Download Applications of Recombinant DNA to Pathologic Diagnosis

CLIN. CHEM.
31/6, 804-811
Applications
(1985)
of Recombinant
C Dan Sauls”2
and C. Thomas
DNA to Pathologic
Caskey’
Recombinant
DNA techniques
are contributing
to the understanding of the pathogeneses
of genetic, neoplastic,
and viral
diseases,
and are used in the diagnosis
of certain genetic
and viral
diseases.
Such
techniques
will have
tion in the future and will play an increasing
laboratory.
The technology
wider
applica-
role in the clinical
of this field rests upon the cleav-
age of DNA by certain enzymes,
restriction endonucleases,
and upon the ability to locate specific sequences
of nucleotides in a cleaved DNA sample by using known fragments of
DNA
labeled
(“probes”)
with
radioisotopes
or biotin.
To
produce
useful probes,
one “clones” multiple copies of the
same DNA fragment in bacteria. The use of DNA probes in
the clinical laboratory
is valuable
in antenatal
diagnosis,
genetic
counseling,
and post-natal
diagnosis
of genetic dis-
eases, especially
metabolism.
genetic
hematologic
DNA
material
probes
in clinical
diseases
and inborn errors of
can also be used
specimens.
DNA probes
fetal status
cancer
Lesch-Nyhan
disease
AdditIonal
Keyphrases:
to detect
viral
heritable
disorders
genetics
viruses
restriction
endonucleases
hemoglobin variants
Huntington’s
disease
Duchenne’s
muscular dystrophy
Ieukemias
Wilms’s tumor
retinoblastoma
.
AIDS
Recombinant
industrial
science.
DNA
research,
technology
basic
.
has significantly
biological
affected
science,
and medical
science
disciplines
and
field utilizes
many
basic
promises
significant
advancements
in laboratory
diagnosis.
It has already
contributed
significantly
to our understanding of the pathogenesis
of inherited
and virally
acquired
diseases.
We present
some examples
of current
applications
in this review,
together with an appropriate
terminology
and illustrations
of the technology.
The
Methods
Principle
of Hybridization
DNA can be readily
for use in molecular
isolated
from
hybridization
strong
acids can destroy
is stable
to extraction
its inherent
procedures,
various
cellular
Glossary
such that adenine pairs with thymidine and quanine pairs with
cytidine
Vol. 31, No. 6, 1985
(or uracil).
Linkage: The linear association of genes on the same DNA
molecule, determined by the distance between two genes. The
closer two genes are on a chromosome, the greater is the
chance of their being inherited together; the further the linkage
distance, the greater the likelihood they will be inherited
independently.
Oncogene:
A gene involved in the process of neoplastic
transformation.
Viral DNA that replicates independently of the DNA of the
host cell. The proteins resulting from plasmid DNA confer
antibiotic resistance to host bacteria.
Point mutation: A change in one nucleotide of a gene, in which one
base replaces another.
Promoter: A segment of DNA involved in binding the enzymes
necessary to initiate transcription of DNA into RNA.
Restriction endonuclease:
An enzyme that can cleave doublestranded DNA at specific sequences of nucleotides, usually at
sequences four to six bases long.
Restriction
fragment:
A DNA fragment generated by the cleavage
of a parent molecule by a restriction endonuclease.
Transcription:
The process that produces a complementary strand
of RNA from DNA.
Plasmid:
Restriction
Endonucleases
Because DNA is a complex polymer
of high molecular
mass, its efficient fragmentation
is required
for detailed
gene study. “Restriction
endonucleases”
are enzymes
capable of cleaving
double-stranded
DNA at specific sequences of
nucleotides, four to six bases long (Figure 1) (1). Cleavage of
the large
DNA
molecule
reproducibly
generates
specific
fragments,
which
can be separated
by electrophoresis
on
agarose gels. The set of fragments
from a specific DNA that
has been cleaved
by action
of a given
restriction
enzyme is
constant
for that DNA.
By use of various
restriction
enzymes
‘Howard
Hughes Medical
Institute,
and R. J. Kleberg,
Jr.,
Center for Human Genetics,
at Baylor College
of Medicine,
Houston, TX 77030.
2Department
of Pathology, The Woman’s
Hospital
of Texas, 7600
Fannin,
Houston,
TX 77054.
Received October 23, 1984; accepted February
13, 1985.
CLINICAL CHEMISTRY,
of Terms
Bacteriophage: Viruses that infect bacteria. Bacteriophage lambda
is commonly used in studies with recombinant DNA.
