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From www.bloodjournal.org by guest on August 3, 2017. For personal use only. RAPID COMMUNICATION Translocation Breakpoints Are Clustered on Both Chromosome 8 and Chromosome 21 in the t(8;21) of Acute Myeloid Leukemia By Jane E. Tighe, Antonio Daga, and Franco Calabi The t(8;21 )(q22;q22) is consistently associated with acute myeloid leukemia (AML) M 2 . Recent data have suggested that breakpoints on chromosome 2 1 are clustered within a single intron of a novel gene, AML 1. just downstream of a region of homology to the runt gene of D melanogaster. In this report, w e confirm rearrangement at the same location in at least 1 2 of 1 8 patients with t(8;21). Further- more, we have isolated recombinant clones spanning the breakpoint regions on both the der(8) and the der(21) from one patient. By using a chromosome 8 probe derived from these clones, we show that t(8;21) breakpoints are also clustered on chromosome 8. 0 1993 by The American Society of Hematology. T A total genomic library from a mutant of the human T-cell line Jurkat (31-13") was prepared by ligation of partially Sau3AI-digested, size-fractionated DNA into X 2001." A partial genomic library of 1 1 to 15 kbp BamHI fragments from patient no. 4 was also prepared in the same vector, following complete digestion and gel fractionation. DNA from a panel of human X rodent somatic hybrids was kindly provided by Lesley Rooke and Dr Nigel Spurr (ICRF, London, UK). An AMLI probe spanning the fifth reported exon was prepared by annealing and extending two partly complementary 74-mers corresponding to nucleotides 1 173 through 1246 and 1300 through 1227 of the published AMLl cDNA sequence.' Library screening and DNA preparation from recombinant clones was according to published procedures. Subcloningwas in pUC9 and pBS/KS+ vectors.' Gel-purified inserts were used for filter hybridization. Labeling was by priming with random 9-men in the presence of [32P-a]dCTP(ICN; 3,000 Ci/mmol). HE t(8;2 l)(q22;q22) is a balanced reciprocal translocation associated with about 6% of all cases of acute myeloid leukemia (AML). Up to 92% of cases with this translocation are classified as French-American-British (FAB) subtype M2, and they have distinctive clinical and morphologic features. ',* The consistency of this association suggests that the t(8;21) has a primary role in leukemogenesis. Alternatively, this translocation may be responsible for conferring only certain characteristics on to this subtype of AML, permitting its distinction from other cases of AML M2. Recently, molecular probes have been reported that map close to the breakpoint on chromosome 2 I 3-6 and a gene has been identified, AMLI, that spans it.' We have previously shown that the predicted protein product of this gene contains a domain of strong homology to runt, a key gene controlling early developmental processes in D melanoguster.' Analysis of four patients showed breakpoints to cluster in an intron placed at the 3' boundary of this domain of homology.' Thus, the translocation may exert its effects by causing the synthesis of a novel chimeric gene product, in which the runt domain is severed from its normal COOH-terminal neighbor(s) and possibly fused to new sequences. In this report, we have investigated 23 patients with AML M2 for rearrangements at the AMLl locus. Furthermore, we report the identification of a chromosome 8 sequence, which is also a preferred breakpoint target. MATERIALS AND METHODS Preparation and Southern analysis of DNA samples was essentially according to published procedures? using 10 pg of each digest per lane and blotting onto Bio-Rad Zeta-Probe GT membranes. Exposure times ranged between 4 and 240 hours at -70°C with intensifying screen. From the MRC Leukaemia Unit, Department of Haematology, Hammersmith Hospital, London, UK. Submitted October 23, 1992; accepted November 18, 1992. J.E.T. is supported by a training fellowship from the MRC, and A.D. by the UK MRC HGMP. Address reprint requests to Franco Calabi, MD,Department of Haematology, RPMS Hammersmith Hospital, Ducane Rd, London W12 ONN, UK. