyotypes. Normally, Bcr gene should occupied a region of about 135 kb on chromosome 22q11.2. Among 55 cases, 41 patients showed Ph positive results and 14 patients showed Ph negative results. However, in addition to the 14 negative cases, some of the Ph positive cases (N=26) also showed abnormality .The CML breakpoints do occur in a very large genomic region on Bcr/Abl fusion gene instead of appearing in the mRNA or protein region. Furthermore, there were four Ph-negative chronic myeloid leukemia cases in which the Bcr/Abl fusion gene was found located on chromosome 9.
Discussions: We were able to identify gene rearrangement in 14 abnormal cases whose the chromosome analysis failed to detect Philadelphia chromosome. Among these 14 cases, the mRNAs of 4.5- and 6.7-kb apparently did not encode the same cytoplasm 160-kD protein. From PCR data, they showed an unusual genomic disorder in the first exon of DNA label. This finding agreed with prior research. Research pointed out that Bcr protein guided the serine/threonine kinase activity; at least two SH2 binding sites were encoded in its first exon. These findings support our assumption of abnormal cases of PCR. Progenitor cell from stem cell may express Bcr/Abl gene rearrangement and may not be evident in m-RNA and protein in cases of chromosome abnormality. According to the literature, the Abl gene was about 225 kb in size but mRNA transcript was only expressed as either a 6- or 7-kb; and genomic sequence contained 20 exons. In particular, the 145-kD ABL protein was already classified being a no receptor tyrosine kinase. The proto-oncogene of Abl encoded a protein; nuclear protein tyrosine kinase was implicated in cell adhesion, cell division, stress response, and processes of cell differentiation. In other words, there were more abnormal rearrangements in Bcr gene than in Abl gene in some of our patient samples. A Long PCR and an extra long PCR successfully identified different breakpoints of these abnormal cases. Especially, there are 6 abnormal cases detected by a long PCR. We found 2 of 6 cases whose breakpoints were very close centromere on chromosome 22 but the Bcr/Abl fusion gene was not found on chromosome 9. This finding supported our assumption and was not founded by prior research.
Conclusions: The change of Bcr/Abl fusion in chromosomal rearrangement and translocation may be caused by malignant transformation. When the malignant transformation occurred, the Abl gene was found right after Bcr gene on chromosome 22 in chronic myeloid leukemia. Both genes were involved in the t (9; 22) translocation (Philadelphia chromosome) which were associated with over 85% of CML, even less than that. However, the rest of percentage of CML may not be found in fusion protein by chromosome analysis, it can be found in gene disorder in different kind of PCR and FISH. In addition, there were rare types of leukemia due to unusual breakpoints that join different exon or intron sets of Bcr. These unusual breakpoints connected to a common subset of the exon of the Abl gene and located at chromosome 9 will cause various types of molecular changes. M-Bcr, m-Bcr or a new breakpoint were found in our patient cases. Thus, these different breakpoints were found possible to appear at different locations in the entire string of genome. These cells will be undetected by techniques that are based on expression of the fusion gene.
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