A Brief Guide to Genetic Diagnosis

Principles

The diagnosis of a genetic disease requires a
systematic approach that takes clinical and
genetic considerations into account. Whereas
clinical medicine tends to classify diseases according
to organ system, age of onset, gender,
or primary method of detection (radiology,
imaging techniques), medical genetics, like
pathology, is oriented towards the basic cause
or lesion, in this case the gene or genes affected
by a relevant genetic change. Genetic diagnosis
is based on an interdisciplinary analysis of all
clinical and laboratory data from a genetic perspective.

Genetic diagnosis, a multistep procedure

The phenotype, which is the clinical manifestations
including individual and family history in
medical terms, is the starting point. The first
decision the medical geneticist must make is
whether a pattern of manifestations can be recognized.
Tools that can assist in this decision include
training and personal experience; appropriate
textbooks and other literature; and online
search systems such as OMIM, MEDLINE,
PubMed, POSSUM, London Dysmorphology
Data Base for congenital malformations, and cytogenetic
databases.

disease pattern can be recognized

If a disease pattern can be recognized, the next
decision concerns the category of disease. Although
difficult to establish in practice, the disease
category is important for the next steps to
be taken. For this purpose, the McKusick catalog
of human genes and diseases, Mendelian Inheritance
in Man (MIM) and its online version
(OMIM) are indispensable. The possibility of
genetic heterogeneity must be considered at
this stage. The term genetic heterogeneity refers
to a phenotype (disease) that has different
causes. A particular phenotype may be caused
by mutations at different loci (locus heterogeneity)
or by different mutant alleles at the same
locus (allele heterogeneity). All genetic diagnostic
procedures should be preceded by genetic
counseling, which properly includes obtaining
the (informed) consent of the persons involved.

Protein truncation test (PTT)

This is a test for frameshift, splice, or nonsense
mutations that leads to a truncated protein due
to an early stop codon created downstream of
the mutation. The truncated protein is detected
in an assay based on an in-vitro translation system.
The translation will be interrupted at a
premature stop codon resulting from the mutation.
The size of the newly translated protein is
determined by gel electrophoresis. PTT detects
the approximate location of the mutation as reflected
by the size of the mutant protein. PTT is
useful in studying genes with frequent nonsense
mutations, such as the APC, BRCA1, and
BRCA2 genes. However, it cannot be applied for
genes with frequent missense mutations.

Detection of Mutations without Sequencing

In addition to the detection of mutations by
different DNA fragments in Southern blots
(p. 62), there are methods based on differences
in the hybridization of mutated and normal segments
of DNA. Incomplete hybridization is
determined by using short segments of singlestranded
DNA (oligonucleotides) with a
sequence complementary to the investigated
region (see A). Other methods are based on
demonstrating incomplete hybridization with
mRNA (see B) or on the fact that a hybridized
segment of normal and mutant DNA is less
stable than normal DNA.

Detection of a point mutation by oligonucleotides

Short segments of DNA (oligonucleotides) are
used to determine whether there is a mutation
in a segment of DNA (1, normal DNA; 2, mutation
from G to A). An oligonucleotide is a synthetically
produced DNA segment about 20 nucleotides
long; its sequence is complementary
to a corresponding segment of the investigated
gene. It hybridizes completely with its complementary
segment (3). If a mutation, here
from G to A (1), is located in this region, hybridization
will not be perfect at this site (mismatch)
(4). On the other hand, an oligonucleotide
that is complementary to the DNA segment
with the mutation will hybridize
completely (allele-specific oligonucleotide,
ASO) (5). This hybridizes incompletely with the
normal DNA (6). By parallel use of both nucleotides,
mutant and nonmutant DNA can be
differentiated. The test results (7) show the hybridization
of mutated DNA and of control DNA
with the allele-specific oligonucleotides (ASO 1
for the control, ASO 2 for the mutation).

Demonstration of a point mutation

Demonstration of a point mutation
by ribonuclease A cleavage
The basis for this method is that a normal DNA
strand hybridizes completely with mRNA from
that region. Completely hybridized DNA and
mRNA are protected from the effects of the
RNA-splitting enzyme ribonuclease A (ribonuclease
protection assay). Hybridization is incomplete
in the area of a mutation. In this region,
mRNA will be cleaved by ribonuclease A
(RNAase A). This can be demonstrated by
Southern blot. There will be two fragments
formed that together correspond to the size of
the completely hybridized fragment