Molecular analysis of the translocation breakpoint in a Philadelphia-positive, bcr-negative ALL patient.
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Alleles↗
Biomedical subjects
G Grosveld
Publications and source records attributed to G Grosveld.
Molecular analysis of chromosome breakpoints.
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Animals↗
The human pim-1 gene is not directly activated by the translocation (6;9) in acute nonlymphocytic leukemia.
In Acute Nonlymphocytic Leukemia (ANLL) specified by a reciprocal translocation (6;9), defined clinical and morphological features are observed. This suggests that genes located near the breakpoints of the translocation chromosomes are involved in the generation of this subtype of leukemia. The human pim-1 gene has been mapped near the t(6;9) breakpoint on chromosome 6. Using somatic cell hybrids we demonstrated that the pim-1 gene remains on chromosome 6. We investigated whether pim-1 plays a role in t(6;9) ANLL. The expression of pim-1 is elevated in two out of three patients with t(6;9) ANLL. However, the pim-1 gene has a size of only 6 kb and using field inversion gel electrophoresis, no chromosomal breakpoint can be detected within a distance of 165 kb from the pim-1 locus. Therefore it seems more likely that the elevated expression is due to the differentiation state of the cells rather than transcriptional activation by the translocation.
Adolescent↗
[Localization of the gene defect in tuberous sclerosis].
Elucidation of the genetic defect in tuberous sclerosis (TS) awaits a precise chromosomal localization. At present two chromosomal regions, 9q34 and 11q23, are candidates for the site of a TS locus. Family studies using polymorphic DNA markers are carried out in other laboratories and in ours and are expected to disclose the existence of one TS gene that is localized on either chromosome 9 or 11, or the involvement of two genes, one on #9 and one on #11. Early postnatal and potentially prenatal diagnosis by means of DNA analysis may be offered to a family with TS after identification of the gene defect, but also after the identification of very closely linked DNA markers.
Chromosomes, Human, Pair 11↗
Cytogenetic and molecular analysis in Philadelphia negative CML.
We studied the clinical, hematologic, cytogenetic and molecular biologic features in four patients with Philadelphia (Ph) negative chronic myeloid leukemia (CML). In all four cases the clinical and hematologic characteristics were indistinguishable from Ph positive CML. Cytogenetic analysis showed a normal karyotype in two patients and chromosomal translocations apparently not affecting chromosome 22 in the other two cases. Southern blot analysis using probes of the bcr region, demonstrated a bcr break-point in all four patients. In situ hybridization with bcr, c-abl, and c-sis probes showed unusual hybridization sites for 5'-bcr and c-abl indicating complex chromosomal rearrangements affecting three different chromosomes in the four patients investigated. Using polymerase chain reaction (PCR) followed by hybridization to oligonucleotide probes specific for the bcr-abl fusion region, the expression of a chimeric bcr-abl mRNA was detected. In these patients we demonstrated that (a) CML with a breakpoint in the bcr region without cytogenetically detectable Ph chromosome is characterized by the same genomic recombination of 5'-bcr and c-abl as CML with standard Ph translocation and (b) unusual localization of 5'-bcr and c-abl sequences caused by complex Ph translocation does not interfere with transcription of the bcr-abl fusion gene.
Adult↗
Characterization of the translocation breakpoint in a patient with Philadelphia positive, bcr negative acute lymphoblastic leukaemia.
Approximately 5% of children and 10-20% of adults with acute lymphoblastic leukaemia (ALL) have a chromosome translocation t(9;22) which at the cytogenetic level appears identical to that in chronic myeloid leukaemia (CML). The t(9;22) translocation was first recognised in CML patients by its 22q- or Philadelphia (Ph) chromosome. While all Ph positive CML patients so far described have a chromosome 22 breakpoint within the breakpoint cluster region (bcr) located in the 3' part of the phl gene, only some Ph positive ALL patients have breakpoints in bcr. We have cloned the breakpoint of the 9q+ chromosome from the DNA of a Ph positive ALL patient in whom there is no breakpoint in the bcr. The non-chromosome 9 sequences of the breakpoint region are shown to be derived from chromosome 22. The breakpoint in chromosome 22 is shown to be the first intron of the phl gene about 66kb upstream of the bcr. Using probes from this intron, rearrangements were detected in the DNA of two out of twelve additional Ph positive, bcr negative ALL patients.
Chromosomes, Human, Pair 22↗
bcr-abl oncogene activation in Philadelphia chromosome-positive acute lymphoblastic leukemia.
Tumor-specific alterations in oncogenes are thought to play a central role in the development of cancer. An example is the consistent fusion of the bcr gene to the c-abl oncogene on the Ph chromosome in CML. The Ph chromosome can also be observed in ALL. About 50% of Ph+ ALL cases, in contrast to CML, do not exhibit chromosomal breakpoints in the major cluster region or mcr (Ph+ mcr- ALL). These cases may have a novel bcr-abl fusion gene instead. We tested this hypothesis in eight Ph+ mcr- ALL patients by amplifying the putative hybrid part of the bcr-abl cDNA, using the polymerase chain reaction method. All cases examined showed the same joining of the first exon of the bcr gene to the c-abl oncogene. Thus, the novel bcr-abl fusion in Ph+ mcr- ALL is the result of a molecularly distinct Ph chromosome. This allows the definition of Ph+ leukemias by their respective bcr-abl oncogene activation. Moreover, the cDNA amplification method we use is a clinically useful tool to screen for bcr-abl oncogene activations in leukemia patients.
