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OCR for page 211
18
Molecular Biology:
Developing DNA Markers of Genolox~c Effects
This chapter briefly discusses the ef-
fect of molecular biology on prenatal diag-
nosis. The assays are discussed in detail
in Chapters 9 and 12.
The ability to obtain DNA from the fetus
has been possible for the past 10 years
through amniocentesis after 16 weeks of
gestation. However, techniques to sample
chorion in the first trimester and to kary-
otype the sample directly without long-
term tissue culture recently became pos-
sible. The chorionic biopsy consists of
aspiration of chorionic villi through the
cervical canal or transabdominally with
ultrasound guidance. Results of chromoso-
mal analysis are available within days,
rather than the weeks required with conven-
tional techniques. Without the need for
cultured preparations, direct karyotyping
could prove amenable to analysis of chromo-
somal aberrations possibly associated
with early loss (1-6 weeks after implanta-
tion). Also, DNA is readily available from
the collected tissue or from cultured cells
derived from the sampling.
DETECTING HERITABLE GENETIC
DAMAGE
One of the most interesting innovations
in prenatal diagnosis is the use of DNA
probes that reveal genetic markers near
211
specific genes (McDonough, 1985) (Table
18-1~. DNA probes have been applied for
prenatal diagnosis of cystic fibrosis,
and predictive testing for the gene for
Huntington's disease began. In addition,
the gene for Duchenne muscular dystrophy
has been sequenced and the so-called "re-
cessive oncogene" responsible for famili-
al predisposition to retinoblastoma was
discovered. The number of probes available
is increasing exponentially.
A genetic marker is a segment of DNA
that lies near a unidentified gene that
is involved in the disease etiology. With
DNA probes and genetic markers, it might
be possible to detect most of the more than
3,000 conditions caused by single-gene
mutations.
Applications of molecular biology to
clinical medicine will change the approach
to diagnosis (McDonough, 1985~. Molecular
diagnosis—even during prenatal life—
is possible with two related techniques
(see Chapter 12 for details):
· Restriction - fragment- length poly-
morphisms. Highly specific restriction
endonucleases cut DNA between particular
base sequences. When altered by mutation,
DNA is severed into fragments of a size
different from normal. The homozygous
and heterozygous states can be differen-
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212
TABLE 1~1 Disorders Diagnosable by Analysis
of Cellular DNA
Validated Uses of DNA Analysis for Diagnosis
Sickl~cell anemia
B-thalessemia
x-thalessem~a
Factor VIII deficiency
Factor IX deficiency
Phenylketonuna
a~-antitrypsin deficiency
Hunungton's disease
Antithromb~n III deficiency
Orn~thine transcarbamylase deficiency
Duchenne muscular dystrophy
Argninosucc~n~c acid dehydrogenase deficiency
Osteogenesis imperfects type II
Congenital adrenal hyperplasia
Probable Uses of DNA Analysis for Diagnosis
Fragile X syndrome
Adult-onset poll ystic kidney disease
Source: McDonough, 1985.
tiated by comparing abnormal fragment size
with normal fragment size. Potentially,
these can be used even if the gene leading
to the disease state is unknown. Abnor-
malities of the genes for hemoglobin (whose
deficiency results in sickle-cell ane-
mia), growth hormone, and 21-hydroxy-
lase (whose deficiency results in congeni-
tal adrenal hyperplasia) are conditions
that can be diagnosed with this approach.
Also, these have been used to diagnose
several other conditions, including
Huntington's disease, phenylketonuria,
factor VIII and factor IX deficiencies,
and §-thalassemia.
· Oligonucleotide probes. When the
precise DNA mutation is known, but the
mutation cannot be discriminated with
a restriction-enzyme cut, oligonucleotide
probes representing the normal and abnor-
mal sequences can be used to identify the
genotypes. As increasing numbers of
normal and mutated gene sequences become
identified, the practicality of this tech-
nique will increase.
DNA technology is versatile. Every mono-
genic disorder potentially is diagnosable
with DNA probes, as increasing numbers
of probes and polymorphisms are recognized
and restriction enzymes are developed.
TOXICI7YDURING PREGNANCY
The explication of the molecular map of
the human genome will be accompanied by
the development of functional correla-
tions that might provide insights into
the basic pathogenesis of most disorders,
including those caused by chemical muta-
gens and physical factors (such as radia-
tion). Within the next decade, many dis-
eases and toxic conditions probably will
be defined in molecular terms and become
subject to diagnosis from a few microliters
of blood (Ward et al., 1983~. DNA probes
might be used to detect chemical or food
contaminants in the body. Tests based on
such DNA probes could replace current as-
says, because of their greater sensitivity
and speed.
Quality control may suffer as DNA
probes are used more widely, particularly
if commercial kits become available. Ac-
curacy is essential, and reliability
could be diminished because of such prob-
lems as incomplete DNA digestion, faulty
hybridization, contamination, and mis-
labeling. Even in the best of hands, inter-
pretation of these tests and associated
family counseling require extensive ex-
perience and commitment. In addition,
the validity of the tests in the absence
of confirmatory assays is a problem. A1-
though revolutionary developments in DNA
probes have enormous potential as biologic
markers in prenatal diagnosis, many chal-
lenges lie ahead.
MARKERS OF EXPOSURE
With the rapid development of monoclonal
antibodies, radioimmunoassays, and mole-
cular genetic technologies, new tech-
niques have been developed to detect toxi-
cants covalently bound to DNA to form
adducts (Wogan and Gorelick, 1985; Perera,
1986; Wogan, 1988~. Many chemicals that
are active carcinogens or mutagens either
are electrophilic or are converted to elec-
trophilic metabolites. These may become
bound to DNA, RNA, orproteins. The conse-
quences of these adducts have not been
clearly demonstrated, but they are
thought to initiate carcinogenesis or
mutagenesis (Wogan and Gorelick, 1985~.
DNA adducts have been measured in blood.
Cord blood has also been used (Daffos et
OCR for page 213
MARKERS OF GENOTOXIC EFFECTS
al., 1985; Reddy and Randerath, 1988~.
Theoretically, it should be possible to
use amniotic cells or chorionic villus
cells to determine the fetal exposure to
genotoxic chemicals. The amount of tissue
required by the assays is large for these
sampling procedures. But improvements
in the laboratory procedures might make
the assays possible on smaller samples.
In humans, assessment of in utero expo-
sure to DNA-damaging agents has been at-
tempted by comparing SCE frequencies in
blood from mothers and their offspring.
A case report from Sweden described in-
creased SCEs in four children of two labor-
atory technicians who worked during preg-
nancy (Funes-Cravioto et al., 1977~. Ar-
dito et al. (1980) compared SCE frequencies
in smoking and nonsmoking mothers and their
infants' cord blood and found that mean
213
SCE frequency was slightly higher in moth-
ers than in the newborns. No difference
was found between frequencies in maternal
or cord blood of smokers and nonsmokers.
In a similar study of smoking mothers and
alcoholic mothers (Seshadri et al., 1982),
the SCE frequency only in drinking mothers
was higher than that in controls ( 13.5
versus 10.95 SCE/cell), but the SCE rate
in their infants was not significantly
increased (9.71 versus 8.95 SCE/cell).
In a separate analysis, neonates with neur-
al tube defects were found to have higher
rates of SCEs than normal babies ( 10.34
versus 8.95 SCE/cell) (Seshadri et al.,
1982~. A systematic study of infants with
normal and reduced birthweights found no
association of growth retardation with
SCEs measured in cord and postnatal blood
(Hatcher and Hook, 1981 b).
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Representative terms from entire chapter:
prenatal diagnosis
