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Late-appearing pseudocentric fission event during chronic myeloid leukemia progression
Cancer Genetics and Cytogenetics 174 (2007) 61e67
Short communication
Late-appearing pseudocentric fission event during chronic myeloid leukemia progression Clelia Tiziana Storlazzia,*, Francesco Albanob, Marija Dencic´-Feketec, Vesna Djordjevic´c, Mariano Rocchia a
Department of Genetics and Microbiology, University of Bari, Via Amendola 165/A, 70126 Bari, Italy b Department of Hematology, University of Bari, Bari, Italy c Institute of Hematology, Clinical Center Serbia, Koste Todorovica 2, 11000 Belgrade, Serbia and Montenegro Received 25 September 2006; received in revised form 13 November 2006; accepted 21 November 2006
Abstract
Pseudocentric fission is a rare event consisting of the splitting of one functional centromere into two new products, of which only one can give rise to a functionally competent kinetochore. We report here a pseudocentric fission event within the D5Z2 alphoid subset disrupting the centromeric region of chromosome 5 in a case of chronic myeloid leukemia (CML) after treatment with imatinib and interferon. The breakage generated unequal partitioning of a-satellite sequences between the two fission products. One product was inserted within the long arm of chromosome 12 at band 14.3, becoming the only functional centromere of chromosome der(5). The other fission product was rearranged to form a sandwich-like dicentricdbut functionally monocentricdchromosome der(6), made up of material from chromosomes 5, 12, and 6. The intercentric distance on der(6) was shown to be largely O20 Mb. To our knowledge, this is the first pseudocentric fission event described in CML. Moreover, our results confirm the susceptibility to breakage of the centromeric region of chromosome 5. Ó 2007 Elsevier Inc. All rights reserved.
1. Introduction The centromere is a functional domain responsible for the correct segregation of eukaryotic chromosomes during cell division. It is composed of centromeric proteins (CENPs) and centromere-specific satellite DNA sequences [1,2]. Primate centromeres consist of AT-rich a-satellite (alphoid) DNA monomers, 171 bp in length, tandemly repeated to constitute large blocks of centromeric heterochromatin [3,4]. In humans, the cores of these blocks are organized to form a higher-order repeat consisting of n monomers almost identically repeated in long DNA arrays [5e7]. Alphoid subsets from different chromosomes show relatively large sequence variation, so that most of these subsets are chromosomespecific. Multiple alphoid subsets can coexist on the same chromosome [8,9]. The relationship between a-satellite DNA and centromere function is not well understood. Indeed, there are several examples of dicentric chromosomes in which one apparently normal centromere is not functional. * Corresponding author: Tel.: þ39-080-5443582; fax: þ39-0805443386. E-mail address: [email protected] (C.T. Storlazzi). 0165-4608/07/$ e see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.cancergencyto.2006.11.009
Furthermore, several human neocentromeres, lacking alphoid DNA sequences, have been recently described in clinical cases [10e12] and, occasionally, in normal individuals [13,14]. Centromere (centric) fission consists of the splitting (or transverse misdivision) of one functional centromere into two new centric chromosomes [15,16]. This fission has been reported in a series of organisms and is represented as an important mechanism of karyotype evolution and speciation [17,18]. Perry et al. [16] have recently reviewed the different kinds of centric fission. Some fissions appear to be preceded by chromosomal rearrangements, such as the preduplication of a centromere or the activation of a new centromere within a preexisting centromere domain [16]. According to these authors, pseudocentric fission differs from the true centric fission because the cleavage within the centromeric region produces no or only one product giving rise to a functionally competent kinetochore. Rare cases of centric fission could be ascribed to this mechanism and generally result in the formation of an isochromosome for one chromosome arm and translocation of the other arm within another chromosome endowed with a functional centromere, resulting in the genesis of a dicentric chromosome [16].
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Chromosomal changes observed in constitutional karyotypes are, obviously, biased by the compatibility with normal embryonic development. This bias does not exist in somatic cells. Indeed, tumors, especially solid tumors, can harbor a large spectrum of chromosomal changes. We report here the fluorescence in situ hybridization characterization of a pseudocentric fission event found in a case of chronic myeloid leukemia (CML), consisting of the breakage within the centromere of chromosome 5 and resulting in the production of only one functional centric product accompanied by a complex three way translocation t(5;6;12). 2. Materials and methods 2.1. Case report In April 2002 a 44-year-old woman presented with splenomegaly, leukocytosis (white blood cell count, 78.3 109/L), and thrombocytosis (platelet count, 1584 109/L). Conventional cytogenetic analysis performed on bone marrow cells showed the following karyotype: 46,XX, t(9;22)(q34;q11)[20]. Reverse transcriptaseepolymerase chain reaction (RT-PCR) analysis showed the b2a2 transcript type, therefore the diagnosis of CML was made. The Sokal and Hasford scores were in the low-risk range; she did not have a sibling donor for stem cell transplantation. The patient, after initial reduction of the white blood cell count obtained with hydroxyurea, started interferon (IFN-a); on August 2002 she had a hematological response without obtaining cytogenetic remission. In September 2002, she started treatment with imatinib (400 mg daily) and in February 2003 she had a minor cytogenetic response. In October 2003, she had a major cytogenetic response, but molecular nested RT-PCR analysis revealed the persistence of BCR-ABL rearrangement. In August 2005, she was in hematological remission. The conventional cytogenetic analysis showed the following karyotype: 46,XX,t(5;6;12)(q?;q?;q?),t(9;22)(q34;q11)[20]. As of writing, she was being treated with imatinib (600 mg daily) and was due to be enrolled in a clinical trial with a new tyrosine kinase inhibitor. 2.2. Cytogenetic analysis Cytogenetic study was performed on unstimulated bone marrow cells by direct preparation, in RPMI 1640 medium with 25% fetal calf serum at 37 C. A modified Giemsa stain technique, previously described as HG (high resolution G-banding analysis) [19], was used for harvesting
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and preparation of the metaphase slides. The karyotypes were described in accordance with ISCN 2005 [20]. 2.3. Multicolor-FISH Multicolor-FISH (M-FISH) was performed in accordance with the manufacturer’s instructions, using the commercially available 24-color SpectraVysion probe (AbbotVysis, Downers Grove, IL) [21]. High-quality metaphase images were captured using a Leica DM-RXA2 epifluorescence microscope equipped with an eight-position automated filter wheel and a cooled charge-coupled device camera (Princeton Instruments, Monmouth Junction, NJ). Six fluorescent images were captured per metaphase, using filter combinations specific for Vysis SpectrumGold, SpectrumAqua, SpectrumGreen, FRed, Red, and 40 ,6diamidino-2-phenylindole (DAPI). Images were processed using Leica CW4000 M-FISH software (Leica Microsystems, Wetzlar, Germany). 2.4. Fluorescence in situ hybridization (FISH) Chromosome preparations from bone marrow cells were hybridized in situ with probes labeled by nick translation. Briefly, 600 ng of labeled probe was used for FISH experiments; hybridization was performed at 37 C in 2 SSC, 50% (vol/vol) formamide, 10% (wt/vol) dextran sulfate, 5 mg Cot-1 DNA (Bethesda Research Laboratories, Gaithersburg, MD), and 3 mg sonicated salmon sperm DNA in a volume of 10 mL. Posthybridization washing was performed at 42 C in 2 SSCe50% formamide (three times), followed by three washes in 0.1 SSC at 60 C. Biotin (Invitrogen Italia, Milan, Italy)-labeled DNA was detected with streptavidineDEAC (Molecular Probes, Milan, Italy). In cohybridization experiments, other probes were directly labeled with fluorescein, Cy3, or Cy5. Chromosomes were identified by DAPI staining. Digital images were obtained using a Leica DMRXA epifluorescence microscope equipped with a cooled CCD camera (Princeton Instruments, Boston, MA). Fluorescence signals of Cy3 (red; New England Nuclear, Boston, MA), fluorescein (green; Fermentas Life Sciences, Milan, Italy), Cy5 (IR; New England Nuclear), and DAPI (blue) were detected using specific filters and were recorded separately as grayscale images. Pseudocoloring and merging of images were performed with Photoshop software (Adobe Systems, San Jose, CA). 2.5. Probes The whole chromosome paints (WCP) used for chromosomes 5, 6, and 12, derived from flow-sorted chromosomes,
= Fig. 1. (A) G-banding, Multicolor-FISH analysis, corresponding DAPI banding, and FISH analyses performed on the case under study. Each column shows normal and derivative chromosomes 5, 6, and 12 relative to the FISH experiments with probes listed at the bottom of the figure. (B) Map of the probes used to map the fission site (arrow) within the centromeric region of chromosome 5. (C) Map of the probes used to locate the insertion site (arrow) of the fission product in chromosome band 12q21.33. The position of the BAC clones is reported according to the UCSC Genome Browser database (Release hg18 [NCBI Build 36.1], March 2006; available at http://genome.ucsc.edu/).
