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Blood, Vol. 94 No. 1 (July 1), 1999:
pp. 283-290
By
From the Leukaemia Research Fund, Paul O'Gorman Centre for Childhood
Leukaemia, Molecular Haematology Unit, Institute of Child Health,
London, UK.
Rearrangements involving the MLL gene at chromosome 11q23
are associated with leukemia and are present in up to 70% of infant leukemias. Loss of heterozygosity (LOH) has been shown for anonymous polymorphic markers at 11q23 in adult leukemias. To study LOH at the
MLL locus, we have identified two new polymorphic
microsatellite markers: a GAA repeat (mllGAAn) in intron 6 of the
MLL gene and a GA (mllGAn) repeat in the 5' flanking
region of the gene, approximately 2 kb upstream of the translation
initiation codon. The heterozygosity index of mllGAAn is 0.54, which
renders it useful for analyzing LOH. We screened two groups of leukemia
patients to study LOH at the mllGAAn marker. Group A (n = 18) was
selected on the basis of presentation before 18 months. Cytogenetic and
reverse transcription-polymerase chain reaction analysis showed that 9 of these 18 children had translocations involving MLL. No LOH
was observed. Group B (n = 36) were randomly selected children who
had presented with leukemia between 1993 and 1994. Cytogenetic analysis
of this group showed a variety of different chromosomal abnormalities.
LOH was shown in 9 of 20 individuals (45%) who were informative.
Microsatellite instability (MSI) was demonstrated in 1 of 18 individuals in group A and 5 of 36 individuals (13.9%) in group B. MSI
and LOH were observed simultaneously in three individuals. Loss of an
allele was confirmed in one individual by fluorescence in situ
hybridization. Individuals with MSI or LOH at mllGAAn were selected for
analysis at anonymous polymorphic markers D11S1364 and D11S1356, which flank the MLL gene. No LOH or MSI was observed at these markers in those individuals who were informative. These results show that LOH
at the MLL gene locus is a common event during leukemogenesis. Furthermore, the presence of MSI at this locus suggests that the region
is a hotspot for genetic instability.
NONRANDOM REARRANGEMENTS in the mixed
lineage leukemia (MLL) gene (also known as ALL-1,
HRX, and HTRX1) at chromosome 11q23 are frequently
observed in leukemia.1 It is estimated that over 70% of
leukemias in infants below 1 year involve rearrangements of MLL,
and such rearrangements define a subgroup of patients who respond
badly to treatment and have a poor prognosis.2-4 The
rearrangements are normally translocations, and over 20 different translocation partners have been identified to date. The most common of
these partners is AF-4 (ALL-1
fusion gene on chromosome 4), but AF-9 and ENL are
also common partners.5-7 Duplications of exons 2-6 or 2-8 of the MLL gene, resulting in "self fusion," and
interstitial deletions of exon 8 have also been
reported.8,9
MLL is a large gene: it spans 100 kb, has at least 35 exons,
and the mRNA is 14.5 kb long.10-12 The MLL protein
comprises 3969 amino acids, has an estimated size of 431 kD, and
appears to comprise a number of distinct functional domains. Near the
amino-terminus are three closely spaced A·T hooks that have been
shown to bind to cruciform DNA and to SAR DNA.12,13
Sequences in this region have also been shown to bind the
leukemia-associated SET protein, which appears to mediate complex
formation between MLL and protein phosphatase 2A.14
Further downstream are two regions that can repress or activate
transcription of reporter genes, respectively. The former has homology
to DNA methyltransferases.13,15 There are also several
regions of homology to the trithorax gene product, which is
involved in positive regulation of homeotic gene transcription in
Drosophila. One of these, located downstream of the
methyltransferase homology region, is predicted to form three plant
homology domain (PHD)-type zinc-finger motifs, which are presumed to
interact with DNA in a sequence-specific manner. Another, located at
the carboxyl terminus of MLL, is named the SET domain, because
it is found in the Drosophila Suppressor of
variegation and Enhancer of zeste gene products
as well as in the protein encoded by trithorax. This
domain has recently been shown to bind sbp (SET
binding protein), which is predicted to be an
antiphosphatase.16 Taken together, these data suggest that
MLL normally makes contact with both proteins and DNA, and