Chromosomal
translocation:
A rearrangement of chromosomal
material such that the chromosome breaks and a fragment of it
is joined to a different chromosome.
Cosmid: Plasmids into which the “cos” site of bacteriophage
lambda has been inserted. A cosmid also allows plasmid
molecules to be inserted into viral coat particles in vitro.
DNA ligase: An enzyme capable of covalently joining two ends of
DNA molecules.
DNA probe: A fragment of single-stranded DNA that can be
labeled with a radioisotope or biotin and hybridized to an
unknown DNA sample to determine whether the probe and the
unknown DNA have sequences of nucleotides in common.
Enhancer: DNA elements that can increase the efficiency of
promoter regions.
Gene: A segment of DNA that codes for a single polypeptide.
Hybridization:
The annealing of two strands of single-stranded
DNA (or RNA) whose sequences of bases are complimentary
sources
reactions.
Exposure
to
information,
but DNA
radical
shifts
in salt
concentration,
and high temperature,
and it can be physically ensconced
in a nitrocellulose
matrix
without
losing
its
ability
to hybridize
by base pairing.
This allows a cloned and
radioactive
DNA
probe
to recognize
precisely
its complementary
base sequence
by hybridization
in a mixture
of
DNA
or RNA isolated
from cellular
sources. This principle
is the
underpinning
of DNA:DNA
(homoduplex)
or
DNA:RNA
(heteroduplex)
hybridization
reactions.
804
Diagnosis
that
recognize
different
internal
sequences,
specific
patterns
can be obtained
for a particular
DNA
More than
140 restriction
endonucleases
are currently available.
Use of a battery
of these endonucleases,
together with establishment
of fragment
overlaps, permits
development
of gene maps.
fragment
sample.
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GLAATT
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Fig. 1. Restriction
in “sticky ends”
endonuclease
cleavage
I
I
I
I
of DNA by Eco RI,
Often it is desirable to clone portions
of DNA directly
from
the genome and study long stretches (20 kb) of DNA. A third
vector
system,
“cosmids,”
makes this possible.
Cosmids
are
produced
from “cos” sites taken from lambda
phage DNA
attached
to portions
of plasmid
material-hence
the name
cosmid.
These systems
can accommodate
genomic
DNA or
cDNA
fragments
of 20-45
kb. The
efficiency
of cosmid
cloning is high, and allows
examination
of longer sequences
of DNA.
Organically
synthesized
DNA probes can also be made if
protein or gene sequence
data are available
for the gene of
interest.
resulting
Southern
of DNA
Probes
technical
The molecular
biologist
has two sources
for isolating
cloned DNA probes: the native DNA in the nucleus of a cell
(genomic DNA), and complementary
DNA (cDNA), made by
enzymically
(with reverse transcriptase)
copying messenger
RNA (mRNA).
DNA probes can be generated
by cloning procedures
(2) by
use of restriction
endonuclease
fragments
inserted
into
bacterial
plasmids.
Plasmids
are circular
DNA molecules
that replicate
independently,
encode antibiotic-resistance
genes, and thus confer
antibiotic
resistance
in the host
bacterium.
Plasmids
carrying
fragments
of foreign DNA can
be constructed
by recombinant
DNA methods by using a
restriction
endonuclease
and DNA ligase.
A restriction
enzyme
can cleave the plasmid
and the
foreign
DNA,
yielding
identical
complementary
singlestranded
junctions
(“sticky ends”), which the DNA ligase
enzymically
links
covalently.
Because
a single molecule
of
the recombinant
plasmid
can be replicated
in 50 to 200
copies
in proliferating
Escherichia
coli, large quantities
of
the cloned DNA can be obtained.
Cloned
DNA inserts are
recovered
in abundance
by being excised
from the recombinant
plasmid
by the
same restriction
enzyme
used in
making
the construction
(Figure
2). Plasmids
efficiently
replicate
with DNA inserts
that are 1000 to 6000 base pairs
(one to six kilobases,
kb) long. Larger
fragments
are not
reliably
replicated.
An alternative
approach,
which makes use of bacteriophage lambda,
allows larger fragments-up
to about 20
kb-to
be efficiently
cloned. The fragments
of DNA
(or
cDNA)
to be cloned are inserted
into modified
phage, which
are then used to infect
the bacteria.
Bacteriophage
replicates in the bacteria
and is subsequently
harvested
by
lysing
the cells.
and isolated
molecule
Foreign
1975, E.