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in nccordunce with 18 U.S.C.section 1734 solely to indicute this fact. 0 1993 by The American Society of Hematology. 0006-4971/93/8103-0039$3.00/0 592 RESULTS By using a probe synthesized on the basis of the published cDNA sequence, clones were isolated from a human genomic library spanning most of the intron centromeric to the fifth reported AMLl exon.7 This is the region where chromosome 21 breakpoints were suggested to cluster in the t(8;21). As the intron contains a single BamHI site, rearrangements within it can be investigated using probes for the two fragments generated by BamHI digestion. Thus, two probes were prepared (Fig 1): the first (21A)detects the telomeric BamHI fragment (normal size of I 1 kbp), is 500 bp long and spans the whole of the fifth exon, extending to the Sac1 site immediately 3' of it. The second (21B)is a 1.1 kbp PstI fragment from the centromeric BumHI fragment (normal size of 20 kbp). A composite probe for the simultaneous detection of both BamHI fragments was prepared by cloning both probes into the same plasmid vector. Southern blotting data were obtained on BamHI digests from 23 patients with AML M2, 18 of whom had been reported to harbor a t(8;21) (Table I and Fig 2). Rearranged bands are clearly visible in 12 of these (67%). In two other patients (nos. 13 and 15) a weak abnormal band is observed, which in the former case is apparently of a very large size (>27 kbp). No rearrangements are seen in the five patients without cytogenetic reports of t(8;2 I). A patient (no. 4) in whom the centromeric AMLl probe detected a novel BamHI fragment of 13 kbp, presumably corresponding to the der(8) chromosome, was selected for further study. A genomic library was prepared from the 11to 15-kbp BamHI fraction. Out of -3 X IO5 recombinants, four hybridizing to the centromeric AMLl probe were isolated - Blood, Vol81, No 3 (February 1). 1993: pp 592-596 From www.bloodjournal.org by guest on August 3, 2017. For personal use only. TRANSLOCATION BREAKPOINTS IN t(8;2 1) AML c-- 2 l q t e r AMLl Ex.5 21 2lcen I * AMLl Ex.6 (runt 3') B 11 kbp - 593 I I B B -- - I - probe 21A + 20 kbp p r o b e 21B 8cen 2lqter AMLl Ex.5 der ( 8 ) I B I I I II B RH R HH sI A - I I B R h JD2 4 - dqter 2lcen - AMLl Ex. 6 der (21) - Eqter 8 B I I R B 8cen R 1 0 . 2 kbp 1 1 . 6 kbp - 1 kbp * probe 8 Fig 1. Schematic map of the t(8;21) breakpoint cluster regions on normal chromosomes 8 and 21 as well as on the der(21) and der(8) chromosomes from patient no. 4. The white bar represents chromosome 8 sequences and the black bar chromosome 21 sequences. The striped boxes on the derivative chromosomes represent sequences whose origin cannot be unambiguously ascertained on the basis of the restriction map. A detailed restriction map is given only for the regions spanned by the clones shown (ie, WD2 and WD5). otherwise only sites relevant to the presented Southern blot analysis are indicated. B, BamHI; E, EcoRI; H, Hindlll; S, Sad. AML 1 Ex. 5 and 6 refer to the fifth and the sixth exons in the published cDNA sequence,' with Ex. 5 encoding the 3-terminal segment of the domain of homology to D melanogaster runt.s and their inserts mapped. The four clones were identical (Fig 1, see