Correspondence 1445 The paternal chromosome 9 and the maternal chromosome 22 are preferentially rearranged in chronic myeloid leukaemia Leukemia (2004) 18, 1445–1448. doi:10.1038/sj.leu.2403404 results also showed that the normal chromosomes 9 and 22 Published online 10 June 2004 were always of maternal and paternal origin, respectively, a finding that was totally consistent with the parental origin of the TO THE EDITOR rearranged chromosomes. However, as the 9 þ and 22 þ cell hybrids (unlike the 9q þ and Ph þ cell hybrids) can also be In the majority of patients with chronic myeloid leukaemia (CML), derived from normal cells where t(9;22) is not present, the a reciprocal chromosome translocation t(9;22)(q34.1;q11.2) origi- parental haplotypes of the normal chromosome 9 and 22 might nates two derived products known as the Philadelphia (Ph) have not been fully reciprocal to the haplotypes of the chromosome and 9q þ following rearrangement at definite BCR 1 rearranged chromosomes. The fact that this did not happen and ABL regions. While breakpoints are well characterised can be explained, regardless of the high tumour mass of the at the cytogenetic and molecular levels, the parental origin of 2–7 patients herein studied, by the selective advantage, in vitro,of the rearranged chromosomes is controversial. To clarify cell hybrids derived from tumour cells with respect to those this issue, we analysed cell hybrids segregating the derived derived from normal cells. Usually, only the first three visible chromosomes and the normal, nonrearranged, chromosomes 9 colonies to appear in a plate are cloned, while only one colony and 22. Cell hybrid panels were created with bone marrow per plate was used for this analysis. Certainly, fast growing cell aspirates from four CML patients following cell fusion with lines are more likely to form visible colonies, a reason why they recipient, Hprt-deficient, rodent cell lines (RAG and AKO-15). are favourably selected by this procedure. The four panels derived from the patients were PCR screened for A simple probability estimate indicates that a translocation amplifying variable markers located in regions across 9q34.1 involving the paternal chromosome 9 and the maternal and 22q11.2 breakpoints (Tables 1 and 2). This allowed us to chromosome 22 represents one-fourth of all potential 9;22 separate hybrid cell lines into different classes per panel: 9 þ translocations and that our findings, in four patients, could have (cells containing chromosome 9 and lacking 9q þ , 22 and Ph), only occurred at random with P ¼ 1/256 ¼ 0.0039. This was 9q þ (containing the 9q þ chromosome and lacking 9, 22 and concordant with previous findings in 15 Ph þ patients, whose Ph), 22 þ (containing chromosome 22 and lacking 9, 9q þ and paternal and maternal chromosomes 9 and 22 were identified Ph) and Ph þ (containing the Ph chromosome and lacking 9, by cytogenetic markers,2 but differed from other reports using 9q þ and 22). All Ph þ lines, when screened by multiplex RT- 5,6 3,4,7 PCR,8 detected BCR-ABL transcripts, subsequently confirmed by cytogenetic markers and others using molecular markers cDNA sequencing, demonstrating the presence of a transcrip- in which the parental origin of the rearranged chromosomes was tionally active BCR-ABL gene. Another cell hybrid class, 9 þ / not coincident with our findings. It has also been proposed, 22 þ , containing both 9q and 22q regions across breakpoints based on theoretical probability estimates, that the participation and shown to be BCR-ABL þ by PCR assays, was also identified of chromosomes 9 and 22 in t(9;22), regardless of the parental and two of these cell lines were used as control in each panel. origin of each chromosome 9 or 22 homologue, is statistically These cell lines must contain, at least, the normal chromosomes more likely to occur between chromosomes of different parental 9 9 and 22 in addition to the Ph chromosome or the 9q þ origin as a strict consequence of chance. This proposition chromosome and the Ph chromosome. Altogether, 53 hybrid implies that similar proportions of t(9;22) would occur by cell lines, derived from four patients, were analysed (Table 3). rearranging the paternal chromosome 9 and the maternal In order to demonstrate the parental origin of each chromo- chromosome 22 on one side, and the maternal chromosome 9 some in the hybrid cell lines derived from CML patients, we and the paternal chromosome 22 on the other. A close screened eight variable regions of chromosome 9 and seven examination of previous reports in which the parental origin of variable regions of chromosome 22 and compared our results both rearranged chromosomes was determined in CML patients with similar assays in DNA extracts from at least one of the showed that most translocations occurred between chromo- patients’ parents and from 9 þ /22 þ hybrid cells. Supplemen- somes