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Table 1 FISH results with probes specific for chromosomes 5, 6, and 12 Probe
Chromosome band
Positiona
FISH results
RP11-1149B6 RP11-630P18 pZ5.1 pGA-16 RP11-717F23 RP11-693D22 RP11-15P2 RP11-172K14 RP11-94G13 RP11-662G12 RP11-938P13 RP11-62D9 RP11-1H14 RP11-207B2 RP11-353I4 RP11-145N7 RP11-42M12 pZ6 RP11-662B13 RP11-111D3 RP11-915L2 RP11-697G4b RP11-653O20b RP11-237B9 RP11-75C8 RP11-1149K21 RP11-347K12 pMR12 RP11-490D11 RP11-158L3 RP11-1025N9 RP11-631N16 RP11-596J18 RP11-947G14 RP11-766N7 RP11-714A22 RP11-677M24 RP11-900F13 RP11-775A14 RP11-1129M3 RP11-108C3 RP11-319O20 RP11-11M4 RP11-356P8 RP11-402N3 RP11-66K23 RP11-1109F11b RP11-936C19 RP11-734K2 RP11-257L20 RP11-101P14
5p11 5p11 cen5 cen5 5q11.1 5q11.1 5q12.3 5q13.3 5q13.3 5q13.3 5q13.3 5q13.3 5q14.1 5q14.2 5q21.3 5q23.1 5q23.2 cen6 6q14.1 6q16.1 6q21 6q21 6q21 6q21 6q21 6q21 6q21 cen12 12q12 12q13.3 12q14.1 12q14.1 12q14.2 12q14.3 12q14.3 12q14.3 12q14.3 12q21.32 12q21.33 12q21.33 12q21.33 12q21.33 12q21.33 12q21.33 12q21.33 12q21.33 12q21.33 12q21.33 12q21.33 12q21.33 12q24.21
chr5:46,271,043e46,420,200 chr5:46,257,085e46,437,323
5, der(6) 5, der(6) 5, der(6), der(5) 5, der(5q) 5, der(5q) 5, der(5q) 5, der(5q) 5, der(5q) 5, der(5q) 5, der(6) 5, der(6) 5, der(6) 5, der(6) 5, der(6) 5, der(6) 5, der(6) 5, der(6) 6, der(6) 6, der(6) 6, der(6) 6, der(6) 6, der(6) 6, der(6) 6 6 6, der(5q) 6, der(5q) 12, der(12) 12, der(12), der(6) 12, der(6) 12, der(6) 12, der(6) 12, der(6) 12, der(6), der(5p) 12, der(5p) 12, der(5p) 12, der(5p) 12, der(5p) 12, der(5p) 12, der(5p) 12, der(5p) 12, der(5p) 12, der(5p) 12, der(5p) 12, der(5p), der(5q) 12, der(5p), der(5q) 12, der(5p), der(5q) 12, der(5q) 12, der(5q) 12, der(5q) 12, der(5q)
chr5:49,469,032e49,642,337 chr5:49,471,589e49,636,940 chr5:66,244,330e66,417,262 chr5:74,490,649e74,673,873 chr5:75,262,889e75,435,337 chr5:75,420,050e75,588,707 chr5:75,880,989e76,065,704 chr5:76,265,696e76,458,308 chr5:77,882,778e78,047,755 chr5:81,661,932e81,821,302 chr5:108,536,733e108,710,503 chr5:122,417,944e122,576,115 chr5:127,176,874e127,327,849 chr6:77,454,027e77,626,192 chr6:98,644,052e98,804,662 chr6:106,588,226e106,736,464 chr6:108,977,579e109,049,832 chr6:109,049,733e109,111,632 chr6:111,231,424e111,383,216 chr6:112,067,211e112,263,337 chr6:112,534,479e112,677,364 chr6:114,822,708e115,001,121 chr12:40,112,780e40,280,202 chr12:55,341,495e55,503,759 chr12:60,054,779e60,236,004 chr12:61,280,203e61,458,292 chr12:63,257,273e63,463,498 chr12:63,398,666e63,571,921 chr12:63,500,649e63,684,874 chr12:63,666,014e63,860,784 chr12:64,436,310e64,612,371 chr12:87,374,561e87,546,806 chr12:87,514,571e87,682,766 chr12:87,682,787e87,833,907 chr12:87,870,352e88,036,536 chr12:88,083,731e88,257,770 chr12:88,092,433e88,268,582 chr12:88,150,192e88,319,567 chr12:88,150,300e88,329,893 chr12:88,156,837e88,311,012 chr12:88,268,642e88,437,492 chr12:88,327,770e88,533,322 chr12:88,319,717e88,508,976 chr12:88,516,411e88,679,636 chr12:115,136,580e115,321,870
a The position of the BAC clones is reported according to the UCSC Genome Browser database (Release hg18 [NCBI Build 36.1], March 2006; available at http://genome.ucsc.edu/). b Fully sequenced BACs, with accession number in parenthesis: RP11-697G4 (AL391646); RP11-653O20 (AL365509); RP11-1109F11 (AC010201).
were a gift of the Sanger Centre (Dr. Nigel Carter). Chromosome 5 partial chromosome painting (PCP) 293 (5p13~5q13) (Fig. 1B) and chromosome 12 PCPs 448 (12q22~qter) and 438 (12pter~12q23) (Fig. 1C) were generated in our laboratory (http://www.biologia.uniba.it/rmc/).
pZ5.1 (D5Z2) and pGA-16 (D5Z1) are probes specific for different alphoid subsets coexisting on chromosome 5 [22,23] (Fig. 1B). pZ5.1 shows additional signals at centromeres of chromosomes 1 and 19 [22] and pGA-16 displays additional signal at the centromere of chromosome 19 [22].