M. Southern
(3) developed
a widely
adopted
procedure
for transferring
electrophoretically
separated
DNA species to nitrocellulose
(Figure
3). Once immobilized
in nitrocellulose,
DNA can be analyzed
for specific
nucleotide
sequences
by use of labeled cloned DNA probes.
The advantage
of Southern’s
approach is that the resolving
power
of gel separation
is efficiently
combined
with
the
specificity
of DNA probes.
This procedure
(3,4) involves use of standard
electrophoretic
agarose
gel techniques.
Treatment
of the doublestanded DNA with dilute sodium hydroxide
effects
strand
separation.
Nitrocellulose
or charged
nylon ifiters are placed
on the gel for strand
transfer
by buffered
salt solution.
Within
hours,
DNA fragments
are transferred
from the gel
to the mtrocellulose
ifiter. Very low-Mr
species (fewer than
300 base pairs) are somewhat
under-represented,
and very
large
fragments
(>15 000 base pairs) often take longer to
transfer
(3,5). As transfer
occurs,
the DNA molecules
bind
to the mtrocellulose.
Hybridization
with
the radioactive
probe is done after the ifiter is washed
free of salt and then
dried under reduced
pressure.
Several
recent improvements
in hybridization
conditions have increased
the sensitivity
of
detection
and decreased
the background.
In
Creation
Transfer
DNA
is cloned
with
high
Dot
(Slot)
Blotting
A second technique
used to probe DNA or RNA samples
is
“dot-blotting”
(6, 7) or “slot-blotting.”
This technique
is used
to detect
the presence,
absence,
or amount
of a genetic
element.
The technique
requires
neither size fractionation
Cleavage
Gene
+
Cleavage
Locus
vvl
4Kb
cules.
Ri
Enzyme
efficiency
in large quantity,
because
a single recombinant
suffices to form thousands
of bacteriophage
mole-
Eco
Sites for Restriction
Iv
1Kb
7.2Kb
$
Sites
Cut
with
v
6Kb
restriction
v
3.5Kb
enzyme
6Kb
Cloned
DNA
insert
or
/
cDNA
0
1Kb
$ Gel
Hybridize
to
P32
DNAwith
cut
Eco
Ri
7.6Kb
-
6Kb
Labelled
Cloned
4Kb
3.5Kb
ReplIcation
in Bacteria
PIaenld
DNA
Fig. 2. Plasmid cloning of foreign DNA or cDNA by annealing of the
ends” produced by Eco RI
“sticky
DNA
1Kb
Probe
DNA+Fluorescent
Stain
Radioautogram
Fig. 3. Southem gel method
from a DNA mixture
(3) for identification of a gene fragment
CLINICAL CHEMISTRY,
Vol. 31, No. 6, 1985
805
nor cleavage
with enzyme.
Samples
of DNA or RNA are
extracted
and dotted onto nitrocellulose
ifiters
or, to save
space, applied as slots 2 mm wide x 5 mm long. Hybridization is carried out as for Southern
blotting,
and probes are
detected
by autoradiography.
With radiolabeled
probes of
high specific
activity,
the resulting
sensitivity
(i.e., the
smallest
detectable
amount)
is about 0.2 to 0.5 pg (6; G.
Buffone,
personal
communication).
This is an especially
valuable
technique
for use with microgram
quantities
of
samples
(6) and for detection
of viral sequences
in a sample
(8-10).
In Situ
Hybridization
A third technique
based upon the use of DNA probes is in
situ hybridization,
used to hybridize
probes to chromosomes
(11,12) or tissue sections (13,14).
Its usefulness
results from
its ability
to localize
relevant
DNA
sequences in larger
structures,
thus linking
biochemistry
with cytogenetics
or
histochemistry.
Genes can be localized
to specific chromosomes by radioactive
probes and the results made visible by
autoradiography
of the
chromosomes.
Similarly,
important
information
about gene expression
can be obtained
in relation to the development
of specific tissues
by identifying
the
presence of mRNA
in histological
sections by autoradiography or fluorescence
microscopy.
Viral
genomes
can be
specifically
localized
within
tissues with this technique
(14).
Tissues are fixed in Carnoy’s
B solution
(ethanollchlorofonn/
acetic acid, 60/30/10
by vol), embedded
in paraffin,
and
sectioned
as usual. The hybridization
is performed
directly
on the cut sections. DNA probes used for in situ cytohybridization (in tissues) can be either radiolabeled
for autoradiography or biotinylated
(13, 14) and coupled to fluorescent
or
immunoenzymic
detection
systems
(Figure
4). The latter
technique
has potential
for anatomical
and clinical
pathology laboratories,
because
no radioisotopes
are required
to
detect the viral genome in tissue sections
or fluid specimens
such as urine.