functions as part of a large multimeric complex of proteins that
regulate transcription of specific target genes, probably by modifying
chromatin structure. In support of this idea, mice that are hemizygous
for a null mutation of the MLL gene show abnormal Hox
gene expression patterns.17
As yet, it is unclear how MLL gene rearrangements contribute to
leukemogenesis. The translocation breakpoints cluster between exons 5 and 8 (exon numbering of Tkachuk et al, 1992).12 Such rearrangements result in synthesis of proteins that have lost the SET
domain, transcription activation domain, and some of the zinc-finger
domain of MLL, but which retain most of the transcription repression domain and the A·T hook region.13,18 The most
common fusion partners, AF-4, AF-9 and ENL, contribute
their own transcription regulation domains to the fusion proteins, but
other partners have disparate functions. It is possible that the fusion
proteins represent gain-of-function mutations that contribute to
leukemogenesis. On the other hand, the loss of some MLL
functions in fusion proteins, and the lack of consistency in the
functions contributed by the fusion partners, have led to the
suggestion that MLL may be a tumor suppressor gene. Corral et
al19 recreated the MLL/AF-9 fusion gene in
transgenic mice by "knock-in" of the human AF-9 gene to
the mouse MLL gene, and found a high incidence of acute myeloid
leukemias in these animals. Similarly, Lavau et al20 found
that retrovirally encoded MLL/ENL fusion protein could
immortalize and transform myeloid cells when used to infect mouse bone
marrow cells enriched in hematopoietic stem cells. These findings show that MLL gene rearrangements are important primary events in
leukemogenesis, but do not distinguish between the hypotheses that the
fusion proteins are gain-of-function mutants, or dominant-negative
mutants that disrupt function of the normal MLL
protein.1 Deletions at 11q23 have been reported in leukemia
but are not a common nonrandom event.21 If MLL is a
tumor suppressor gene, then loss of heterozygosity (LOH) at this locus
would be expected to be a common event in the development of leukemias
and, possibly, other types of cancer. In a genome-wide study of
microsatellite markers in adults with leukemia, LOH at 11q23 was
reported in 14% of patients.22 The investigators proposed
that there was a tumor suppressor gene in that region and suggested
MLL as a candidate, but the markers used flanked MLL.
Ideally, to determine whether there is LOH at the MLL locus, a
marker within that locus should be used.
To determine whether LOH at the MLL locus is a common event in
childhood leukemia, we identified a polymorphic GAA repeat in the
breakpoint cluster region of the MLL gene. We have shown 45%
LOH and 13.9% MSI in a group of randomly selected children with
leukemia. We also analyzed this marker in a group of patients selected
by presentation before 18 months. MSI was detected in 1 of 18 patients.
Despite a high incidence of gross MLL rearrangements in this
latter group, no LOH was detected.
Patients.
Patients attended the Childhood Leukaemia Clinic at Great Ormond Street
Hospital NHS Trust (London, UK) unless otherwise stated. DNA was
prepared from bone marrow at presentation and at remission. We studied
two groups of patients. Members of group A were selected on the basis
of presentation before 18 months. Such patients have a high incidence
of MLL rearrangements and we wished to determine whether they
also exhibited a high incidence of more subtle abnormalities at the
MLL locus, irrespective of whether they had an 11q23
translocation. Group B patients were selected by sequential admissions
in 1993 and 1994 to the Childhood Leukaemia Clinic at Great Ormond
Street Hospital NHS Trust. They were therefore not selected on the
basis of any phenotypic or genotypic criteria. In group A, sample A16 was generously provided by E. Grace (Department of Cytogenetics, Royal
Hospital for Sick Children, Edinburgh Sick Children NHS Trust,
Edinburgh, UK) and sample A17 was generously provided by M. McKinley
(Oxford Medical Genetics Laboratories, The Churchill Hospital, Oxford
Radcliffe Hospital NHS Trust, Oxford, UK). Details of the karyotypes of
these samples were generously provided by Dr Christine Harrison
(Leukaemia Research Fund UKCCG ALL Karyotype Database, Royal Free
Hospital Medical School, London, UK). DNA was prepared by the salt
precipitation method,23 unless otherwise stated.
Isolation of PAC clones.
