TO THE EDITOR
In the majority of patients with chronic myeloid leukaemia (CML), a reciprocal chromosome translocation t(9;22)(q34.1;q11.2) originates two derived products known as the Philadelphia (Ph) chromosome and 9q+ following rearrangement at definite BCR and ABL regions.1 While breakpoints are well characterised at the cytogenetic and molecular levels, the parental origin of the rearranged chromosomes is controversial.2, 3, 4, 5, 6, 7 To clarify this issue, we analysed cell hybrids segregating the derived chromosomes and the normal, nonrearranged, chromosomes 9 and 22. Cell hybrid panels were created with bone marrow aspirates from four CML patients following cell fusion with recipient, Hprt-deficient, rodent cell lines (RAG and AKO-15). The four panels derived from the patients were PCR screened for amplifying variable markers located in regions across 9q34.1 and 22q11.2 breakpoints (Tables 1 and 2). This allowed us to separate hybrid cell lines into different classes per panel: 9+ (cells containing chromosome 9 and lacking 9q+, 22 and Ph), 9q+ (containing the 9q+ chromosome and lacking 9, 22 and Ph), 22+ (containing chromosome 22 and lacking 9, 9q+ and Ph) and Ph+ (containing the Ph chromosome and lacking 9, 9q+ and 22). All Ph+ lines, when screened by multiplex RT-PCR,8 detected BCR-ABL transcripts, subsequently confirmed by cDNA sequencing, demonstrating the presence of a transcriptionally active BCR-ABL gene. Another cell hybrid class, 9+/22+, containing both 9q and 22q regions across breakpoints and shown to be BCR-ABL+ by PCR assays, was also identified and two of these cell lines were used as control in each panel. These cell lines must contain, at least, the normal chromosomes 9 and 22 in addition to the Ph chromosome or the 9q+ chromosome and the Ph chromosome. Altogether, 53 hybrid cell lines, derived from four patients, were analysed (Table 3).
In order to demonstrate the parental origin of each chromosome in the hybrid cell lines derived from CML patients, we screened eight variable regions of chromosome 9 and seven variable regions of chromosome 22 and compared our results with similar assays in DNA extracts from at least one of the patients’ parents and from 9+/22+ hybrid cells. Supplementary Tables 1, 2, 3, 4, 5, 6, 7 and 8 show the chromosome haplotypes found in the different cell hybrid classes in all panels. A summary of our results is presented in Table 4 for each of the four hybrid cell classes/per panel/per marker in which the parental origin (maternal or paternal) of the alleles is indicated. Of the 15 variable markers, 14 proved to be informative in 489 of 535 PCR assays, indicating that the paternal chromosome 9 and the maternal chromosome 22 are preferentially involved in the characteristic t(9;22)(q34.1;q11.2) rearrangement of CML. These results also showed that the normal chromosomes 9 and 22 were always of maternal and paternal origin, respectively, a finding that was totally consistent with the parental origin of the rearranged chromosomes. However, as the 9+ and 22+ cell hybrids (unlike the 9q+ and Ph+ cell hybrids) can also be derived from normal cells where t(9;22) is not present, the parental haplotypes of the normal chromosome 9 and 22 might have not been fully reciprocal to the haplotypes of the rearranged chromosomes. The fact that this did not happen can be explained, regardless of the high tumour mass of the patients herein studied, by the selective advantage, in vitro, of cell hybrids derived from tumour cells with respect to those derived from normal cells. Usually, only the first three visible colonies to appear in a plate are cloned, while only one colony per plate was used for this analysis. Certainly, fast growing cell lines are more likely to form visible colonies, a reason why they are favourably selected by this procedure.
A simple probability estimate indicates that a translocation involving the paternal chromosome 9 and the maternal chromosome 22 represents one-fourth of all potential 9;22 translocations and that our findings, in four patients, could have only occurred at random with P=1/256=0.0039. This was concordant with previous findings in 15 Ph+ patients, whose paternal and maternal chromosomes 9 and 22 were identified by cytogenetic markers,2 but differed from other reports using cytogenetic markers5, 6 and others using molecular markers3, 4, 7 in which the parental origin of the rearranged chromosomes was not coincident with our findings. It has also been proposed, based on theoretical probability estimates, that the participation of chromosomes 9 and 22 in t(9;22), regardless of the parental origin of each chromosome 9 or 22 homologue, is statistically more likely to occur between chromosomes of different parental origin as a strict consequence of chance.9 This proposition implies that similar proportions of t(9;22) would occur by rearranging the paternal chromosome 9 and the maternal chromosome 22 on one side, and the maternal chromosome 9 and the paternal chromosome 22 on the other. A close examination of previous reports in which the parental origin of both rearranged chromosomes was determined in CML patients showed that most translocations occurred between chromosomes of different parental origin, but the number of cases was very small and, in one t(9;22), both paternal chromosomes were rearranged.5 Moreover, these few cases reported to date and our own data do not indicate that the two putative types of t(9;22) with chromosomes of different parental origin might be equally likely because there seems to be a higher incidence of translocations involving the paternal chromosome 9 and the maternal chromosome 22 than those resulting from a parental reciprocation.
These analyses, however, must take into consideration that the data might not be strictly comparable due to the different approaches used for determining the parental origin of chromosomes involved in t(9;22). Our results were provided by screening regions that are closely placed, both proximally and distally, to cytogenetic breakpoints. This makes it highly unlikely that our data could be biased by somatic recombination between breakpoint regions and these markers as it might occur between breakpoint regions and the very distant cytogenetic markers previously used for identifying the parental origin of chromosomes 9 and 22.2, 5, 6 Moreover, as our molecular markers were also allocated outside the BCR and ABL genes, our selection criterion was unrelated to the presence of polymorphisms in these loci. This excluded the possibility that our results could be biased by this variability as it might have been the case of previous studies in which patient selection relied on the presence of polymorphic CGG repeats at the 5′-unstranslated BCR region,3 endonuclease cleavage sites inside the M-BCR4 and ABL polymorphic alleles.7 Moreover, our own data were not affected by any selection bias with respect to the establishment of hybrid cell panels because cell fusion experiments were successful with all patients that this was attempted.
The fact that the paternal chromosome 9 and the maternal chromosome 22 are preferentially involved in t(9;22) was initially associated to imprinting,2 although monoallelic expression of BCR and ABL was clearly ruled out.10, 11 Thus, the mechanisms responsible for the distinctive parental participation of chromosomes 9 and 22 in t(9;22) remains to be elucidated.
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This work was supported by Instituto Nacional de Câncer (INCA) and Fundação Ary Frauzino (Rio de Janeiro, Brazil). We are grateful to Drs Arthur Moellman and Claudete Klumb, to other members of the staff of the Haematology Service-INCA and the Bone Marrow Transplantation Centre-INCA for samples and clinical data. We are grateful to Claudio Vieira da Silva for technical support and to Amersham Biosciences for lending the MegaBACE Genetic profiler software. This work was approved by the local Committee of Medical Ethics in accordance with the guidelines of the Helsinki declaration.