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pZ6 and pMR12 are alphoid probes specific for chromosomes 6 and 12, respectively. For further details on the centromeric probes, see: http://www.biologia.uniba.it/rmc/ 5-alfoidi/-alfoidi.html. Appropriate bacterial artificial chromosome (BAC) clones from the RPCI-11 de Jong BAC library (http:// bacpac.chori.org/) were selected to characterize breakpoints on chromosomes 5, 6, and 12 (Table 1). The location of these clones was based on the UCSC Genome Browser (Release hg18 [NCBI Build 36.1], March 2006; available at http://genome.ucsc.edu/cgi-bin/hgGateway?db5hg18). All the clones were first used in FISH experiments on normal metaphases, to confirm their predicted chromosomal localization.
3. Results G-banding analysis of bone marrow metaphase revealed a karyotype 46,XX,t(5;6;12)(q?;q?;q?),t(9;22)(q34;q11) [20] (data not shown). M-FISH confirmed the t(9;22) translocation (data not shown) and a complex t(5;6;12) rearrangement (Fig. 1A). WCP and alphoid probes specific for chromosomes 5, 6, and 12 were then used in FISH experiments, to characterize the respective derivative chromosomes. Probes pGA-16 and pZ5.1, recognizing the two alphoid subsets that make up the centromeric core of chromosome 5 [22], were also used to characterize the translocation. The subset identified by pGA-16 is shared by chromosomes 5 and 19. The one recognized by pZ5.1 is shared by chromosomes 1, 5, and 19. pGA-16 yielded FISH signals on chromosomes der(5) and normal 5, in addition to cross-hybridization signals on both 19 homologs. pZ5.1 yielded a split signal on der(5) and der(6) (in addition to the expected signals on normal 5, 1, and 19) (Fig. 1A). The signal intensity of pZ5.1 on der(5) was significantly fainter than der(6) (Fig. 1A), disclosing an uneven split of this subset between the two derivative chromosomes. DAPI staining, used in the FISH procedure to produce a chromosome banding, frequently makes the centromeric satellite blocks bright, as apparent in the last column of Figure 1A, where the arrows point to the two portions of the centromere of chromosome 5 located on der(5) and der(6). The centromeric heterochromatin appears to be equally distributed between the two chromosomes. To further characterize this centromeric fission, partial chromosome paint probe PCP 293 (5p13~5q13), showing a splitting signal between chromosomes der(5) and der(6), was cohybridized with BAC clones mapping close to the pericentromeric region of chromosome 5. In detail: RP11-630P18, specific for band 5p11, was mapped on der(6), and RP11-717F23, mapping at 5q11, yielded FISH signals on der(5) (Fig. 1A). These results are presented diagrammatically in Figure 1B. In addition, pZ5.1 was used in cohybridization experiments with PCPs
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448 (12q22~qter) and 438 (12pter~12q23) (Fig. 1C). PCP 448 hybridized to the long arm of der(5) (Fig. 1A). PCP 438, however, gave hybridization signals on der(5) (short and long arm), der(6), and der(12). Panels of appropriate BAC clones, reported in Table 1, were then used to refine the rearrangement. In detail: (i) the inserted region within chromosome der(5) extends from 5q10, representing the centromere fission point, to 5q13.3; (ii) the chromosome 5 insertion point was defined as occurring within the overlapping region between BAC clones RP11-94G13 and RP11-662G12 (chr5:75,420,050e75, 435,337, ~15.3 kb), because both BACs gave a splitting signal between der(5) and der(6) (data not shown); (iii) this breakpoint disrupted the SV2C gene, encoding for the synaptic vesicle protein 2C; (iv) the insertion point of chromosome 5 material [der(5)] on the chromosome 12 long arm was defined as occurring at chr12:88,268,642e88,329,893 (~61.1 kb) because of the splitting signals yielded by the two overlapping BACs RP11-1109F11 and RP11-402N3 (Fig. 1A and 1C); (v) this breakpoint was located between the two undisrupted genes DUSP6 (dual specificity phosphatase 6) and WDR51B (WD repeat domain 51B). In conclusion, incorporating the FISH analysis, the karyotype can be described as 46,XX,t(9;22)(q34;q11.2), der(5)t(5;6;12)(12q14.3/12q21.33::5q10/5q13.3::12 q21.33/12q?ter::6q21/6q?ter),der(6)t(5;6;12)(6pter/ 6q21::12q12/12q14.3::5q31.1/5q13.3::5p10/5pter), der(12)t(5;6;12)(12pter/12q12::5q31.1/5qter)(9;22) (q34;q11.2)[20].
4. Discussion In the present study, we investigated a complex cancer rearrangement involving chromosomes 5, 6, and 12 and featuring a pseudocentric fission event occurring within the a-satellite array of chromosome 5. The rearrangement, found in a case of CML after treatment with interferon and imatinib, has been characterized in detail using appropriate painting probes and BAC clones. The relevant results are presented in Figure 1. The breakage within the alphoid subset D5Z2, as shown by a FISH experiment with pZ5.1 probe, generated unequal partitioning of the corresponding D5Z2 a-satellite subset, between the two fission products on der(5) and der(6). The portion on der(6) appeared inactivated. The inactivation status was inferred by the absence of any primary constriction at this locus on der(6). The primary constriction was quite evident in the region harboring the normal chromosome 6 centromere (Figure 1). Immunohistochemical analysis of functional centromeres using anti-CENP antibodies could not be performed due to lack of unfixed material. As far as we know, however, location discrepancy between primary constriction and specific immunostaining of the kinetochore proteins (CENP-A and CENP-C) has never been reported. Literature data on centric fission have
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shown that both products of the fission can retain the centromeric function [15,24e26]. Several factors can affect retention of the functionality. If the fission generates a dicentric chromosome, then the distance between the two centromeres is crucial. Two close centromeres tend to correctly coorient the sister chromatids, and therefore both functional centromeres can be stably retained [15]. In the present case, the centromere 5 fission product was inserted far from the centromere of der(6) (~58 Mb); thus, this derivative chromosome could be highly unstable if both centromeres were functional [27]. Similar occurrence in constitutional karyotypes can lead to a strong selection against imbalances generated by the instability. Consequently, the finding that the cell population was very homogeneous could suggest that the inactivation of the alphoid block on der(6) was concomitant to the rearrangement. The presence of a single hybridization signal of BAC clones mapping very close to the chromosome 5 centromere excluded the possibility of a centromere duplication prior to the fission event. Taken together, all this evidence leads us to conclude that the fission event we have described is a pseudocentric fission event, according to the definition by Perry et al. [16]. Cases of therapy-related MDS (t-MDS) and AML (tAML) with centromeric and pericentromeric breakage at several chromosomes, including chromosome 5, have been previously reported as associated with treatment with alkylating agents or associated