hlD2). While one end of the map is colinear to that of unrearranged AMLI for at least 8 kbp, the remaining differs. Southern blotting analysis of a panel of human X rodent somatic hybrids using a probe made from this region (probe 8, Fig 1) confirmed that the latter mapped to chromosome 8 (data not shown). In DNA from patient no. 4, the same probe hybridizes to a normal IO-kbp BamHI fragment, in addition to the same rearranged 13 kbp BamHI previously detected with the AMLl probe. Patients' Southern blots were reanalyzed with the chromosome 8 probe. Ten of 18 patients show clear rearrangements on BamHI digests, and a further four on EcoRI digests (total of 78%). Based on BamHI digests, all of these patients have rearrangements at the AMLI locus, with the exception of two (nos. 7 and 18 in Table I), for which we presume that a rearranged BumHI fragment is present, but comigrates with - the normal, unrearranged allele. Patient no. 5 gives a weak abnormal BamHI band of apparently very large size (>27 kbp). However, rearrangement is clearly confirmed by EcoRI analysis. Of the four patients not rearranged with the chromosome 8 probe, two (nos. I I and 12) were also not rearranged with the chromosome 21 probes, while the remaining two (nos. 13 and 15) gave weak and/or questionable rearranged bands. When rearranged BamHI fragments are detected with both chromosome 2 1 and chromosome 8 probes, they comigrate in all but two, possibly three, cases (nos. 10, 16, and possibly 5). Comigration indicates that the probes are on the same translocation derivative chromosome [ie, either the der(8) or the der(21)I and implies that, with respect to the centromere, the breakpoints fall on opposite sides of each chromosome probe. Conversely, lack of comigration suggests that the probes are on different reciprocals. Hence, by this argument, From www.bloodjournal.org by guest on August 3, 2017. For personal use only. 594 TIGHE, DAGA, AND CALABI Table 1. Summary of the Southern Blotting Analysis of AML M2 Patients Chrom. 21 Rearrangement % (abs. no.)t'8;21) Metaphases Patient No. 1 100 (12) 2 3 4 5 100 (10) Location t Size (enr)' 14.0(B) 20 kbp/? Chrom. 8 Rearrangement Size (enz)' Location t NR (B) 10.5 (R) 18.0(B) 18.0(6) 13.0(B) 227.0(B) 10.0(R) C 75 (1 5) 18.0(B) 18.0(B) 13.0(B) 22.0(B) 20 kbp/c 20 kbp/c 20 kbp/c 20 kbp/? 6 80(12) 17.5(6) 20 kbp/? NR (B) 8.0 (R) C 7 8 9 100 (IO) 89 (8) 80 (16) NR (B) 15.0(B) 15.0(6) 20 kbp/c 20 kbp/c 20 kbp/? 20.0(B) 15.0(B) t 10 100 (17) 19.0(B) 20 kbp/t + 9 0 (27) + + 11 12 13 14 15 16 17 18 100 (12) 95 (20) 83 (5) 100 (8) + 100 (20) 19 0 20 21 22 23 0 0 0 0 - NR (6) NR (6) - >27.0(B) 20 kbp/? 1 1 kbp/tS 20 kbp/? 20 kbp/t 20 kbp/c 20 kbp/c 9.0 (B) 8.5 (B) 23.0(B) 26.5 (B) NR (B) - NR (B) NR (B) NR (B) NR (B) NR (6) - NR (B) 9.5 (R) 1 1.5 (B) NR (B) NR (6. R) NR (B. R) 9.0 (B) NR (B. R) 9.0 (B) 26.5 (B) 20.0(B) NR (6) NR (B) NR (B) NR (B) NR (B) t t t ? t C t t t t t - - +, Abbreviations: t(8;21)' metaphases present, but numbers unknown; NR, no rearranged band detected with 278 on BamHl digests. Restriction enzyme detecting a rearrangement: B, &"I; R, EcoRI. t Inferred location of translocation breakpoint: c, centromeric to probe 8/2 I B ; t, telomeric to probe 8/2 7B; ?, unknown. For AML 7, the normal size of the rearranged BamHl fragment is also indicated. $ Rearranged BamHl fragment with 27A, ie, breakpoint telomeric to 27B, but inferred to be centromeric to 27A. by the sequential use of the two AMLl probes and by the assumption that BamHI rearrangements detected with probe 8 would most likely be telomeric to the probe, because of its position next to the centromeric BamHI site, we can provisionally assign the position of the breakpoints