of different parental origin, but the number of cases was tary Tables 1–8 show the chromosome haplotypes found in the very small and, in one t(9;22), both paternal chromosomes were different cell hybrid classes in all panels. A summary of our rearranged.5 Moreover, these few cases reported to date and our results is presented in Table 4 for each of the four hybrid cell own data do not indicate that the two putative types of t(9;22) classes/per panel/per marker in which the parental origin with chromosomes of different parental origin might be equally (maternal or paternal) of the alleles is indicated. Of the 15 likely because there seems to be a higher incidence of variable markers, 14 proved to be informative in 489 of 535 PCR translocations involving the paternal chromosome 9 and the assays, indicating that the paternal chromosome 9 and the maternal chromosome 22 than those resulting from a parental maternal chromosome 22 are preferentially involved in the reciprocation. characteristic t(9;22)(q34.1;q11.2) rearrangement of CML. These 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 chromo- Correspondence: Dr HN Seua´nez, Genetics Division, Instituto somes involved in t(9;22). Our results were provided by ˆ ´ Nacional de Cancer, Rua Andre Cavalcanti, 37, 4th floor, 20231- screening regions that are closely placed, both proximally and 050 Rio de Janeiro, RJ, Brazil; Fax: þ 55 21 3233 1423; E-mail: [email protected] distally, to cytogenetic breakpoints. This makes it highly unlikely Received 15 December 2003; accepted 20 April 2004; Published that our data could be biased by somatic recombination online 10 June 2004 between breakpoint regions and these markers as it might occur Leukemia Correspondence 1446 Table 1 Amplimers used for the amplification of chromosome 9 markers Amplimers Amplimer Cytogenetic Size of amplified Type of repeat Annealing Maximum reference allocation of fragment (bp) temperature (1C) heterozygosity amplified marker Mfd135CA* GDB: 180704 9q31.1 Dinucleotide 53 0.8380 Mfd135GT 116–150 C3B2-1* 9q31.3 105–137 Dinucleotide 52 0.8840 C3B2-2 GDB: 185718 Mfd 178CA* GDB: 180718 9q31.3 Dinucleotide 51 0.8030 Mfd178GT 187–203 Mfd94CA* 9q33.1 135–159 Dinucleotide 55 0.8050 Mfd94GT GDB: 180558 Mfd77CA* GDB: 180555 9q33.2 Dinucleotide 55 0.5530 Mfd77GT 89–97 GSN.PCR1.1* 9q33.2 111–147 Dinucleotide 53 0.7610 GSN.PCR1.2 GDB: 178525 1627-1* GDB: 185720 9q33.3 Dinucleotide 55 0.8100 1627-2 136–154 DBH.PCR2.1* 9q34.2 235–280 Dinucleotide 65 0.7238 DBH.PCR2.2 GDB: 196477 *Sense primer 50 labelled with 6-FAM. bp ¼ base pairs; GDB ¼ Genome Database (www.gdb.org). Table 2 Amplimers used for the amplification of chromosome 22 markers Amplimers Amplimer Cytogenetic Size of amplified Type of repeat Annealing Maximum reference allocation of fragment (bp) temperature (1C) heterozygosity amplified marker F8VWFP.PCR4.1** GDB: 277263 22q11.1 Tetranucleotide 53 F8VWFP.PCR4.2 329–349 0.6100 TOP1P2.PCR1.1** 113–155 Dinucleotide 50 0.9200 TOP1P2.PCR1.2 GDB: 188759 22q11.2 CYP2D8P.PCR2.1** GDB: 180365 22q12.3 Dinucleotide 58 CYP2D8P.PCR2.2 108–130 0.8000 MB-1F** 217–219 Dinucleotide 53 0.4712 MB-1R GDB: 270294 22q12.3 IL2RB.PCR1.1** GDB: 188757 22q13.1 Dinucleotide 51 125–135 0.9100 IL2RB.PCR1.2 TG-01** GDB: 180405 22q13.1 Dinucleotide 56 0.9100 TG-02B 149–163 CYP2D8P.PCR1.1** GDB: 179878 22q13.3 Dinucleotide 51 CYP2D8P.PCR1.2 98–116 0.8000 **Sense primer 50 labelled with NED. bp ¼ base pairs; GDB ¼ Genome Database (www.gdb.org). TOP1P2.PCR1.1/1.2 amplify a marker that is distally located (bp 23 485 119–3 485 250) with respect to BCR (bp 21 847 702–21 982 691) according to http://www.ensembl.org/Multi/blastview. between breakpoint regions and the very distant cytogenetic the M-BCR4 and ABL polymorphic alleles.7 Moreover, our own markers previously used for identifying the parental origin of data were not affected by any selection bias with respect to the chromosomes 9 and 22.2,5,6 Moreover, as our molecular establishment of hybrid cell panels because cell fusion experi- markers were also allocated outside the BCR and ABL genes, ments were successful with all patients that this was attempted. our selection criterion was unrelated to the presence of The fact that the paternal chromosome 9 and the maternal polymorphisms in these loci. This excluded the possibility that chromosome 22 are preferentially involved in t(9;22) was our results could be biased by this variability as it might have initially associated to imprinting,2 although monoallelic expres- been the case of previous studies in which patient selection sion of BCR and ABL was clearly ruled out.10,11 Thus, the relied on the presence of polymorphic CGG repeats at the 50- mechanisms responsible for the distinctive parental participation unstranslated BCR region,3 endonuclease cleavage sites inside of chromosomes 9 and 22 in t(9;22) remains to be elucidated.