Of the various
potential
immunoenzymic
systems,
peroxidase
(EC 1.11.1.7)-anti-peroxidase
and alkaline phosphatase
(EC 3.1.3.1)-anti-alkaline
phosphatase
(15) are the most widely used. Biotin,
covalently
linked to
triphosphates
of thymidine,
uridine,
or cytidine
(16), works
most effectively
as a “recorder”
molecule
if it is separated
by
about 1 nm from the nucleotide
by an 11- to 16-carbon
spacer-arm
(13). Biotinylated
nucleotides
are then incorporated into the DNA
probe by using the enzyme
DNAdirected DNA polymerase
(EC 2.7.7.7). This same procedure
can be used to incorporate
radiolabeled
nucleotides,
generating highly radioactive
probes, with specific activities exceeding i0 counts/mm
per microgram.
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Fig. 4. Immunoenzymic
detection
complementary
biotinylated
probe,
tion of a visually detected product
806
CLINICAL CHEMISTRY,
Heritable
Disease
Recombinant
DNA technology
is currently
being applied
in clinical
medicine
to heritable,
neoplastic,
and infectious
diseases.
The diagnosis
and understanding
of inherited
diseases have benefitted
from the use of restriction
endonucleases and DNA probes. DNA
or RNA samples
can be
prepared
from biopsies of chorionic villus
or from amniocytes obtained
by amniocentesis.
Fibroblasts
cultured
from
persons suspected of having an inherited
disease can be used
antenatally.
Diagnosis
by use of this material
and DNA
hybridization
is limited
only by the availability
of cloned or
synthetically
derived
probes. Recently
developed
probes
include ones for Lesch-Nyhan
trait (17), a1-antitrypsin
(18),
phenylketonuria
(19), and deficiencies
of argininosuccinate
lyase (20), clotting
factor IX (21), and insulin (22).
Lesch-Nyhan
disease results from a deficiency
of hypoxanthine
phosphoribosyltransferase
(HPRT, EC 2.4.2.8), an
essential enzyme in purine metabolism.
Such patients show
mental retardation,
extrapyrainidal
choreoathetoid
spasticity, and a compulsive
tendency to self-mutilation.
Hyperuricemia and increased
urinary
excretion
of uric acid are
constant findings.
A recently
developed
probe for the HPRT
gene (17) can be hybridized
to fibroblast
DNA (Figure
5, A
and B) to screen for deletions
of all or a portion
of the gene
responsible
for the Lesch-Nyhan
syndrome. This probe can
also be used in cases of Lesch-Nyhan
syndrome
in which the
only abnormality
of the mutant
gene is a point mutation
(i.e., a single nucleotide
substitution).
The HPRT probe can
also be used to study the mRNA
of the mutant
gene (Figure
SC) (23). Sequencing
procedures
(24,25)
are then used to
determine
the incorrect
base substitution.
In some genetic
diseases
the site of the mutation
is
constant.
Knowledge
of the normal
sequence
at the site of
the sickle cell mutation
allowed
Chang and Kan (26) to use
restriction
endonuclease
Mstll for diagnosis. The sickle cell
mutation
involves a single base change in a single codon,
from GAG to GTG. Mstll
recognizes
the base sequence
containing
GAG, but not GTG. Treatment
of DNA with
MatH therefore
provides
fragments
that are of different
lengths
for sickle cell homozygotes
(SA), heterozygotes
(SA),
and normal
homozygotes
(AA): homozygotes
of sickle cell
disease (SS) show fragments
containing
1350 base pairs, AA
(normals)
1150, and sickle cell heterozygotes
(SA) both sizes
(26).
Knowledge
of the normal sequence
at a mutation
site also
allows organically
synthesized
probes to be used for diagnosis. Nucleotides
19 bases long have been synthesized
for the
mutation
site of the normal (A) and the sickle gene (5) and
can discriminate
between
the normal and affected individuals(27).
Synthesized
probes are also useful for studying
diseases
where the amino
acid substitution
is known
but gene
structure
data are unavailable.
By this method Woo et al.
(28) have been able to distinguish
between
the M and Z
alleles in a1-antitrypsin
deficiency.
