MAFB in t(14;20) Myeloma
featuring hyperdiploidy with 14q+ chromosomes. FISH
analysis of the SACHI cells identified the donor chromo-
somes of the IgH loci as 8q24 and 20q11 loci (Taniwaki et
al., unpublished observation). The presence of
t(14;20)(q32;q11) in SK-MM-1 was demonstrated by
spectral karyotyping analysis.1) Twenty-two patients with
MM, whose percentage of tumor cells exceeded 13.6% of
the total nucleated cells in bone marrow aspirates, were
included in this study after their informed consent had
been obtained.
Information for yeast artificial chromosomes (YACs),
bacterial artificial chromosomes (BACs) and P1 artifi-
cial chromosomes (PACs) mapped to the 20q11 locus
was obtained through the homepages of the MIT White-
Sanger Centre Human Genome Data Base (http://
purchased from Research Genetics Inc. (Huntsville, AL)
and their contig was constructed on the basis of the pres-
ence or absence of the distinct STS markers. The PAC
contig was constructed according to the sequence overlap
and on the basis of hybridization results with PAC-end
probes prepared with the bubble-PCR method when
sequence data were not available.14)
Chromosome preparation for DCFISH analyses has
been described previously.4, 9, 10) Each PAC/BAC/YAC
DNA itself was labeled with either Spectrum Orange or
Spectrum Green using a nick translation kit (Vysis Inc.,
purchased from Fujisawa Pharmaceutical Co., Osaka)
according to the manufacturer’s instruction. BAC417P24
DNA purchased from BAC/PAC Resources (Oakland,
CA) was used as a probe derived from the IgH constant
region at the 14q32 locus. To determine the chromosomal
loci of specific signals on the metaphase spreads, we used
whole chromosome painting (WCP) probes which could
detect chromosomes 2, 14, 20 and 22 (Vysis) when
needed. Hybridization and washing protocols were the
same as those available from the company (Vysis) on the
internet. Each slide was counterstained with 4′,6-diamino-
2-phenylindole dihydrochloride (DAPI). For the interphase
DCFISH used for the patient marrow samples, signals
containing PAC644L1/BAC417P24 fusions were evalu-
ated in 100 nuclei per slide. The cut-off index to assess
fusion signals was determined by evaluating peripheral
blood mononuclear cell samples derived from five normal
volunteers.
the probe was 479 bp. Expressed sequenced tag (EST)
clones, mapped telomeric to SACHI’s breakpoint, were
purchased from Research Genetics Inc. and their inserts
were used as probes. The quality and amount of each
RNA were assessed by subsequent hybridization with
β-ACTIN probe.
RESULTS AND DISCUSSION
The chromosome 20q11 locus is known to be rear-
ranged or deleted in various types of human malignancies.
It has attracted the attention mainly of hematologists
because a region extending from 20q11.2 to 20q12 is com-
monly deleted in myeloid leukemias, suggesting the exis-
tence of an as yet unidentified tumor suppressor gene
within this region.15–17) Accordingly, YAC contigs have
already been constructed by several groups and were
available when we focused on t(14;20), which is occasion-
ally encountered in human MM. In order to determine the
approximate locations of the 20q11 breakpoints of SK-
MM-1 and SACHI cell lines, we decided to use DCFISH
analysis. In brief, two Mega-YACs’ DNAs located
between loci D20S884 and D20S96 were labeled (Fig.
1A), one with Spectrum Green and the other with Spec-
trum Orange, and hybridized to metaphase spreads of the
two cell lines. When the green and red signals were split
onto different chromosomes, we could determine the loca-
tions of the breakpoints between the two YACs, which
were used as probes. This strategy resulted in the eventual
confirmation of SK-MM-1’s breakpoint within y953C12
itself and that of SACHI between y953C12 and y808C5
(Fig. 1, A, B and Fig. 2, A, B). These YACs were then
mapped at the 20q11.2–12 locus. To narrow down the
breakpoints, PAC/BAC contigs encompassing the region
between the D20S174 and PLC1 gene loci, whose loca-
tions were mapped to centromeric and telomeric ends of
y953C12 and y808C5, respectively, were constructed
based on the sequence information provided by Sanger
Centre Human Genome Data Base. As for the PAC/BAC
clones whose complete sequences were not available, we
confirmed each overlap by means of Southern hybridiza-
tion analyses using PAC/BAC-end probes obtained with
the bubble-PCR method,9) shown as open circles in Fig. 1.
Further FISH analyses using PAC/BAC probes could nar-
row down the locations of these two breakpoints to within
PACs 600E6 for SK-MM-1 and 191L6 for SACHI cells,
as shown in Fig. 1. This means that the distance between
the breakpoints was no more than 680 kb (460–680 kb),
as estimated from the PAC/BAC data with respect to size
and sequence.
Northern blot analysis was performed as described pre-
viously.7, 14) Total RNA was isolated with the guanidine iso-
thiocyanate/cesium chloride ultracentrifugation method.
Human MAFB probes were amplified from the PAC
RPCI4-644L1 by PCR, as this gene has no intron, by using
a primer pair, F5′ (5′-GACCGCTTCTCCGACGACCA-
3′) and R6′ (5′-CCCTCTCGCTCAAGTCAAAC-3′), whose
sequences were reported by Wang et al.15) The size of
In an attempt to make sure that these 20q11 breakpoints
were juxtaposed to the IgH gene at the chromosome 14q32
locus as a result of t(14;20), we next employed DCFISH
using either y808C5 or PAC644L1, which were mapped
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