Pools of the de Jong RPCI1 human PAC library,25 obtained
from HGMP (Cambridge, UK), were screened by polymerase chain reaction (PCR) using primers corresponding to sequences in exon 8 of the MLL gene (sense primer:
5'-gagctccttatagatgaagagg-3'; antisense primer
5'-tcctatccgatcctgagcagta-3'). PCR cycling conditions were as follows: 95°C, 1 minute; 55°C, 1 minute; 72°C, 1 minute,
25 cycles. DNA was prepared from positive PACs using maxiprep columns (Qiagen, Crawley, UK). PAC DNA was digested with multiple restriction enzymes according to the manufacturer's conditions (GIBCO-BRL, Paisley, UK), electrophoresed on 0.8% (wt/vol) agarose gels, and transferred to Hybond-N+ nylon membranes (Amersham, Little
Chalfont, UK) by Southern blotting. Oligonucleotides
corresponding to sequences in exon 1 (5'-gatggcgcacagctgtcggtgg-3'), exon 3 (5'-gcagactagtgctccggcagagcc-3'), and exon 11 (5'-acacccagtttattctccaacacag-3') of the MLL gene
were labeled with Microsatellite analysis.
The forward primer was labeled with Cytogenetics.
Bone marrow samples obtained at presentation were processed by standard
methods and the GTG-banded karyotypes were described according to
ISCN.28 Whole chromosome paints (Cambio, UK)
and locus-specific probes for BCR/ABL (Appligene Oncor, Durham,
UK) and TEL/AML-1 (Vysis, Richmond, UK) were processed
according to the manufacturers' instructions. Fixed cells from bone
marrow aspirates were available for fluorescent in situ hybridization (FISH) analysis of the MLL locus from patients
B22, B32, and B34, who demonstrated loss at mllGAAn. A
digoxygenin-labeled, locus-specific probe for the MLL locus
(Appligene Oncor) was prepared according to the manufacturer's
instructions and FISH was performed as described previously.29 Cells were viewed using a CCD camera
(Photometrics, Tuscon, AZ) and Smartcapture software (Digital
Scientific, Cambridge, UK).
Isolation of MLL PAC clones.
To identify polymorphic microsatellite markers, a human genomic PAC
library was screened by PCR to obtain clones spanning the MLL
gene locus. The PCR primers corresponded to sequences in exon 8 of the
MLL gene. Three positive PACs were identified: dJ217a21,
dJ167k13, and dJ59j2. To characterize these PACs, and to check for
integrity, they were digested with a number of restriction enzymes,
Southern blotted, and hybridized to labeled oligonucleotides complimentary to sequences in exons 1, 3, and 11 of the MLL
gene. As shown in Fig 1, dJ217a21 extends
from approximately 6 kb upstream of MLL exon 1 to downstream of
exon 8, dJ59j2 extends from upstream of exon 3 to downstream of exon
37, and dJ167k13 extends from upstream of exon 7 to downstream of exon
37.
Identification of polymorphisms.
A 5.7-kb HindIII-NotI fragment containing part of
MLL exon 1 and upstream (centromeric) sequences, and a 3.7-kb
TaqI fragment spanning from intron 5 to intron 8, were
subcloned into pBluescript SK+ and sequenced. As shown in
Fig 1, four repetitive elements were identified: a CA repeat, a GA
repeat, and a TTTTA repeat in the HindIII-NotI
fragment, and a GAA repeat in the TaqI fragment. These
repetitive elements were amplified from 50 normal individuals and
members of two families to identify which, if any, were polymorphic. The CA and TTTTA repeats were not polymorphic. The GA repeat upstream of exon 1 (mllGAn) had three alleles, two
of which were rare (<5%) and one of which was predominant in the
population analyzed. The heterozygosity index of this polymorphism was
0.06. The GAA repeat located in intron 6 (mllGAAn) had three common
alleles, designated a, b and c; the frequency of these alleles is 0.5, 0.43, and 0.07 respectively (Fig 2). The heterozygosity index of this
polymorphic marker was 0.54 and the alleles were in Hardy-Weinberg
equilibrium in the normal population. The nucleotide sequence of the
region of intron 6 of the MLL gene containing the GAA repeat
has been lodged with the EMBL Nucleotide Sequence Database (accession
no. AJ131191).
LOH and microsatellite instability (MSI) analysis.