DNA, Neoplasm↗
Molecular characterization of the testis specific c-abl mRNA in mouse.
The c-abl gene encodes a protein tyrosine kinase and is transcribed from at least two promoters giving rise to transcripts of two size classes of approximately 5 and 6 kb in length. These mRNAs only differ in their most 5' exon and encode proteins of similar size but with different N-termini. In the mouse testis an additional abundant c-abl mRNA of 4 kb is detected. This mRNA was shown to be expressed in the haploid male germ cells of the adult mouse. Here we describe the cloning and molecular characterization of a cDNA representing the testis specific c-abl transcript. We show that the 4 kb c-abl mRNA arises from alternative polyadenylation of an RNA transcribed from the same promoter as the 5 kb mRNA. The site of polyadenylation is unusual in this shorter transcript as it is not preceded by the highly conserved hexanucleotide AAUAAA. The use of this polyadenylation site removes 1.2 kb of 3' sequences present in the somatic c-abl mRNAs, but does not affect the main open reading frame of the transcript. Using in situ hybridization on whole testis sections it is shown that the 4 kb c-abl mRNA is most abundant in the elongating spermatids.
Animals↗
Unique fusion of bcr and c-abl genes in Philadelphia chromosome positive acute lymphoblastic leukemia.
The Philadelphia (Ph) chromosome, the product of t(9:22), is the cytogenetic hallmark of chronic myelogenous leukemia. The c-abl oncogene on chromosome 9 is translocated to the Ph chromosome and linked to a breakpoint cluster region (bcr), which is part of a large bcr gene. This results in the formation of a bcr-c-abl fusion gene, which is transcribed into an 8.5 kb chimeric mRNA encoding a 210 kd bcr-c-abl fusion protein. The Ph chromosome is also found in acute lymphoblastic leukemia (Ph+ ALL). Although the c-abl is translocated and a new 190 kd c-abl protein has been identified, no breakpoints are observed in the bcr (Ph+bcr- ALL). Here we show that in Ph+bcr- ALL, breakpoints in chromosome 22 occur within the same bcr gene, but more 5' of the bcr. Cloning of a chimeric bcr-c-abl cDNA demonstrates that the fusion gene is transcribed into a 7 kb mRNA, encoding a novel fusion protein.
Base Sequence↗
The role of the Philadelphia translocation in chronic myelocytic leukemia.
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Amino Acid Sequence↗
Comparison of the human and mouse PIM-1 cDNAs: nucleotide sequence and immunological identification of the in vitro synthesized PIM-1 protein.
Pim-1 is a putative oncogene which was discovered as a target for transcriptionally activating proviral insertions in T-cell lymphomas in mice. Its genomic and cDNA sequence have been published (Selten et al., 1986, Cell 46, 603-611). We have isolated and sequenced pim-1 cDNA clones from the human leukemia cell line K562 (h-pim-1). Comparison of human and mouse cDNA sequences reveals that both contain: (i) a GC rich leader sequence, (ii) an open reading frame encoding a 313 amino acid protein (94% conserved) showing obvious homology to protein kinases and (iii) a 1.3 kb 3' untranslated region with two polyadenylation signals and five copies of the mRNA destabilizing motif ATTTA. "In vitro" translation experiments show that both mouse and human cDNA derived RNA can translate into a protein of the expected size. Their identity was confirmed by immuno-precipitation with antisera raised against synthetic pim-1 oligopeptides.
Amino Acid Sequence↗
Persistence of chronic myelocytic leukemia despite deletion of rearranged bcr/c-abl sequences in blast crisis.
We report on a Ph+ chronic myelocytic leukemia (CML) case, cytogenetically characterized by the occurrence of a second Philadelphia (Ph) chromosome in lymphoid blast crisis of T cell lineage. In situ hybridization analyses showed a deletion of translocated c-abl sequences, present on the Ph during chronic state, from both Ph in acute state. Moreover, Southern blot analyses of blastic cells exhibited a rearrangement within bcr, but a deletion of 5' bcr sequences, and Northern blots failed to detect the hybrid 8.5 kb bcr/c-abl transcript usually observed in Ph+ CML.
Base Sequence↗
Molecular analysis of both translocation products of a Philadelphia-positive CML patient.
