Haematologica 2003 - Supplements

034
Instability of Pericentromeric Heterochromatin and
Associated Gene Duplication in Multiple Myeloma
(MM).
J. Sawyer, G. Tricot, J. Lukacs, R. Lichti Binz, E. Tian, B.
Barlogie, J. Shaughnessy.
Myeloma Institute for Research and Therapy, University of
Arkansas for Medical Sciences, Little Rock, AR 72205
Chromosome instability is a hallmark of cytogenetic progression
in MM. One of the largest subsets of structural chromosome
anomalies in the progression of MM involves the duplication of
all or part of chromosome 1q. We investigated chromosome 1q
associated duplications in 35 patients which showed 1q
aberrations by G-banding. Spectral karyotyping (SKY) and
locus-specific fluorescence in situ hybridization (FISH) were
used to help further define these aberrations. FISH probes for the
pericentromeric heterochromatin (sat II/III sequences) at 1q12
were used in conjunction with locus specific probes for BCL9
and IL6Ra, which map to 1q21. G-banding analysis identified
the most frequent aberrations as whole-arm or “jumping”
translocations of 1q (21cases). The second most frequent
aberrations were partial duplications of the 1q12~32 region (16
cases). FISH analysis of the partial duplications revealed both
direct and inverted sequential duplications of the 1q12~32 region,
which were associated with up to three copies of the satII/III
sequences on a single chromosome arm. Inverted duplications
(five cases) resulted in larger numbers of duplicated genes with
up to five copies of BCL9 and/or IL6Ra per chromosome arm.
Subsequent whole chromosome duplications of both normal and
abnormal chromosomes 1 in hyperdiploid cells resulted in up to
12 copies of BCL9 and IL6Ra in a metaphase spread. SKY
detected cryptic insertions of non-homologous chromosome
segments next to the pericentric 1q12 regions in three cases. In
two of these cases duplications of BCL9 and IL6Ra were found.
In one case, however, the insertion/translocation of satII/III next
to the c-myc gene region resulted in the duplication of multiple
copies of the c-myc gene. A surprising finding in this study was
that, not only does the 1q12~24 region undergo sequential
duplication at the original chromosome 1q locus, but also
undergoes sequential duplication after being translocated to nonhomologous
chromosomes. In four cases duplications of multiple
copies of satII/III were found translocated, and in two cases the
evolution of subclones showed the sequential duplications of
satII/III and extra copies of BCL9 subsequent to the translocation.
No evidence of duplication of BCL9 or IL6Ra on nonhomologous
chromosomes was found without the presence of
satII/III sequences. We have found an association of duplication
of sat II/III sequences and the subsequent duplication of BCL9,
IL6Ra, and c-myc in MM. The behavior of the tandemly repeated
satellite DNA sequences and transposon-derived repeats in the
pericentromeric region may be affected by the methylation status
of the cell. The characteristic pattern of decondensation of the
pericentromeric heterochromatin at 1q12 found in many of these
cases suggests hypomethylation of the satII/III sequences could
be one factor which facilitates instability and duplication of this
region.
035
Hypomethylation of Chromosome 1 Satellite 2
Sequences and Hypermethylation of p16 Gene in
Multiple Myeloma and Amyloidosis
Guangzhi Qu, Tammy L. Price-Troska, Gregory J. Ahmann,
Philip R. Greipp, Morie A. Gertz, Robert A. Kyle, Melanie
Ehrlich, Rafael Fonseca
Division of Hematology, Mayo Clinic, Rochester, MN 55901;
*Human Genetics Program,Tulane Medical School, New Orleans,
LA 70112
Introduction: Epigenetic regulation of gene expression and its
interrelated effects on chromatin organization seem to play
critical roles in the genesis of human neoplasias.
Hypomethylation of pericentromeric satellite sequences of
chromosome 1 (Chr1) predisposes to Chr1 rearrangements in the
vicinity of centromere. This has been observed in the ICF
syndrome (immunodeficiency, centromere instability, and facial
anomaly) and in a human pro-B cell line treated with 5-
azadeoxycytidine. In multiple myeloma (MM), Chr1 aberrations
in the pericentromeric region are found in 40% of patients, and
Chr1 rearrangements confer negative prognostic implications. In
contrast, hypermethylation of the p16 promoter, which is linked
to silencing of this tumor suppressor gene on 9p21, has been
found in 30-40% of examined human MM cases.
Hypermethylation of p16 gene is associated with increased cell
proliferation and shorter patient survival. We thereby
hypothesized that altered DNA methylation may contribute to the
pathogenesis of MM.
Patients and methods: By Southern blot analysis, we detected
satellite hypomethylation using a CpG methylation-sensitive
enzyme to digest DNA (Bst BI) followed by hybridization with a
Chr1-specific satellite 2 probe. Human sperm DNA served as the
hypomethylation standard, and human lung and brain DNA as
hypermethylation standards. We also looked for p16 methylation
using a methylation sensitive PCR after sodium bisulfite
modification. Among the 12 patients with primary plasma cell
neoplasms studied, 8 had MM, 3 light chain associated
amyloidosis and 1 smoldering MM. DNA was purified from
CD138+ plasma cells. We also studied 5 human MM cell lines;
SK-MM-1, OPM-2, MM-1, JJN3, and KP16.
Results: Overt hypomethylation was detected in all five human
MM cell lines. Cytogenetic data were available for four of the
five cell lines, all of which showed chr1 pericentromeric
rearrangement. All 12 patient samples showed Chr1 satellite 2
hypomethylation. The degrees of hypomethylation in both cell
lines and patients’ specimen were equivalent to that in human
sperm DNA, in which many tandem repeats are hypomethylated.
In contrast, 3 out of 12 patients had hypermethylation of p16 gene
(25%), including one with amyloidosis and two with MM.
Discussion: Our study suggests that plasma cells may be
epigenetically prone to Chr1 pericentromeric rearrangements
mediated through DNA hypomethylation of Chr1 satellite
sequences. However, satellite hypomethylation and p16
hypermethylation gene silencing can co-exist, both of which
could contribute to carcinogenesis independently.
S103