We used the mllGAAn marker to investigate whether LOH at the
MLL locus was a common event in two groups of patients with
leukemia. The first group (group A) were selected on the basis of
presentation before 18 months of age. Half the members of group A had a
rearrangement of the MLL gene previously identified by
cytogenetic analysis (Table 1). The second
group (group B) were patients selected randomly, by sequential
admission, from children presenting with acute leukemia from 1993 to
1994. Cytogenetic analysis showed a number of different chromosomal
abnormalities in this group but no translocations at 11q23
(Table 2). DNA prepared from bone marrow
taken at presentation and after remission was amplified by PCR, and the
presentation and remission samples for each individual were compared to
ascertain whether the leukemic samples exhibited LOH or MSI. One sample
in group A was heterozygous at presentation but homozygous at
remission, indicating there was MSI. No LOH was detected in this group.
Analysis of other polymorphic microsatellite markers.
To determine the extent of LOH at other markers, we analyzed the
flanking markers D11S1364 and D11S1356 in all samples in which the
mllGAAn haplotypes were different at presentation and remission (Fig 3B
and C, and Table 3). No LOH or MSI was
identified at these markers in those samples which were informative,
indicating that LOH is confined to a region between them. As part of a
different study, some patients were analyzed for microsatellite markers at the TEL locus on chromosome 12. Samples A15, B9, B12, B15, B27, B32, and B35 were analyzed for D12S89; and samples A15, B9, B12,
B22, B32, B34, and B35 were analyzed for D12S98. No LOH or MSI was
detected (data not shown).
Cytogenetic analysis.
Conventional cytogenetics and FISH analysis was performed to determine
whether the LOH observed correlated with cytogenetic abnormalities.
G-banding of chromosomes from patients in group B showed no
translocations involving MLL. However, sample B9, in which
partial LOH of the mllGAAn polymorphism was observed, had an
interstitial deletion of 11q, which is likely to involve the 11q23
region (Table 3). FISH analysis was performed on three samples (B22,
B32, and B34,) in which LOH at mllGAAn had been observed. Between 89 and 104 interphase cells and 16 and 24 metaphase cells from each
patient were analyzed for loss of MLL signal. Patient B22 had
loss of signal in 28 of 101 (27.7%) interphase cells
(Fig 4B) and 2 of 16 (12.5%) metaphase
cells (Fig 4D). The percentage of cells with loss of an MLL
allele may be an underrepresentation because there is often loss of
leukemic cells during cell culture. No significant loss of signal was
observed in samples B32 and B34.
Comparison of genotype and phenotype.
Four informative children in group B had acute myeloid leukemia, and of
these 3 (75%) had LOH. In contrast, out of 20 informative children in
group B with acute lymphoblastic leukemia, 6 (30%) had LOH. The level
of MSI was not different in children with AML and ALL.
We have shown in this report that 45% of patients in a randomly
selected group of children with leukemia exhibit LOH at a polymorphic
marker within the MLL gene. The rate of LOH was highest in
patients with AML (75%), and although the sample size was small (n = 4), this may indicate that LOH at MLL is a common event in the
development of AML. LOH is common in some solid tumors but rarer in
leukemia. LOH in childhood leukemia has been described at the
TEL gene at chromosome 12p21, particularly when an
AML/TEL fusion gene is present on the other
allele30 and also at chromosome 9p21 where the p16 tumor
suppressor gene is located.31,32 LOH at chromosome 6p has
also been described in childhood ALL.33
We are grateful to E. Grace and M. McKinley for providing samples, and
to Christine Harrison for providing cytogenetic data on those samples.
Submitted June 24, 1998; accepted March 8, 1999.
Supported by the Leukaemia Research Fund. I.G. was the recipient of a
Royal Society/NATO Postdoctoral Fellowship.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Julie C. Webb, PhD, Leukaemia Research
Fund, Paul O'Gorman Centre for Childhood Leukaemia, Molecular
Haematology Unit, Institute for Child Health, 30 Guilford St, London
WC1N IEH, UK.
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Related Letter in Blood Online:
This article has been cited by other articles:
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TOP
ABSTRACT
INTRODUCTION
32P-ATP (Amersham) using T4
polynucleotide kinase (GIBCO-BRL), as recommended by the manufacturer,
and hybridized to Southern-blotted PAC DNA under stringent conditions,
as described previously.
70°C. The reactions were repeated three times independently.
), Positions of small repetitive elements. E, EcoRI;
X, Xho; T, Taq I. *No restriction digestion at this
site due to overlapping Dam methylation. cen, centromere; tel,
telomere; bcr, breakpoint cluster region.