By this approach,
heterozygotes
can be accurately
differentiated
from nonnal
individuals
at the genetic level. Orkin
and Markham
(29)
have successfully
used synthetic
probes to diagnose
a
thalassemia
variant
in which a single base change leads to
abnormal
processing of J3-globin mRNA.
Gene probes, both synthesized
and cloned, provide powerful tools for genetic disease diagnosis,
but they require
a
specific knowledge
of the gene or enzyme involved.
Another
ATGCA
I
Applications
of a DNA sequence
by use of a
with alkaline phosphatase produc-
Vol. 31, No. 6, 1985
approach,
restriction
fragment
length
polymorphisms
(RFLPs),
is applicable
to illnesses
where
information
about
the gene, or even about the chromosome
involved, is lacking
‘A
C
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FIg. 5. Malysis
of Lesch-Nyhan
(A) Deletion of exons 6,7,8,
muta ons by method of Southern
and 9 (P.JK849);
and Northern
(2
exons 7,8, and 9 (RJK3487);
and complete gene (RJK853). The DNA was digested with endonuclease Pst. (B) Partial
by new BgI I fragments
with exon-specific
probes. (C) The dl5erent mRNA charactaristicsof three Leech-Nyhan mutants,
gene duplication,exons 2 and 3, are detected
shown by Northern analysis. All patients lacked HPRT enzymlc activity
(Figure
6) (30). The sites recognized
by restriction
enzymes
are interspersed
throughout
the human genome. Differences
between
individuals
in the type, number,
and position
of
these sites provide an abundant
source of genetic markers
for linkage
studies.
DNA
fragments
of variable
size are
therefore
generated
when DNAs from different
individuals
are cleaved with the same restriction
enzyme and identified
by the Southern
method with a single probe.
These variations
in fragment
size, RFLP, are inherited.
In
families
where a genetic disease is expressed,
it is possible,
A.
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as
V
V
V
aA
7.6Kb
B.
AS
Mother
AS
SS
AA
Father
Child
Child
Fetus1
13.0
7.6
Fig. 6. Restriction fragment length polymorphism linked to the sickle cell
gene
(A) Diagram of restriction sites in DNA fiarwcing a p5 and p5 gene. The p-globln
genes are found in 13 and 7 kb restriction fragments,
respectively.
(0) Southern
analysis of DNA digested with the indicated endonuclease
and probed with the
globin
gene (boxed area). The globin genotypes
of the patients
by the RFLP linkage with the A and S genes
predicted
and fetus
are
by using an appropriate
probe, to link the occurrence
of a
particular
RFLP pattern
with the disease gene. By performing RFLP
studies
on large numbers
of family
members,’
affected and unaffected,
the degree of probability
that the’
abnormal
RFLP pattern
will occur when the disease gene is’
present can be statistically
assessed. This probability
dofines the degree of “linkage”
between
the disease gene and
the occurrence
of the abnormal
fragment
pattern.
This
knowledge
can be used in subsequent
RFLP
studies
to
determine
which family
members
are asymptomatic
carriers of the gene and can, in some cases, be extended
to
antenatal
diagnosis.
Thus RFLP
studies
are useful
for
genetic
counseling
as well as disease diagnosis.
This approach
has been successfully
exploited
with several hemoglobinopathies,
including
the /3-thalassemias
(3133). The f3-globin gene cluster,
located on the short arm of
chromosome
11
is represented
by several
different
genetic
abnormalities
in the clinical
group of p-thalassemiss, including
abnormalities
in RNA transcription
(34),
RNA processing
(36), and RNA translation
(37). Phillips
et
al. (38) used RFLPs
to identifr
polymorphisms
associated
with the y.globin genes and used these in the prenatal
diagnosis
of sickle cell anemia
and p.thalassemia
mutations. This has been a successful
prenatal
diagnostic
technique in more than 100 families
(39).
Probes to known genes or probes to DNA sequences of no
known
function
(“anonymous
sequences”)
can be used in
RFLP linkage
studies. RFLP
techniques
combined
with in
situ chromosomal
hybridization
can determine
which chromosome carries the fragment
closely linked
to the disease
gene.
Recently,
Gusella
et al. (40) screened
Venezuelan
and
American
families
with
Huntington’s
disease
for RFLP
linkages.