with respect to the probes (see Table 1). At least 15 of 16 breakpoints on chromosome 21 fall within the 20-kbp BamHI fragment. As this fragment corresponds to -*I3 of the region this is significant (P< .01). At least seven breakpoints can be mapped to the 1 1 .4-kbp segment between probe 2 1B and the centromeric BamHI site. While the rearranged bands are mostly of similar intensity to the normal ones, in agreement with the cytogenetic data showing that most metaphases contain translocated as well as normal chromosomes 8 and 2 1 (Table l), the rearranged EcoRI fragment detected in one patient by the chromosome 8 probe is proportionately much weaker than that detected by the AMLl probe (Fig 2, compare A1 and Fl). This may be caused by preferential loss, in this case, of the der(21) chromosome. As in the case of the AMLl probes, no rearranged bands are found with the chromosome 8 probe in the five patients without the t(8;2 1). Furthermore, only a single, normal band was observed in a series of at least 14 random (not AML) samples (data not shown). Thus, the additional bands in t(8;2I) patients are unlikely to be caused by polymorphism. The chromosome 8 probe was used to isolate genomic clones spanning the normal locus. In addition to the normal 10-kbp fragment, a probe made from these clones detects a 16.4-kbp band in BamHI digests of DNA from patient no. 4, presumably corresponding to the der(2 1) reciprocal of the der(8) chromosome. A clone containing this rearranged fragment was obtained from the library made from this patient (Fig 1). A comparison of restriction maps gives molecular evidence that the translocation is balanced; ie, it does not entail any significant net loss of genomic material. - DISCUSSION The data presented in this report show that t(8;21) breakpoints in AML M2 are clustered, not only on chromosome 21, but also on 8. The cluster region on chromosome 21 spans a maximum of -25 kbp, with a strong preference for its centromeric two thirds, while that on chromosome 8 spans up to 15 kbp, with a preference for its telomeric half. - From www.bloodjournal.org by guest on August 3, 2017. For personal use only. TRANSLOCATION BREAKPOINTS IN t(8;21) AML 595 Chromosome 21 probes A 1 2 3 4 B 5 6 7 8 c 9 10 11 12 23- 9.4 - 6.6 - Chromosome 8 probe Fig 2. Representative Southem blots from t(8;21) AML M2 patients. The numbers at the top of each lane refer to the patient number, as given in the table. A through E, 8amHl digests; F, EcoRl digests; A, combined 21A and 218 probes; B and C, 218 probe, D through F, 8 probe. Panels A and D, as well as B and E, represent the same blot, analyzed sequentially with the two probes. A double, leftward-pointing arrowhead corresponds to the normal (unrearranged)fragment. The bands in lane C1 look slightly retarded because of an artifact (compare the top band with the adjacent lanes), as confirmed in subsequent experiments. Panel F has been overexposed so as to show the weak a b m a l band in lane 1. D 23 l . 94. 2 3 4 E 5 6 7 8 ----- F 6 1 23- 66 The cluster region on chromosome 2 1 corresponds to one intron that marks a significant position in the AMLl gene, ie, the 3' end of the region of homology to the runt gene of D melanogaster.' By analogy with this, as well as with other translocations, it can be expected that the cluster region on chromosome 8 also resides within one intron, thus producing a highly consistent exon-exon fusion. Preliminary experiments have identified a short region of strong sequence conservation between humans and rodents within chromosome 8 immediately centromeric to the breakpoint in patient no. 4. This is an obvious candidate for an exon, although no transcripts have as yet been