multiplex fluorescence
in situ
hybridization over
G-banding include improved accuracy in the detec-
tion and characterization of translocations and an
appreciable shortening of the time required for
performance of an chromosome analysis. Disadvan-
tages of the method include an inability to detect
paracentric inversions (those not involving the
centromere) or insertions involving a single chromo-
some arm and decreased accuracy in the detection of
small duplications and small deletions (La Beau
1996), including those giving rise to the microdele-
tion syndromes (Table 10.2).
Centromeric repeat probes hybridize to
chromosome-specific, tandemly repeated DNA
sequences present at the centromeres. Because the
DNA sequences are repeated, a number of copies of
the probe bind to the respective centromere, result-
ing in an intense fluorescence signal. For this rea-
son, centromeric repeat probes are especially useful
in the enumeration of specific chromosomes. For
instance, trisomy 21 can be efficiently evaluated in
cultured lymphocytes using a centromeric repeat
probe for chromosome 21. If trisomy is present,
three chromosomes will fluoresce in metaphase cells
and three fluorescent domains will be present in
interphase nuclei. A number of specific chromo-
somes can be enumerated if multiple probes, each
labeled with a different fluorochrome, are used.
This technique is called multicolor fluorescence
in
situ
hybridization.
Another use for multicolor fluorescence
in situ
hybridization is in the evaluation of chromosome
microdeletions. Microdeletions are not detected by
metaphase G-banding and are only sometimes seen
using high-resolution prometaphase G-banding.
They are readily identified using multicolor fluores-
cence
in situ
hybridization. For such investigations
two probes are used. One probe is either a centro-
meric repeat probe for the chromosome of interest or
a locus-specific probe for an uninvolved site on the
chromosome. The other probe is locus-specific for
DNA at the site of the microdeletion. In a
metaphase chromosome preparation, the first probe
will hybridize to both copies of the chromosome of
interest. If the suspected microdeletion is present,
the second probe will hybridize to only one of the
two homologous chromosomes. Similarly, if inter-
phase nuclei are studied, two fluorescent domains
will mark the hybridization of the first probe but the
second probe will show only a single fluorescent
domain.
DNA MUTATION
As used in this chapter, DNA mutation refers to
an alteration in DNA that is too small to be detected
by microscopy. The enormous variation in DNA
sequences found among humans (and other species)
arises from the accumulation of mutations over time.
Much of the variation is found only sporadically in
the population. Sometimes, however, a particular
gene variant may be present with a frequency that
exceeds that attributable to
de novo
mutational pro-
duction of the variant. Such gene variants are called
alleles and the genetic locus is said to show
polymorphism.
Mutations are caused by nucleotide substitution,
deletion or insertion of one or a few nucleotides,
deletion, fusion, duplication or insertion of large
nucleotide sequences, and expansion of trinucleotide
repeat sequences (Table 10.3; Reddy and Housman
1997). Single nucleotide substitutions are the most
frequent cause of mutation. When they involve
coding DNA, they are also further categorized
according to the effect of the mutation on the
transcription of the DNA. Silent mutations, which
usually involve the third base in a codon, do not
result in a change of the amino acid specified by the
involved codon. Missense mutations do cause a
change in the amino acid specification. Nonsense
mutations convert amino acid-specifying codons into
stop codons, resulting in truncation of the protein
product. Stop codon mutations have the opposite
effect, converting a stop codon into one specifying
an amino acid, thereby resulting in an elongated
protein product.
Mutations usually cause disease by reducing the
synthesis of a normal gene product or by replacing
the synthesis of the normal gene product with a
modified product that is not fully functional (Ravine
and Cooper 1997). Reduced synthesis of a normal
product results from mutations that cause a defect in
promoter function, a disruption of gene structure
Genetic Disease
10-3
Table 10.2
Microdeletion syndromes
Chromosome Disorder
7
Williams syndrome
15,maternal
Angelman syndrome
15,paternal
Prader-Willi syndrome
22
CATCH-22 syndrome
