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Leukemia: Laboratory Evaluation - Molecular
Genetics
Overview:
Chromosomal aberrations are rather gross changes reflecting
molecular abnormalities. DNA probes against a selected gene locus
can be used to identify sequences of specific proteins, or clonal
rearrangements of specific cell types.
In this way we identify segments of DNA unique to a cell
type (such as the sequence for the T cell receptor gene) or
unique to a novel protein (such as the p210 tyrosine protein
kinase produced by the bcr/c-abl fusion gene only in
CML).
The finding of a DNA rearrangement is evidence that a clonal
population exists, and it offers clues as to the lineage of the
clonal population. Clonal population = leukemia, variant population=
reactive/infectious lymphocytosis.
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Process of DNA Analysis:
DNA is extracted from cells and the long (high MW) DNA
strands are cleaved or cut into fragments of varying length
depending on the restriction enzyme's site of action.
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These fragments of DNA are then electrophoresed,
separating the fragments by size. Next the fragments are
transferred to a nylon support mesh and hybridized with the
DNA probe (DNA complementary to the sequence you are looking
for). The probe is labeled (usually radioactive) allowing it
to be visualized.
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If a clonal population of cells exists (suggesting
leukemia), all cells within that clone will have identical
DNA rearrangements. Thus restriction enzymes will cut all
the DNA into pieces of the same size. These will all migrate
the same distance on the gel electrophoresis and thus will
be in the same location when identified with a DNA
probe.
In reactive situations multiple lymphocytes are stimulated each
having a different rearrangement. Expansion of the reactive lymphocytes
results in innumerable relatively small clones. Since each clone
has a different rearrangement, restriction enzymes will cut the
DNA from each clone into sightly different sized pieces. These
pieces will electrophorese as a smear so that no rearrangement
bands are observed on Southern hybridization.
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Bam HI is a restriction enzyme. In this example,
two different sized DNA fragments result from processing with
the same enzyme, following a gene rearrangement.
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Main Points
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Reactive
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Leukemia
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Multiple lymphocytes, innumerable small clones
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One clonal population
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Different size strands by restriction enzymes
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Same length strands by restriction enzymes
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Smear on electrophoresis
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Band on electrophoresis
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In the "Normal BM" column, note the light smearing
of the DNA due to small numbers of cells each with different rearrangements
of their DNA. No diiscrete band is formed.
The patient's marrow contains normal as well as leukemic cells,
thus the presence of both germline (G) and rearranged (R) DNA.
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Practice Question #1
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An 11-year-old boy comes to you because of a cough and
fever. His spleen is enlarged, but no lymphadenopathy was
noted. His WBC was 42.0 x10 /L with 60% immature vs.
reactive lymphocytes. The marrow aspirate was a dry tap.
Cytochemical and immunologic studies were consistent with a
lymphoid origin for the cells.
What is the best diagnosis based on the DNA probe studies
of the peripheral blood shown at right?
A. c/w reactive viral lymphocytosis
B. c/w pertussis lymphocytosis
C. c/w a pro-B cell Acute Lymphoblastic Leukemia
D. c/w Acute Lymphoblastic Leukemia, L3 (Burkitt's)
E. c/w T-cell Acute Lymphoblastic Leukemia
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Another molecular genetic technique, PCR (Polymerase Chain Reaction),
can detect minute quantities of abnormal DNA allowing for the
early detection of residual leukemia following therapy. One problem
with this technique may be that it can be overly sensitive, magnifying
small reactive clones, or even the DNA from single cells.
In summary, molecular genetic studies can
detect clonal populations, can provide evidence of
lineage through the identification of DNA rearrangements
associated with a particular cell type, and can aid in
the diagnosis of specific disorders through the
identification of DNA rearrangements that result in unique
gene fusions as in the bcr/c-abl fusion product of chronic
myelogenous leukemia.
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