with mutations of TP53 [28,29]. Whole arm translocations (WATs) have been described in multiple myeloma [30] and in solid tumor cell lines [31]. However, no centric fission events could be disclosed in any of these cases. Centric fission events in human cancer are well documented only in cases of head and neck and skin carcinomas [32,33], where they result in the formation of isochromosomes and WATs. None of the cases described resembles a pseudocentric fission event, however, either because of the presence of a WATwith derivative chromosomes showing hybrid centromeres (a-satellite sequences of both the chromosomes involved in the translocation) or because of the formation of an isochromosome for one arm and the loss of the other arm after centromeric misdivision. Martins et al. [33] described seven cases of centric fission with different breakpoints located within the centromere of chromosome 5. In one case, the breakage occurred within D5Z2 (both fission products were positive for pZ5.1), as in the case here described. Four of their cases disclosed a breakpoint within one of the two alphoid subsets flanking D5Z2 (i.e. only one fission product positive for pZ5.1, but both positive for the pancentromeric probe). The remaining two cases were undetermined. Our results confirm previous evidence regarding the susceptibility of the centromeric region of chromosome 5 to breakage [28,31,33]. Puechberty et al. [23] reported that both the short- and long-arm proximal sides of the centromere of chromosome 5 were found to contain blocks of a-satellite sequences bordered by repetitive L1 elements. Recent
studies have identified a dynamic role for L1 elements in mediating instability, resulting in complex chromosomal rearrangements in AML [34] and in the progression of CML [35]. Roman-Gomez et al. [35] showed that the methylation status at L1 retrotransposon promoter was lower in CML blast crisis than in the chronic phase, leading to activation of both ORF1 sense and MET gene antisense transcription, associated with high levels of BCR-ABL and DNMT3b4 transcripts. The reactivation of L1 elements plays a role in the progression and clinical behavior of CML, so it could be speculated that it may also affect the stability of the centromeric region of chromosome 5. In addition, hypomethylation of the centromeric heterochromatin could be responsible for centromeric decondensation and separation. Its involvement in centric fission, however, remains speculative [16]. Some authors hypothesize that centromeric instability could be due to the recombination between homologous chromosome regions containing integrated viral DNA sequences [31] or to exposure to alkylating agents, as in t-MDS and t-AML [28,29]. Nevertheless, our patient has no clinical history of viral infection and was treated only with interferon and imatinib. Further studies are needed to elucidate whether this kind of treatment could play a role in centromeric instability leading to fission events and, consequently, in the progression of CML. Acknowledgments This work has been supported by AIRC (Associazione Italiana per la Ricerca sul Cancro) and the MIUR (Ministero dell’Istruzione, dell’Universita` e della Ricerca). The authors thank Dr. Richard Lusardi for language revision of the manuscript. References [1] Amor DJ, Kalitsis P, Sumer H, Choo KH. Building the centromere: from foundation proteins to 3D organization. Trends Cell Biol 2004;14:359e68. [2] Lamb JC, Birchler JA. The role of DNA sequence in centromere formation. Genome Biol 2003;4:214. [3] Maio JJ. DNA strand reassociation and polyribonucleotide binding in the African green monkey, Cercopithecus aethiops. J Mol Biol 1971;56:579e95. [4] Manuelidis L. Chromosomal localization of complex and simple repeated human DNAs. Chromosoma 1978;66:23e32. [5] Willard HF, Waye JS. Hierarchical order in chromosome-specific human alpha satellite DNA. Trends Genet 1987;3:192e8. [6] Romanova LY, Deriagin GV, Mashkova TD, Tumeneva IG, Mushegian AR, Kisselev LL, Alexandrov IA. Evidence for selection in evolution of alpha satellite DNA: the central role of CENP-B/pJa binding region. J Mol Biol 1996;261:334e40. [7] Lee C, Wevrick R, Fisher RB, Ferguson-Smith MA, Lin CC. Human centromeric DNAs. Hum Genet 1997;100:291e304. [8] Waye JS, England SB, Willard HF. Genomic organization of alpha satellite DNA on human chromosome 7: evidence for two distinct alphoid domains on a single chromosome. Mol Cell Biol 1987;7: 349e56.
C.T. Storlazzi et al. / Cancer Genetics and Cytogenetics 174 (2007) 61e67 [9] Choo KH, Vissel B, Nagy A, Earle E, Kalitsis P. A survey of the genomic distribution of alpha satellite DNA on all the human chromosomes, and derivation of a new consensus sequence. Nucleic Acids Res 1991;19:1179e82. [10] Amor DJ, Choo KH. Neocentromeres: role in human disease, evolution, and centromere study. Am J Hum Genet 2002;71:695e714. [11] Warburton PE. Chromosomal dynamics of human neocentromere formation. Chromosome Res 2004;12:617e26. [12] Italiano A, Attias R, Aurias A, Perot G, Burel-Vandenbos F, Otto J, Venissac N, Pedeutour F. Molecular cytogenetic characterization of a metastatic lung sarcomatoid carcinoma: 9p23 neocentromere and 9p23~p24 amplification including JAK2 and JMJD2C. Cancer Genet Cytogenet 2006;167:122e30. [13] Amor DJ, Bentley K, Ryan J, Perry J, Wong L, Slater H, Choo KH. Human centromere repositioning ‘‘in progress.’’. Proc Natl Acad Sci U S A 2004;101:6542e7. [14] Ventura M, Weigl S, Carbone L, Cardone MF, Misceo D, Teti M, D’Addabbo P, Wandall A, Bjo¨rck E, de Jong P, She X, Eichler EE, Archidiacono N, Rocchi M. Recurrent sites for new centromere seeding. Genome Res 2004;14:1696e703. [15] Perry J, Nouri S, La P, Daniel A, Wu Z, Purvis-Smith S, Northrop E, Choo KH, Slater HR. Molecular distinction between true centric fission and pericentric duplication-fission. Hum Genet 2005;116: 300e10. [16] Perry J, Slater HR, Choo KH. Centric fission: simple and complex mechanisms. Chromosome Res 2004;12:627e40. [17] Kolnicki RL. Kinetochore reproduction in animal evolution: cell biological explanation of karyotypic fission theory. Proc Natl Acad Sci U S A 2000;97:9493e7. [18] Todd NB. Karyotypic fissioning and canid phylogeny. J Theor Biol 1970;26:445e80. [19] Novak A, Kruskic M, Ludoski M, Jurukovski V. Rapid method for obtaining high-quality chromosome banding in the study of hematopoietic neoplasia. Cancer Genet Cytogenet 1994;74:109e14. [20] ISCN 2005: an international system for human cytogenetic nomenclature (2005). In: Shaffer LG, Tommerup N, editors. Basel: S. Karger, 2005. [21] Speicher MR, Gwyn Ballard S, Ward DC. Karyotyping human chromosomes by combinatorial multi-fluor FISH. Nat Genet 1996;12: 368e75. [22] Finelli P, Antonacci R, Marzella R, Lonoce A, Archidiacono N, Rocchi M. Structural organization of multiple alphoid subsets coexisting on human chromosomes 1, 4, 5, 7, 9, 15, 18, and 19. Genomics 1996;38:325e30. [23] Puechberty J, Laurent AM, Gimenez S, Billault A, Brun-Laurent ME, Calenda A, Marcais B, Prades C, Ioannou P, Yurov Y, Roizes G. Genetic and physical analyses of the centromeric and pericentromeric regions of human chromosome 5: recombination across 5cen. Genomics 1999;56:274e87.