Restriction
digests
of DNA
from affected
and
normal
family
members
were probed with anonymous
sequences to uncover
associations
between
the Huntington’s
disease gene and various different
probes. Although
Gusella
had originally
estimated
that as many as 100 anonymous
sequence
probes might
have to be tested to find a reliable
linkage
(41), fortuitously,
the twelfth
probe (“G8”) provided
CLINICAL CHEMISTRY,
Vol. 31, No. 6,
1985
807
evidence of linkage;
in situ hybridization
then localized
G8
to chromosome
4(40). The degree of linkage
between G8 and
the Huntington’s
gene is quite high. Additional
studies will
doubtless lend to improved
knowledge
of linkage by isolating the DNA adjacent
to the G8 clone and closer to the
Huntington’s
gene. Continued
development
should lead to a
clinically
useful method
for detection
of the Huntington’s
gene (41) in families
at risk. This example
illustrates
the
power of RFLP linkage
studies applied
to genetic diseases
about which the nature of the protein and gene defects is
completely
unknown.
Given the high degree of RFLP reported
for both phenylketonuria
(43) and Lesch-Nyhan
syndrome
(43), RFLP
analysis
of affected families
should improve
carrier
detection and prenatal
diagnosis
for both diseases.
The gene for Duchenne’s
muscular
dystrophy
has been
mapped
to band 21 of the short arm of the X chromosome
(Xp2l),
and probes are available
that map to that region
(44). Prenatal
diagnosis for Duchenne’s
is predictably
going
to be available
as probes with tighter
linkage
to the Duchenne’s gene become available.
Neoplastic
Disease
Recombinant
DNA
techniques
have
not yet made a
formal
impact
upon the clinical
diagnosis
of neoplastic
disease but this seems inevitable.
Already
these techniques
have expanded
the scope of understanding
of the pathogenetic nature
of the neoplastic
process. The discovery
and
current
understanding
of oncogenes is a case in point.
The concept of oncogenes derives from two independent
lines of investigation.
Studies of chemically
induced carcinogenesis identified
specific genetic elements
capable of inducing neoplastic
transformation
in fibroblasts
that took up
DNA from transformed
cells (45-47).
Second, genes with
transforming
properties,
found in certain retroviruses
(46,
48), were termed oncogenes.
Both lines of study make it
clear that the genes are not in themselves
a sufficient
condition
for transformation
but require
a given biologic
context
to operate-in
keeping
with the idea that cancer
occurs as a multistep
process.
Oncogenes
correspond
to naturally
occurring
genes in the
host genome, “proto-oncogenes,”
which seem to be important
in the control
of embryonic
development,
differentiation,
and cell growth.
Some proto-oncogenes
produce
proteins
similar
to components
of receptors
for growth factors such as
epidermal
growth
factor (49) and platelet-derived
growth
factor (50). More than 30 oncogenes have been identified
(46).
Neoplastic
transformation
may occur when the amounts
of genetic transcripts
from oncogenes are abnormally
high
(46). Gene amplification
(the production
of multiple
copies of
the same gene) and abnormal
regulation
of gene transcription are two ways this might happen. An oncogene inserted
into host DNA in the region of a powerful
“promoter”
or
“enhancer”
could result in high proportions
of transcription
with correspondingly
increased
amounts
of gene product.
Similarly,
proto-oncogenes
may be juxtaposed
to such a
region secondary
to chromosomal
translocation
(51). This is
thought
to be the mechanism
by which,
e.g., the myc
oncogene is activated
in Burkitt
lymphoma.
Alternatively,
transformation
may result
if a point mutation
in a protooncogene
produces
an amino acid substitution
in a critical
position
in its gene (i.e., rasH) (46). Altered
protein
might
disrupt
critical
processes
in the cell, leading
to features of
neoplastic
transformation:
e.g., altered adhesion properties,
abnormal
growth,
and shape. Circumstantial
evidence supports these general
mechanisms.
For some time we have known
that many
neoplastic
808
CLINICAL
CHEMISTRY,
Vol. 31, No. 6, 1985
diseases are associated
with chromosomal
aberrations
(52,
53), including
Burkitt
lymphoma,
Philadelphia
(Ph) chromosome-positive
cases of chronic
myelocytic
leukemia,
acute promyelocytic
leukemia,
retinoblastoma,
and Wilma’s
tumor.
Recombinant
techniques
are extending
an understanding
of the chromosomal
aberrations
to the level of
molecular
genetics
(51, 54). For other tumors,
molecular
genetic abnormalities
have been identified
where
no previous cytogenetic
lesion was known.
Chromosomal
translocation
is relevant
to induction
of
neoplasia
in Burkitt
lymphoma
and most cases of chronic
myelocytic
leukemia.