identified in a limited sample of cell lines. While the vast majority of patients with t(8;2 1) show concordant rearrangements with both the chromosome 8 and the AMLl probes, a minority of patients score negative for rearrangement either with the first or with both probes. We cannot completely exclude at present that in these cases a rearrangement has gone undetected for technical reasons (ie, a low proportion of t(8;2 1) positive cells, or the comigration ofa rearranged with a normal fragment]. However, assuming that, as in the case ofAML1,a transcription unit does indeed span the breakpoints on chromosome 8, it will be particularly interesting to investigate whether these variant breakpoints occur in different functional regions of the same units or in separate, nearby genes. ACKNOWLEDGMENT We thank Dr Mark Vickers and particularly Dr David M. Swirsky for providing patient samples; Lesley Rooke and Dr Nigel Spurr for DNA from human X rodent hybrids; Dr Dwij Rudra for technical help; and Prof Lucio Luzzatto for encouragement. REFERENCES I. Swirsky D, Hoyle C Morphology and cytochemistry of specific chromosome abnormality in acute myeloid leukaemia, in Scott CS (ed):Leukaemia Cytochemistry and Diagnosis: Principles and Practice. Chichester, UK, Ellis Honvood, 1989 2. Mitelman F, Heim S: Cancer Cytogenetics. New York, NY, Lis, 1987 3. Shimizu K, Ichikawa H, Miyoshi H,Ohki M, Kobayashi H, Maseki N, Kaneko Y. Molecular assignment of a translocation breakpoint in acute myeloid leukaemia with t(8;21). Genes Chrom Cancer 3: 163, 1991 4. Gao J, Erickson P, Gardiner K, Le Beau MM, Dim MO, Patterson D, Rowley J, Drabkin H: Isolation of a yeast artificial chro- From www.bloodjournal.org by guest on August 3, 2017. For personal use only. 596 mosome spanning the 8;2 1 translocation breakpoint t(8;2 l)(q22;q22.3) in acute myelogenous leukaemia. Proc Natl Acad Sci USA 88:4882, 1991 5. Kearney L, Watkins P, Young B, Sacchi N: DNA sequences of chromosome 21-specific YAC detect the t(8;21) breakpoint of acute myelogenous leukemia. Cancer Genet Cytogenet 57: 109, 1991 6. Yaspo M-L, Theophile D, Aurias A, Crkte N, Crhu-Goldberg N, Bastard C, Suberville A-M, Valensi F, Viguier F, Fkrger R, Sinet P-M, Delabar J-M Molecular analysis of 12 patients with the t(8;21) translocation and M2 acute myelogenous leukaemia. Genes Chrom Cancer 5:166, 1992 7. Miyoshi H, Shimizu K, Kozu T, Maseki N, Kaneko Y, Ohki M: t(8;21) breakpoints on chromosome 21 in acute myeloid leukemia TIGHE, DAGA, AND CALABI are clustered within a limited region of a single gene, AMLI. Proc Natl Acad Sci USA 88:10431, 1991 8. Daga A, Tighe J, Calabi F Leukaemia-Drosophila homology. Nature 356:484, 1992 9. Sambrook J, Fritsch E, Maniatis T: Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY, Cold Spring Harbor Laboratory, 1989 IO. Alcover A, Alberini C, Acuto 0,Clayton L, Transy C, Spagnoli G, Moingeon P, Lopez P, Reinherz E: Interdependence of CD3-Ti and CD2 activation pathways in human lymphocytes-T. EMBO J 7: 1973, 1988 1 1. Kam J, Matthes H, Gait M, Brenner S: A new selective phage cloning vector, X 2001, with sites for XbaI, BumHI, HindIII, EcoRI, SstI and XhoI. Gene 32:217, 1984 From www.bloodjournal.org by guest on August 3, 2017. For personal use only. 1993 81: 592-596 Translocation breakpoints are clustered on both chromosome 8 and chromosome 21 in the t(8;21) of acute myeloid leukemia JE Tighe, A Daga and F Calabi Updated information and services can be found at: http://www.bloodjournal.org/content/81/3/592.full.html Articles on similar topics can be found in the following Blood collections Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. 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