(such as mutations leading to frameshifts), or a
decrease in RNA transcription. Mutations that give
rise to RNA that is not processed or translated
properly will also reduce the synthesis of normal
product. The severity of the disease caused by a
mutation varies depending upon whether the
mutation results in a reduction in gene product
synthesis or the production of a modified product,
upon the degree of dysfunction or the modified
product, and upon the gene dosage (Table 10.4). In
addition, there is considerable between-family and
between-individual variability in the severity of
disease caused by the same mutation (Wolf 1997).
Molecular diagnostic studies
A mutation produces an abnormal segment of
DNA. If transcription of the DNA is possible, the
messenger RNA (mRNA) transcribed will be abnor-
mal as will the protein that is translated. The
laboratory diagnosis of a genetic disease caused by a
mutation is accomplished by demonstration of the
abnormal DNA, of the abnormal mRNA, or of the
abnormal protein product. Subsequent sections of
this chapter (Genetic disorders of metabolism,
Genetic disorders of plasma proteins, and Genetic
disorders of blood cells) describe ways in which
some genetic diseases can be diagnosed by evalua-
tion of protein products. The techniques used to
evaluate mRNA and DNA for evidence of a
mutation are referred to as molecular diagnostic
studies.
The first step for many molecular diagnostic
studies is amplification of the nucleic acid target
sequences by use of the polymerase chain reaction
(PCR). This step, which generates a large amount
of target DNA, is included as a means of achieving
or improving the detectability of the analytic signal
(Eisenstein 1990, Ferrari
et al.
1996). PCR uses
two oligonucleotide primers, one that binds at or
near the 3’ end of the target sequence on the sense
strand of the DNA and one that binds at or near the
3’ end of the target sequence on the antisense strand
of the DNA. A reaction mixture containing the
DNA and an excess of the primers is heated to a
temperature at which the double-stranded DNA
dissociates into single-stranded DNA. Upon cool-
ing, the primers hybridize to their complementary
sequences on the single DNA strands. Also present
in the reaction mixture is a thermostable DNA
polymerase (e.g., the
Taq
polymerase). Starting
from the ends of the bound primers, the polymerase
catalyzes the synthesis of new DNA strands using
the original DNA strands as the templates. The
mixture is then heated and cooled again followed by
a second round of primer-directed DNA synthesis.
This cycle is repeated numerous times with the
amount of target DNA doubling with each cycle.
After a few cycles, the major synthetic product is a
segment of DNA that contains only the target
sequence, that is, the DNA between and including
the primer binding sites.
mRNA sequences can also be amplified using a
variant of PCR called reverse transcriptase-PCR
(RT-PCR). In this technique, DNA that is comple-
mentary to the mRNA in a sample is synthesized
using a reverse transcriptase. The mRNA-DNA
product is then used as the nucleic acid source for
the fist round of the PCR. The primer for the
antisense target sequence hybridizes to the single-
stranded DNA leading to the synthesis of double-
stranded DNA. Subsequent cycles of the reaction
amplify this DNA.
PCR-based diagnostic techniques.
Besides
amplifying DNA for subsequent methodologic steps,
Genetic Disease
10-4
Table 10.3
Trinucleotide repeat syndromes
Repeat Location in gene
Gene product
Disorder
CAG open reading frame
huntingtin
Huntington’s disease
ataxin-1,2,3,6
spinocerebellar ataxia type 1, 2, 3, 6 respectively
atrophin
dentatorubral-pallioluysian atrophy
androgen receptor
spinobulbar muscular atrophy
CTG 3’ flanking region
myotonin
myotonic dystrophy
GAA intron
frataxin
Friedreich’s ataxia
CGG 5’ flanking region
FMR-1
fragile X syndrome