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[24] Dallapiccola B, Mastroiacovo P, Gandini E. Centric fission of chromosome no. 4 in the mother of two patients with trisomy 4p. Hum Genet 1976;31:121e5. [25] Fryns JP, Bulcke J, Hens L, Van den Berghe H. Balanced transmission of centromeric fission products in man. Hum Genet 1980;54: 127e8. [26] Wevrick R, Earnshaw WC, Howard-Peebles PN, Willard HF. Partial deletion of alpha satellite DNA associated with reduced amounts of the centromere protein CENP-B in a mitotically stable human chromosome rearrangement. Mol Cell Biol 1990;10:6374e80. [27] Higgins AW, Gustashaw KM, Willard HF. Engineered human dicentric chromosomes show centromere plasticity. Chromosome Res 2005;13:745e62. [28] Andersen MK, Christiansen DH, Pedersen-Bjergaard J. Centromeric breakage and highly rearranged chromosome derivatives associated with mutations of TP53 are common in therapy-related MDS and AML after therapy with alkylating agents: an M-FISH study. Genes Chromosomes Cancer 2005;42:358e71. [29] Andersen MK, Pedersen-Bjergaard J. Increased frequency of dicentric chromosomes in therapy-related MDS and AML compared to de novo disease is significantly related to previous treatment with alkylating agents and suggests a specific susceptibility to chromosome breakage at the centromere. Leukemia 2000;14:105e11. [30] Sawyer JR, Tricot G, Mattox S, Jagannath S, Barlogie B. Jumping translocations of chromosome 1q in multiple myeloma: evidence for a mechanism involving decondensation of pericentromeric heterochromatin. Blood 1998;91:1732e41. [31] Padilla-Nash HM, Heselmeyer-Haddad K, Wangsa D, Zhang H, Ghadimi BM, Macville M, Augustus M, Schrock E, Hilgenfeld E, Ried T. Jumping translocations are common in solid tumor cell lines and result in recurrent fusions of whole chromosome arms. Genes Chromosomes Cancer 2001;30:349e63. [32] Jin Y, Jin C, Salemark L, Martins C, Wennerberg J, Mertens F. Centromere cleavage is a mechanism underlying isochromosome formation in skin and head and neck carcinomas. Chromosoma 2000;109: 476e81. [33] Martins C, Jin Y, Jin C, Wennerberg J, Hoglund M, Mertens F. Fluorescent in situ hybridisation (FISH) characterisation of pericentromeric breakpoints on chromosome 5 in head and neck squamous cell carcinomas. Eur J Cancer 1999;35:498e501. [34] Hejlik DP, Nagarajan L. Deletion of 5q in myeloid leukemia cells HL-60: an L1 element-mediated instability. Cancer Genet Cytogenet 2005;156:97e103. [35] Roman-Gomez J, Jimenez-Velasco A, Agirre X, Cervantes F, Sanchez J, Garate L, Barrios M, Castillejo JA, Navarro G, Colomer D, Prosper F, Heiniger A, Torres A. Promoter hypomethylation of the LINE-1 retrotransposable elements activates sense/antisense transcription and marks the progression of chronic myeloid leukemia. Oncogene 2005;24:7213e23.
Short communication
Late-appearing pseudocentric fission event during chronic myeloid leukemia progression Clelia Tiziana Storlazzia,*, Francesco Albanob, Marija Dencic´-Feketec, Vesna Djordjevic´c, Mariano Rocchia a
Department of Genetics and Microbiology, University of Bari, Via Amendola 165/A, 70126 Bari, Italy b Department of Hematology, University of Bari, Bari, Italy c Institute of Hematology, Clinical Center Serbia, Koste Todorovica 2, 11000 Belgrade, Serbia and Montenegro Received 25 September 2006; received in revised form 13 November 2006; accepted 21 November 2006
Abstract
Pseudocentric fission is a rare event consisting of the splitting of one functional centromere into two new products, of which only one can give rise to a functionally competent kinetochore. We report here a pseudocentric fission event within the D5Z2 alphoid subset disrupting the centromeric region of chromosome 5 in a case of chronic myeloid leukemia (CML) after treatment with imatinib and interferon. The breakage generated unequal partitioning of a-satellite sequences between the two fission products. One product was inserted within the long arm of chromosome 12 at band 14.3, becoming the only functional centromere of chromosome der(5). The other fission product was rearranged to form a sandwich-like dicentricdbut functionally monocentricdchromosome der(6), made up of material from chromosomes 5, 12, and 6. The intercentric distance on der(6) was shown to be largely O20 Mb. To our knowledge, this is the first pseudocentric fission event described in CML. Moreover, our results confirm the susceptibility to breakage of the centromeric region of chromosome 5. Ó 2007 Elsevier Inc. All rights reserved.
1. Introduction The centromere is a functional domain responsible for the correct segregation of eukaryotic chromosomes during cell division. It is composed of centromeric proteins (CENPs) and centromere-specific satellite DNA sequences [1,2]. Primate centromeres consist of AT-rich a-satellite (alphoid) DNA monomers, 171 bp in length, tandemly repeated to constitute large blocks of centromeric heterochromatin [3,4]. In humans, the cores of these blocks are organized to form a higher-order repeat consisting of n monomers almost identically repeated in long DNA arrays [5e7]. Alphoid subsets from different chromosomes show relatively large sequence variation, so that most of these subsets are chromosomespecific. Multiple alphoid subsets can coexist on the same chromosome [8,9]. The relationship between a-satellite DNA and centromere function is not well understood. Indeed, there are several examples of dicentric chromosomes in which one apparently normal centromere is not functional. * Corresponding author: Tel.: þ39-080-5443582; fax: þ39-0805443386. E-mail address: [email protected] (C.T. Storlazzi). 0165-4608/07/$ e see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.cancergencyto.2006.11.009
Furthermore, several human neocentromeres, lacking alphoid DNA sequences, have been recently described in clinical cases [10e12] and, occasionally, in normal individuals [13,14]. Centromere (centric) fission consists of the splitting (or transverse misdivision) of one functional centromere into two new centric chromosomes [15,16]. This fission has been reported in a series of organisms and is represented as an important mechanism of karyotype evolution and speciation [17,18]. Perry et al. [16] have recently reviewed the different kinds of centric fission. Some fissions appear to be preceded by chromosomal rearrangements, such as the preduplication of a centromere or the activation of a new centromere within a preexisting centromere domain [16]. According to these authors, pseudocentric fission differs from the true centric fission because the cleavage within the centromeric region produces no or only one product giving rise to a functionally competent kinetochore. Rare cases of centric fission could be ascribed to this mechanism and generally result in the formation of an isochromosome for one chromosome arm and translocation of the other arm within another chromosome endowed with a functional centromere, resulting in the genesis of a dicentric chromosome [16].