For Burkitt
lymphoma,
often associated with a translocation
(55, 56) of the distal long arm of
chromosome
8 to one of three other chromosomes
(2, 14, or
22), the breakpoint
of the translocated
fragment
is consistently at band 24 of the long arm of chromosome
8. DallaFavera et al. (57), studying
somatic
cell hybrids
containing
portions
of chromosome
8 from Burkitt
lymphoma
cell lines,
used a DNA probe for the oncogene c-myc that was homologous with the transforming
gene of avian myelocytomatosis
virus (v-myc).
Their Southern
blot analyses
confirmed
that
c-myc is situated
on chromosome
8. Taub et al. (58) and
others (59) further
refined the c-myc location
to the band 24
translocation
site. The majority
of cases of Burkitt
lymphoma involve
a translocation
from chromosome
8 to the
immunoglobulin
heavy chain loci on the long arm of chromosome
14 (58), but a significant
minority
are associated
with
translocations
to the immunoglobulin
kappa
light
chain gene on the short arm of chromosome
2 (51, 60).
Some have hypothesized
(51,58-60)
that such translocations may bring about neoplastic
transformation
by juxtaposing the c-myc gene on chromosome
8 next to a promoter
or enhancer
region for immunoglobulin
genes on chromosomes 14, 2, or 22. This would
bring about an abnormal
regulation
of the oncogene and subsequent
neoplastic
transformation
of the cell.
The case for Ph chromosome-positive
patients
with chronic myelocytic
leukemia
is analogous.
Patients
with the Ph
chromosome
constitute
about 92% of the adult patients
with
chronic myelocytic
leukemia
(62); they have a better prognosis than Ph chromosome-negative
patients.
The translocation involved
is from chromosome
9 to, most commonly,
chromosome
22. Such translocations
were studied
by DeKlein et al. (61), using in situ chromosome
hybridization;
they localized
the oncogene
c-abl to the breakpoint
on the
long arm of chromosome
9. The c-abl oncogene is homologous to the transforming
gene of Abelson murine leukemia
virus; thus, the hypothesis
of abnormal
regulation
of an
oncogene at a translocation
breakpoint
can be invoked here
also.
A 15/17 translocation
has been identified
in patients
with
acute promyelocytic
leukemia
(M3 subtype)
of either the
common hypergranular
type or microgranular
variant
(51,
62-64).
Evidence
implicating
specific
oncogenes
in this
disease awaits further
studies.
Oncogenes
appear to act dominantly
when activated
by
translocations.
However,
recessive
tumorigenic
alleles may
be operative
in at least two childhood
cancers.
Childhood
retinoblastoma
(65, 66) and Wilma’s tumor
(67) have both
hereditary
and sporadic
forms. Hereditary
transmission
of
retinoblastoma
is autosomal
dominant,
but sporadic
cases
are associated
with a cytogenetic
deletion in the long arm of
chromosome
13. The presumptive
locus for the retinoblastoma gene is termed
Rb-i
(66). Wilma’s
tumor
is also
sometimes
associated
with abnormalities
of the short arm of
chromosome
11. Several groups (65, 68-71) have now compared normal tissue DNA and tumor DNA by Southern blot
analyses after digestions
with various restriction
endonucle-
ases and the use of cloned DNA probes to genes in the
vicinities
of the known
chromosomal
deletions.
The results
tend to support the hypothesis
that a recessive
mutant gene
is involved in some sporadic cases for both tumors.
Expression
of a recessive tumorigenic
gene requires that
it be present as the only allele in the tumor
tissue. This
could occur if the wild-type
or normal
allele
were lost
through
mitotic non-disjunction
or recombination
(65, 69,
71). The mutant
allele could then be duplicated
locally
to
produce a homozygous
state. In Wilma’s
tumor,
gene locus
deletions
have been demonstrated
when there is no visible
cytogenetic
abnormality
(68-70).
Viral
genomic
probes are providing
intriguing
associations between
other neoplasms
and several types of viruses.
Probes of Epstein-Barr
virus
DNA
(72, 73) have added
stronger
evidence
on the association
of this virus
and
lymphomas.
Hochberg
et al. (72) demonstrated
by gel separation-blotting
procedures
the presence of the viral genome
in brain lymphoma
tissue but not in adjacent
uninvolved
brain. Lancaster
et al. (74), using similar
methods,
demonstrated a relationship
between papilloma
virus infection
and
dysplasias
of the uterine
cervix.
Lass and associates
(75)
found the papilloma
virus genome
in conjunctival
papillomas.