The breakpoint regions of both translocation products of the (9;22) Philadelphia translocation of CML patient 83-H84 and their normal chromosome 9 and 22 counterparts have been cloned and analysed. Southern blotting with bcr probes and DNA sequencing revealed that the breaks on chromosome 22 occurred 3' of bcr exon b3 and that the 88 nucleotides between the breakpoints in the chromosome 22 bcr region were deleted. Besides this small deletion of chromosome 22 sequences a large deletion of chromosome 9 sequences (greater than 70 kb) was observed. The chromosome 9 sequences remaining on the 9q+ chromosome (9q+ breakpoint) are located at least 100 kb upstream of the v-abl homologous c-abl exons whereas the translocated chromosome 9 sequences (22q-breakpoint) could be mapped 30 kb upstream of these c-abl sequences. The breakpoints were situated in Alu-repetitive sequences either on chromosome 22 or on chromosome 9, strengthening the hypothesis that Alu-repetitive sequences can be hot spots for recombination.
Adolescent↗
Additional c-abl/bcr rearrangements in a CML patient exhibiting two ph1 chromosomes during blast crisis.
Recent data suggest that two human genes, c-abl on chromosome 9 and bcr on chromosome 22, are involved in the generation of Ph1-positive CML. To examine a possible role of these sequences in transition from chronic towards blastic phase, rearrangements within bcr were analysed in 4 patients with Ph1-positive CML during chronic and acute phase. In 3 patients bcr rearrangements were identical in both phases, while in a fourth patient with duplicated Ph1 an amplified additional bcr fragment was detected in acute phase. Northern blot analysis of blast cells of the latter patient showed a novel 10.3 kb RNA species that replaced the altered 8 kb RNA transcript usually found in Ph1-positive CML.
Adolescent↗
The chronic myelocytic cell line K562 contains a breakpoint in bcr and produces a chimeric bcr/c-abl transcript.
In the DNAs of all Ph1-positive chronic myelocytic leukemia patients studied to date, a breakpoint on chromosome 22 (the Ph1 chromosome) can be demonstrated with a probe from the bcr (breakpoint cluster region). Although the K562 cell line was established from cells of a chronic myelocytic leukemia patient, we have been unable to detect the Ph1 chromosome by cytogenetic means. Employing a probe from the 5' region of bcr, we have cloned an amplified Ph1 breakpoint fragment from K562. This demonstrates that K562 contains multiple remnants of a Ph1 chromosome with a breakpoint within bcr and thus may serve as a model system for the study of Ph1-positive chronic myelocytic leukemia at a molecular level. The isolation of bcr cDNA sequences shows that parts of bcr encode a protein. Employing K562, we demonstrate the presence of an abnormally sized mRNA species hybridizing to c-abl and to a bcr cDNA probe, indicating the possible consequence of the Ph1 translocation on a transcriptional level in chronic myelocytic leukemia. The isolation and sequencing of a cDNA containing the breakpoint area of this mRNA provide further evidence for its chimeric structure. Cloning of large stretches of chromosomal DNA flanking bcr and c-abl sequences in K562 and identification of the exons participating in the formation of the chimeric mRNA shows that a splice of at least 99 kilobases is made to fuse the 3' bcr exon to the 5' c-abl exon. Furthermore two chimeric cDNAs were isolated containing chromosome 9 sequences that map 43.5 kilobases downstream from the K562 breakpoint. These chromosome 9 sequences neither hybridize to the 8.5-kilobase chimeric c-abl mRNA nor to normal c-abl mRNAs in Hela cells and probably represent incorrect splicing products present in the K562 cell line.
Amino Acid Sequence↗
The role of the Philadelphia translocation in chronic myelocytic leukemia.
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Base Sequence↗
Evidence of a new chimeric bcr/c-abl mRNA in patients with chronic myelocytic leukemia and the Philadelphia chromosome.
The hallmark of chronic myelocytic leukemia is the presence of the Philadelphia chromosome (Ph1). In recent studies, we obtained data that strongly suggested the involvement of an oncogene, c-abl, in this type of leukemia. This oncogene, normally located on chromosome 9, is translocated to chromosome 22 as a result of the Ph1 translocation. In addition, we identified a region on chromosome 22, the breakpoint cluster region (bcr), which contains the chromosomal breakpoint in all patients with chronic myelocytic leukemia who are positive for Ph1. Recent studies have suggested that the bcr is part of a gene that is truncated as a consequence of the Ph1 translocation. The deleted part of this gene could be replaced by c-abl sequences; to test this hypothesis we analyzed the RNA of five patients with chronic myelocytic leukemia. All five had chimeric bcr/c-abl messenger RNA, suggesting that the deleterious effects of this disease can be associated with an abnormal chimeric protein encoded by the bcr and the c-abl oncogene.
Adult↗
C-abl and bcr are rearranged in a Ph1-negative CML patient.
Chromosomal analysis of a patient with chronic myelocytic leukemia (CML) revealed a translocation (9;12) (q34;q21) without a detectable Philadelphia chromosome (Ph1). Using molecular approaches we demonstrate (i) a rearrangement within the CML breakpoint cluster region (bcr) on chromosome 22, and (ii) a joint translocation of bcr and c-abl oncogene sequences to the derivative chromosome 12. These observations support the view that sequences residing on both chromosome 9 (c-abl) and 22 (bcr) are involved in the generation of CML and suggest that a subset of Ph1-negative patients may in fact belong to the clinical entity of Ph1-positive CML.
Adult↗