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Chromosomal changes observed in constitutional karyotypes are, obviously, biased by the compatibility with normal embryonic development. This bias does not exist in somatic cells. Indeed, tumors, especially solid tumors, can harbor a large spectrum of chromosomal changes. We report here the fluorescence in situ hybridization characterization of a pseudocentric fission event found in a case of chronic myeloid leukemia (CML), consisting of the breakage within the centromere of chromosome 5 and resulting in the production of only one functional centric product accompanied by a complex three way translocation t(5;6;12). 2. Materials and methods 2.1. Case report In April 2002 a 44-year-old woman presented with splenomegaly, leukocytosis (white blood cell count, 78.3 109/L), and thrombocytosis (platelet count, 1584 109/L). Conventional cytogenetic analysis performed on bone marrow cells showed the following karyotype: 46,XX, t(9;22)(q34;q11)[20]. Reverse transcriptaseepolymerase chain reaction (RT-PCR) analysis showed the b2a2 transcript type, therefore the diagnosis of CML was made. The Sokal and Hasford scores were in the low-risk range; she did not have a sibling donor for stem cell transplantation. The patient, after initial reduction of the white blood cell count obtained with hydroxyurea, started interferon (IFN-a); on August 2002 she had a hematological response without obtaining cytogenetic remission. In September 2002, she started treatment with imatinib (400 mg daily) and in February 2003 she had a minor cytogenetic response. In October 2003, she had a major cytogenetic response, but molecular nested RT-PCR analysis revealed the persistence of BCR-ABL rearrangement. In August 2005, she was in hematological remission. The conventional cytogenetic analysis showed the following karyotype: 46,XX,t(5;6;12)(q?;q?;q?),t(9;22)(q34;q11)[20]. As of writing, she was being treated with imatinib (600 mg daily) and was due to be enrolled in a clinical trial with a new tyrosine kinase inhibitor. 2.2. Cytogenetic analysis Cytogenetic study was performed on unstimulated bone marrow cells by direct preparation, in RPMI 1640 medium with 25% fetal calf serum at 37 C. A modified Giemsa stain technique, previously described as HG (high resolution G-banding analysis) [19], was used for harvesting
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and preparation of the metaphase slides. The karyotypes were described in accordance with ISCN 2005 [20]. 2.3. Multicolor-FISH Multicolor-FISH (M-FISH) was performed in accordance with the manufacturer’s instructions, using the commercially available 24-color SpectraVysion probe (AbbotVysis, Downers Grove, IL) [21]. High-quality metaphase images were captured using a Leica DM-RXA2 epifluorescence microscope equipped with an eight-position automated filter wheel and a cooled charge-coupled device camera (Princeton Instruments, Monmouth Junction, NJ). Six fluorescent images were captured per metaphase, using filter combinations specific for Vysis SpectrumGold, SpectrumAqua, SpectrumGreen, FRed, Red, and 40 ,6diamidino-2-phenylindole (DAPI). Images were processed using Leica CW4000 M-FISH software (Leica Microsystems, Wetzlar, Germany). 2.4. Fluorescence in situ hybridization (FISH) Chromosome preparations from bone marrow cells were hybridized in situ with probes labeled by nick translation. Briefly, 600 ng of labeled probe was used for FISH experiments; hybridization was performed at 37 C in 2 SSC, 50% (vol/vol) formamide, 10% (wt/vol) dextran sulfate, 5 mg Cot-1 DNA (Bethesda Research Laboratories, Gaithersburg, MD), and 3 mg sonicated salmon sperm DNA in a volume of 10 mL. Posthybridization washing was performed at 42 C in 2 SSCe50% formamide (three times), followed by three washes in 0.1 SSC at 60 C. Biotin (Invitrogen Italia, Milan, Italy)-labeled DNA was detected with streptavidineDEAC (Molecular Probes, Milan, Italy). In cohybridization experiments, other probes were directly labeled with fluorescein, Cy3, or Cy5. Chromosomes were identified by DAPI staining. Digital images were obtained using a Leica DMRXA epifluorescence microscope equipped with a cooled CCD camera (Princeton Instruments, Boston, MA). Fluorescence signals of Cy3 (red; New England Nuclear, Boston, MA), fluorescein (green; Fermentas Life Sciences, Milan, Italy), Cy5 (IR; New England Nuclear), and DAPI (blue) were detected using specific filters and were recorded separately as grayscale images. Pseudocoloring and merging of images were performed with Photoshop software (Adobe Systems, San Jose, CA). 2.5. Probes The whole chromosome paints (WCP) used for chromosomes 5, 6, and 12, derived from flow-sorted chromosomes,
= Fig. 1. (A) G-banding, Multicolor-FISH analysis, corresponding DAPI banding, and FISH analyses performed on the case under study. Each column shows normal and derivative chromosomes 5, 6, and 12 relative to the FISH experiments with probes listed at the bottom of the figure. (B) Map of the probes used to map the fission site (arrow) within the centromeric region of chromosome 5. (C) Map of the probes used to locate the insertion site (arrow) of the fission product in chromosome band 12q21.33. The position of the BAC clones is reported according to the UCSC Genome Browser database (Release hg18 [NCBI Build 36.1], March 2006; available at http://genome.ucsc.edu/).
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Table 1 FISH results with probes specific for chromosomes 5, 6, and 12 Probe
Chromosome band
Positiona
FISH results
RP11-1149B6 RP11-630P18 pZ5.1 pGA-16 RP11-717F23 RP11-693D22 RP11-15P2 RP11-172K14 RP11-94G13 RP11-662G12 RP11-938P13 RP11-62D9 RP11-1H14 RP11-207B2 RP11-353I4 RP11-145N7 RP11-42M12 pZ6 RP11-662B13 RP11-111D3 RP11-915L2 RP11-697G4b RP11-653O20b RP11-237B9 RP11-75C8 RP11-1149K21 RP11-347K12 pMR12 RP11-490D11 RP11-158L3 RP11-1025N9 RP11-631N16 RP11-596J18 RP11-947G14 RP11-766N7 RP11-714A22 RP11-677M24 RP11-900F13 RP11-775A14 RP11-1129M3 RP11-108C3 RP11-319O20 RP11-11M4 RP11-356P8 RP11-402N3 RP11-66K23 RP11-1109F11b RP11-936C19 RP11-734K2 RP11-257L20 RP11-101P14
5p11 5p11 cen5 cen5 5q11.1 5q11.1 5q12.3 5q13.3 5q13.3 5q13.3 5q13.3 5q13.3 5q14.1 5q14.2 5q21.3 5q23.1 5q23.2 cen6 6q14.1 6q16.1 6q21 6q21 6q21 6q21 6q21 6q21 6q21 cen12 12q12 12q13.3 12q14.1 12q14.1 12q14.2 12q14.3 12q14.3 12q14.3 12q14.3 12q21.32 12q21.33 12q21.33 12q21.33 12q21.33 12q21.33 12q21.33 12q21.33 12q21.33 12q21.33 12q21.33 12q21.33 12q21.33 12q24.21
chr5:46,271,043e46,420,200 chr5:46,257,085e46,437,323
