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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
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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,
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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.
-
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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.
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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
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