Human
T-cell leukemia-lymphoma
virus type I (HTLV-I)
is the first retrovirus
to be convincingly
associated
with a
human
malignancy
(76), although
other retroviruses
have
an established
association
with several animal
neoplasms.
Evidence from recombinant
methods
and standard virologic
techniques
has implicated
HTLV-I
in the pathogenesis
of a
subtype of T-cell leukemia
(76). This adult illness manifests
splenomegaly,
hypercalcemia,
and skin disorders
(77). Geographic
concentrations
occur in Japan,
South America,
the
Caribbean,
and some pai-ts of Africa. Dot blot procedures
involving
2 zg of DNA can efficiently
screen for one viral
genome in fewer than 106 molecules
(76). This technique
is
therefore useful both for diagnostic studies and the epidemiological surveys necessary
to pinpoint high-density
locales.
Infectious
Disease
DNA probes are now available
for many viral genomes,
including
hepatitis
B, Epstein-Barr,
herpes simplex types I
and II, papova-viruses
(BK, JC, and 5V40),
papilloma
viruses
of several subtypes, adenoviruses,
HTLV,
and others. Recombinant
techniques
allow direct access to the viral
genome. Not only can the presence of viral nucleic acid be
assessed, but also information
regarding
the state of the
genome can be obtained.
By using appropriate restriction
endonucleases
and Southern
blotting,
one can determine
whether viral DNA is present in a cell as an episome (viral
DNA separate from host DNA) or is integrated
into the host
genome (78). if the viral DNA is integrated,
partial
viral
genomic
probes can be used to determine
what portions
of
viral DNA have been inserted
and which if any have been
deleted.
Such information
is not possible with standard
culture procedures
and may elucidate
the mechanisms
of
quiescent
states in viral life cycles. Diagnostic
virology
makes use of available
probes for rapid determination
of the
presence of pathogenic
viruses, dot blot or slot blot hybridization being probably
the most efficient type of procedure
for this. Only microgram
quantities
of sample (host DNA)
are required
to detect picogram
quantities
of viral DNA (G.
Buffone, personal communication).
Southern
blotting
analysis
must be applied
after restriction enzyme
digestion,
to determine
whether
the viral
genome has been integrated
into the host DNA, but this
requires
larger
amounts
of DNA.
The meaning
of viral
integration
into the host genome relative
to pathogenic
states is not altogether
clear at this point. Detection
of viral
nucleic acid in a sample
is not synonymous
with active
production
of virus particles
within
the cell. Thus DNA
hybridization
methods
for viral detection,
based on use of
genomic digests, must be assessed carefully.
Production
of
virions-detectable
by immunochemical
methods
or electron microscopy-is
therefore
more reliable direct evidence
of viral replication.
An interesting
application
of hybridization
technology
relative
to viral diseases is in situ cytohybridization
(13,14).
Even with
radiolabeled
probes this procedure
is not as
sensitive as dot blot analysis for indicating
the presence of a
viral genome, but it makes possible the direct tissue localization of virus material.
Weller et al. (79) used this technique to demonstrate
hepatitis
B virus in hepatocytes,
and
Grinnell
et al. (78) could localize the JC subtype
of papovavirus to specific sites in patients
dying from progressive
multifocal
leukoencephalopathy.
This
capability,
like
immunocytochemistry,
allows
a biochemical
approach
to
tissue sections-of
critical
importance
when biopsy material
is small and cannot be divided for culture, biochemical,
and
morphologic
studies.
DNA
in situ cytohybridization
can
even be performed
with fixed and paraffin-embedded
material, and combined with immunoenzymic
detection systems.
In cases of suspected
herpes
encephalitis
or progressive
multifocal
leukoencephalopathy,
this approach allows rapid
specific
diagnosis
with minimum
material
and technical
time.
HTLV-ffl has been suggested
as the etiologic
agent
in
cases of acquired
immunodeficiency
syndrome
(AIDS)
(8084).
Antibodies
against
envelope
and core antigens
of
HTLV-ffl
have been shown to have value in the diagnosis
of
this disease (82,83,85).
Commercial
enzyme-linked
immunoabsorbant
and radioimmunoassay
systems
for detecting
HTLV-IU
are now under development
(86). Recombinant
DNA techniques
may thus offer future diagnostic
utility
(87).
This work was supported by Medical
Service Program
Grant
Corp. Dev. 01-3 from the March of Dimes Birth Defects Foundation,
Metropolitan
Houston Chapter,
to C.T.C., and by the Howard
Hughes
Medical
Institute.
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