46
Chromosome, Microdissection and Microcloning
isolated sequences fall within the region
encompassed by the deletion. For ex-
ample, using a chromosome 9p–speci±c
microdissection probe showed chromo-
some 9p anomaly resulting from two
translocations [t(9; 18)(p12; p11.2) and t(1;
9)(p12; p12)] in polycythemia vera patients.
Similarly, chromosome microdissection
and reverse painting of aberrant chromo-
some 2 revealed a 2q33–q35 deletion in
lung hypoplasia.
6.5
Investigation of Other Specialized
Chromosomal Structures
Recombinant
libraries
from
microdis-
sected chromosomes can be very useful
in genetic analysis of specialized chro-
mosome structures, in genomic sequenc-
ing projects, and for the characterization
of disease-related genetic loci. Special-
ized chromosome regions are particularly
amenable to microdissection and cloning.
These regions include common struc-
tures such centromeres and telomeres and
the regions adjacent to these structures,
short arms of acrocentric chromosomes,
translocations, and fragile sites. Also, the
structural basis of light and dark Giemsa
banding as well as of the banding patterns
observed with newer staining methods can
be investigated.
In addition to chromosome band–speci-
±c libraries (probes), microdissection in
combination with reverse-painting fluo-
rescence
in situ
hybridization (FISH) has
proven to be a very effective method to
identify the origin of less common but im-
portant structures such as homogeneous
staining regions (HSR), marker chromo-
somes, ring chromosomes, small acces-
sory chromosomes (SACs), and directed
isolation
and
mapping
of
microsatel-
lite sequences.
Regions of gene ampli±cation (homoge-
neous staining regions) are relatively large
structures that can be microdissected eas-
ily for cloning and molecular analysis. For
example, the chromosomal origin of HSR
in neuroblastoma was identi±ed as coam-
pli±cation of chromosome bands 2p13 and
2p24 by chromosome microdissection and
FISH. A microdissection-speci±c probe
isolated from HSR in cells of transitional-
cell carcinoma of the bladder revealed
a new region (1p31–p32) that is ampli-
±ed in primary tumors in addition to the
previously known regions involving am-
pli±cations in chromosomes 6p22, 7p11,
9p23–pter.
In myelodysplastic syndromes (MDS),
karyotypic aberrations identify subgroups
of patients with distinct clinical–morpho-
logical features and can be relevant in
risk assessment of developing leukemia.
Often, conventional cytogenetic analysis
is not suf±ciently informative due to
the presence of numerous unrecogniz-
able markers. Microdissection of these
markers followed by reverse-paint nor-
mal karyotypes may resolve questions
regarding their origin in cases of MDS
in patients previously treated for cancer.
Ring chromosomes are another exam-
ple of a rare constitutional abnormality
with inconsistent phenotypic features. The
ring chromosomes were characterized us-
ing microdissection in combination with
degenerate nucleotide-primed polymerase
chain reaction (PCR) and reverse paint-
ing (micro-FISH). This method made it
possible to determine the chromosomal
origin of the ring chromosomes in detail
in several diseases and thus to compare
the phenotypes of similar cases. Reverse
painting of microdissected chromosome
6 probe onto normal metaphase spreads
revealed a small deletion of the termi-
nal region of 6q26qter in cases of acute
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