5, der(6) 5, der(6) 5, der(6), der(5) 5, der(5q) 5, der(5q) 5, der(5q) 5, der(5q) 5, der(5q) 5, der(5q) 5, der(6) 5, der(6) 5, der(6) 5, der(6) 5, der(6) 5, der(6) 5, der(6) 5, der(6) 6, der(6) 6, der(6) 6, der(6) 6, der(6) 6, der(6) 6, der(6) 6 6 6, der(5q) 6, der(5q) 12, der(12) 12, der(12), der(6) 12, der(6) 12, der(6) 12, der(6) 12, der(6) 12, der(6), der(5p) 12, der(5p) 12, der(5p) 12, der(5p) 12, der(5p) 12, der(5p) 12, der(5p) 12, der(5p) 12, der(5p) 12, der(5p) 12, der(5p) 12, der(5p), der(5q) 12, der(5p), der(5q) 12, der(5p), der(5q) 12, der(5q) 12, der(5q) 12, der(5q) 12, der(5q)
chr5:49,469,032e49,642,337 chr5:49,471,589e49,636,940 chr5:66,244,330e66,417,262 chr5:74,490,649e74,673,873 chr5:75,262,889e75,435,337 chr5:75,420,050e75,588,707 chr5:75,880,989e76,065,704 chr5:76,265,696e76,458,308 chr5:77,882,778e78,047,755 chr5:81,661,932e81,821,302 chr5:108,536,733e108,710,503 chr5:122,417,944e122,576,115 chr5:127,176,874e127,327,849 chr6:77,454,027e77,626,192 chr6:98,644,052e98,804,662 chr6:106,588,226e106,736,464 chr6:108,977,579e109,049,832 chr6:109,049,733e109,111,632 chr6:111,231,424e111,383,216 chr6:112,067,211e112,263,337 chr6:112,534,479e112,677,364 chr6:114,822,708e115,001,121 chr12:40,112,780e40,280,202 chr12:55,341,495e55,503,759 chr12:60,054,779e60,236,004 chr12:61,280,203e61,458,292 chr12:63,257,273e63,463,498 chr12:63,398,666e63,571,921 chr12:63,500,649e63,684,874 chr12:63,666,014e63,860,784 chr12:64,436,310e64,612,371 chr12:87,374,561e87,546,806 chr12:87,514,571e87,682,766 chr12:87,682,787e87,833,907 chr12:87,870,352e88,036,536 chr12:88,083,731e88,257,770 chr12:88,092,433e88,268,582 chr12:88,150,192e88,319,567 chr12:88,150,300e88,329,893 chr12:88,156,837e88,311,012 chr12:88,268,642e88,437,492 chr12:88,327,770e88,533,322 chr12:88,319,717e88,508,976 chr12:88,516,411e88,679,636 chr12:115,136,580e115,321,870
a The position of the BAC clones is reported according to the UCSC Genome Browser database (Release hg18 [NCBI Build 36.1], March 2006; available at http://genome.ucsc.edu/). b Fully sequenced BACs, with accession number in parenthesis: RP11-697G4 (AL391646); RP11-653O20 (AL365509); RP11-1109F11 (AC010201).
were a gift of the Sanger Centre (Dr. Nigel Carter). Chromosome 5 partial chromosome painting (PCP) 293 (5p13~5q13) (Fig. 1B) and chromosome 12 PCPs 448 (12q22~qter) and 438 (12pter~12q23) (Fig. 1C) were generated in our laboratory (http://www.biologia.uniba.it/rmc/).
pZ5.1 (D5Z2) and pGA-16 (D5Z1) are probes specific for different alphoid subsets coexisting on chromosome 5 [22,23] (Fig. 1B). pZ5.1 shows additional signals at centromeres of chromosomes 1 and 19 [22] and pGA-16 displays additional signal at the centromere of chromosome 19 [22].
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pZ6 and pMR12 are alphoid probes specific for chromosomes 6 and 12, respectively. For further details on the centromeric probes, see: http://www.biologia.uniba.it/rmc/ 5-alfoidi/-alfoidi.html. Appropriate bacterial artificial chromosome (BAC) clones from the RPCI-11 de Jong BAC library (http:// bacpac.chori.org/) were selected to characterize breakpoints on chromosomes 5, 6, and 12 (Table 1). The location of these clones was based on the UCSC Genome Browser (Release hg18 [NCBI Build 36.1], March 2006; available at http://genome.ucsc.edu/cgi-bin/hgGateway?db5hg18). All the clones were first used in FISH experiments on normal metaphases, to confirm their predicted chromosomal localization.
3. Results G-banding analysis of bone marrow metaphase revealed a karyotype 46,XX,t(5;6;12)(q?;q?;q?),t(9;22)(q34;q11) [20] (data not shown). M-FISH confirmed the t(9;22) translocation (data not shown) and a complex t(5;6;12) rearrangement (Fig. 1A). WCP and alphoid probes specific for chromosomes 5, 6, and 12 were then used in FISH experiments, to characterize the respective derivative chromosomes. Probes pGA-16 and pZ5.1, recognizing the two alphoid subsets that make up the centromeric core of chromosome 5 [22], were also used to characterize the translocation. The subset identified by pGA-16 is shared by chromosomes 5 and 19. The one recognized by pZ5.1 is shared by chromosomes 1, 5, and 19. pGA-16 yielded FISH signals on chromosomes der(5) and normal 5, in addition to cross-hybridization signals on both 19 homologs. pZ5.1 yielded a split signal on der(5) and der(6) (in addition to the expected signals on normal 5, 1, and 19) (Fig. 1A). The signal intensity of pZ5.1 on der(5) was significantly fainter than der(6) (Fig. 1A), disclosing an uneven split of this subset between the two derivative chromosomes. DAPI staining, used in the FISH procedure to produce a chromosome banding, frequently makes the centromeric satellite blocks bright, as apparent in the last column of Figure 1A, where the arrows point to the two portions of the centromere of chromosome 5 located on der(5) and der(6). The centromeric heterochromatin appears to be equally distributed between the two chromosomes. To further characterize this centromeric fission, partial chromosome paint probe PCP 293 (5p13~5q13), showing a splitting signal between chromosomes der(5) and der(6), was cohybridized with BAC clones mapping close to the pericentromeric region of chromosome 5. In detail: RP11-630P18, specific for band 5p11, was mapped on der(6), and RP11-717F23, mapping at 5q11, yielded FISH signals on der(5) (Fig. 1A). These results are presented diagrammatically in Figure 1B. In addition, pZ5.1 was used in cohybridization experiments with PCPs
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448 (12q22~qter) and 438 (12pter~12q23) (Fig. 1C). PCP 448 hybridized to the long arm of der(5) (Fig. 1A). PCP 438, however, gave hybridization signals on der(5) (short and long arm), der(6), and der(12). Panels of appropriate BAC clones, reported in Table 1, were then used to refine the rearrangement. In detail: (i) the inserted region within chromosome der(5) extends from 5q10, representing the centromere fission point, to 5q13.3; (ii) the chromosome 5 insertion point was defined as occurring within the overlapping region between BAC clones RP11-94G13 and RP11-662G12 (chr5:75,420,050e75, 435,337, ~15.3 kb), because both BACs gave a splitting signal between der(5) and der(6) (data not shown); (iii) this breakpoint disrupted the SV2C gene, encoding for the synaptic vesicle protein 2C; (iv) the insertion point of chromosome 5 material [der(5)] on the chromosome 12 long arm was defined as occurring at chr12:88,268,642e88,329,893 (~61.1 kb) because of the splitting signals yielded by the two overlapping BACs RP11-1109F11 and RP11-402N3 (Fig. 1A and 1C); (v) this breakpoint was located between the two undisrupted genes DUSP6 (dual specificity phosphatase 6) and WDR51B (WD repeat domain 51B). In conclusion, incorporating the FISH analysis, the karyotype can be described as 46,XX,t(9;22)(q34;q11.2), der(5)t(5;6;12)(12q14.3/12q21.33::5q10/5q13.3::12 q21.33/12q?ter::6q21/6q?ter),der(6)t(5;6;12)(6pter/ 6q21::12q12/12q14.3::5q31.1/5q13.3::5p10/5pter), der(12)t(5;6;12)(12pter/12q12::5q31.1/5qter)(9;22) (q34;q11.2)[20].
4. Discussion In the present study, we investigated a complex cancer rearrangement involving chromosomes 5, 6, and 12 and featuring a pseudocentric fission event occurring within the a-satellite array of chromosome 5. The rearrangement, found in a case of CML after treatment with interferon and imatinib, has been characterized in detail using appropriate painting probes and BAC clones. The relevant results are presented in Figure 1. The breakage within the alphoid subset D5Z2, as shown by a FISH experiment with pZ5.1 probe, generated unequal partitioning of the corresponding D5Z2 a-satellite subset, between the two fission products on der(5) and der(6). The portion on der(6) appeared inactivated. The inactivation status was inferred by the absence of any primary constriction at this locus on der(6). The primary constriction was quite evident in the region harboring the normal chromosome 6 centromere (Figure 1). Immunohistochemical analysis of functional centromeres using anti-CENP antibodies could not be performed due to lack of unfixed material. As far as we know, however, location discrepancy between primary constriction and specific immunostaining of the kinetochore proteins (CENP-A and CENP-C) has never been reported. Literature data on centric fission have
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shown that both products of the fission can retain the centromeric function [15,24e26]. Several factors can affect retention of the functionality. If the fission generates a dicentric chromosome, then the distance between the two centromeres is crucial. Two close centromeres tend to correctly coorient the sister chromatids, and therefore both functional centromeres can be stably retained [15]. In the present case, the centromere 5 fission product was inserted far from the centromere of der(6) (~58 Mb); thus, this derivative chromosome could be highly unstable if both centromeres were functional [27]. Similar occurrence in constitutional karyotypes can lead to a strong selection against imbalances generated by the instability. Consequently, the finding that the cell population was very homogeneous could suggest that the inactivation of the alphoid block on der(6) was concomitant to the rearrangement. The presence of a single hybridization signal of BAC clones mapping very close to the chromosome 5 centromere excluded the possibility of a centromere duplication prior to the fission event. Taken together, all this evidence leads us to conclude that the fission event we have described is a pseudocentric fission event, according to the definition by Perry et al. [16]. Cases of therapy-related MDS (t-MDS) and AML (tAML) with centromeric and pericentromeric breakage at several chromosomes, including chromosome 5, have been previously reported as associated with treatment with alkylating agents or associated with mutations of TP53 [28,29]. Whole arm translocations (WATs) have been described in multiple myeloma [30] and in solid tumor cell lines [31]. However, no centric fission events could be disclosed in any of these cases. Centric fission events in human cancer are well documented only in cases of head and neck and skin carcinomas [32,33], where they result in the formation of isochromosomes and WATs. None of the cases described resembles a pseudocentric fission event, however, either because of the presence of a WATwith derivative chromosomes showing hybrid centromeres (a-satellite sequences of both the chromosomes involved in the translocation) or because of the formation of an isochromosome for one arm and the loss of the other arm after centromeric misdivision. Martins et al. [33] described seven cases of centric fission with different breakpoints located within the centromere of chromosome 5. In one case, the breakage occurred within D5Z2 (both fission products were positive for pZ5.1), as in the case here described. Four of their cases disclosed a breakpoint within one of the two alphoid subsets flanking D5Z2 (i.e. only one fission product positive for pZ5.1, but both positive for the pancentromeric probe). The remaining two cases were undetermined. Our results confirm previous evidence regarding the susceptibility of the centromeric region of chromosome 5 to breakage [28,31,33]. Puechberty et al. [23] reported that both the short- and long-arm proximal sides of the centromere of chromosome 5 were found to contain blocks of a-satellite sequences bordered by repetitive L1 elements. Recent
studies have identified a dynamic role for L1 elements in mediating instability, resulting in complex chromosomal rearrangements in AML [34] and in the progression of CML [35]. Roman-Gomez et al. [35] showed that the methylation status at L1 retrotransposon promoter was lower in CML blast crisis than in the chronic phase, leading to activation of both ORF1 sense and MET gene antisense transcription, associated with high levels of BCR-ABL and DNMT3b4 transcripts. The reactivation of L1 elements plays a role in the progression and clinical behavior of CML, so it could be speculated that it may also affect the stability of the centromeric region of chromosome 5. In addition, hypomethylation of the centromeric heterochromatin could be responsible for centromeric decondensation and separation. Its involvement in centric fission, however, remains speculative [16]. Some authors hypothesize that centromeric instability could be due to the recombination between homologous chromosome regions containing integrated viral DNA sequences [31] or to exposure to alkylating agents, as in t-MDS and t-AML [28,29]. Nevertheless, our patient has no clinical history of viral infection and was treated only with interferon and imatinib. Further studies are needed to elucidate whether this kind of treatment could play a role in centromeric instability leading to fission events and, consequently, in the progression of CML. Acknowledgments This work has been supported by AIRC (Associazione Italiana per la Ricerca sul Cancro) and the MIUR (Ministero dell’Istruzione, dell’Universita` e della Ricerca). The authors thank Dr. Richard Lusardi for language revision of the manuscript. References [1] Amor DJ, Kalitsis P, Sumer H, Choo KH. Building the centromere: from foundation proteins to 3D organization. Trends Cell Biol 2004;14:359e68. [2] Lamb JC, Birchler JA. The role of DNA sequence in centromere formation. Genome Biol 2003;4:214. [3] Maio JJ. DNA strand reassociation and polyribonucleotide binding in the African green monkey, Cercopithecus aethiops. J Mol Biol 1971;56:579e95. [4] Manuelidis L. Chromosomal localization of complex and simple repeated human DNAs. Chromosoma 1978;66:23e32. [5] Willard HF, Waye JS. Hierarchical order in chromosome-specific human alpha satellite DNA. Trends Genet 1987;3:192e8. [6] Romanova LY, Deriagin GV, Mashkova TD, Tumeneva IG, Mushegian AR, Kisselev LL, Alexandrov IA. Evidence for selection in evolution of alpha satellite DNA: the central role of CENP-B/pJa binding region. J Mol Biol 1996;261:334e40. [7] Lee C, Wevrick R, Fisher RB, Ferguson-Smith MA, Lin CC. Human centromeric DNAs. Hum Genet 1997;100:291e304. [8] Waye JS, England SB, Willard HF. Genomic organization of alpha satellite DNA on human chromosome 7: evidence for two distinct alphoid domains on a single chromosome. Mol Cell Biol 1987;7: 349e56.
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