J
Am Acad Child Adolesc Psychiatry,38:9, 1200-02 September 1999
by David Ward, Ph.D., Patricia Bray-Ward, Ph.D., and Paul J. Lombroso, M.D.
It
is perhaps surprising to those not intimately involved in the field of
genetics to realize that the total number of human chromosomes was not
clearly established until 1956. However, over the past 43 years, cytogenetics
has undergone a spectacular evolution involving both cytological and molecular
techniques. A major breakthrough was the development in 1970 of the first
chromosomal banding technique, called quinicrine banding. This provided
the ability to identify and enumerate the 46 individual human chromosomes
as well as to detect translocations, inversions, regions of amplification,
deletions, rearrangements, insertions, and other chromosomal abnormalities.
Very quickly, a number of additional banding techniques were developed.
Today, Giemsa banding of metaphase chromosomes is the gold standard for
karyotypic analysis. The
advent of recombinant DNA cloning techniques in the mid 1970s
provided an approach to identify genes associated with specific genetic
disorders. Cloning made available chromosome-specific repetitive DNA probes
and single-copy sequence probes that could be hybridized to chromosomes
to detect genetic changes at the molecular level. The development of chromosome-specific
libraries by flow cytometry, the introduction of nonisotopically labeled
probes, and the advent of methods to suppress hybridization signals from
ubiquitous, repetitive sequences, rapidly led to high-throughput gene
mapping. Furthermore, the development of interphase cytogenetics permitted
the analysis for extra chromosomal copies, loss of heterozygosity, and
translocations in interphase nuclei as well as metaphase chromosomes.
One
of the steps in isolating a gene of interest is to know exactly where
it lies on the human genome. Fluorescence detection methods, which rapidly
superseded isotopic detection, are the most sensitive and efficient means
for detecting mapping.Furthermore, the development of interphase cytogenetics
permitted the analysis for extra chromosomal copies, loss of heterozygosity,
and translocations in interphase nuclei as well as metaphase chromosomes.
One of the steps in isolating a gene of interest is to know exactly where
it lies on the human genome. Fluorescence detection methods, which rapidly
superseded isotopic detection, are the most sensitive and efficient means
for detecting hybridized probes. Fluorescence in situ hybridization (FISH)
of single-sequence probes is used today for clinical testing as well as
for research (Fig.
1A). Increasingly, clinical cytogenetic laboratories are using
disease-specific FISH probes in confirmational cytogenetic screening for
many microdeletion syndromes with recognizable phenotypes, e.g., cri-du-chat,
DiGeorge, and Williams syndromes.
FISH
has become an essential element in positional cloning strategies by providing
a bridge between 2 data sets: the clinical data on patients with characteristic
cytogenetic abnormalities and the information on the physical organization
of the human genome derived from the human genome project. The approximate
position of a chromosomal abnormality in a patient is first described
by cytogenetic band or by the distance from the p telomere. Using public
databases that are now available at a touch of a personal computer, human
genomic DNA clones are identified that have a similar physical location
and known genetic linkage association, providing the options of testing
patient DNA by analysis of neighboring genetic linkage markers or using
genomic DNA clones for further testing of patient chromosomes by FISH.
These
technologies have led to an intensive search for characteristic chromosomal
changes in individuals with neuropsychiatric disorders. The search for
the gene(s) within microdeletion syndrome regions has been successful
in several cases. For example, Williams syndrome is now known to result
from a deletion on chromosome 7, while Angelman and Prader-Willi syndromes
are most often caused by deletions on chromosome 15 .It is likely that
most neuropsychiatric disorders are due to single-base mutations that
are undetectable by in situ techniques. However, even a single translocation
case can lead to the identification of the disease-causing gene through
the identification of clones mapping to the translocation breakpoint.
This permits subsequent mutation analysis of patients with no detectable
cytogenetic abnormality. Reported chromosomal changes seen in individuals
with autism include small duplications (Xp22.322.2), small deletion (1q43),
and larger deletions (5q15-q22.3 or 18q). There is an increased incidence
of autism in individuals with 15q11.2-q13 duplications (Prader-Willi region).
Molecular analysis of such breakpoints has not yet led to the identification
of an autism gene, although this is being pursued in several laboratories.
Similarly, an increased incidence of schizophrenia has been reported to
be associated with abnormalities on X chromosome as well as with a variety
of autosomal rearrangements.
Another
advantage of FISH is that several spectrally distinct fluorophores can
be used simultaneously. By hybridizing sets of chromosome-specific DNA
probes, each labeled with a different combination of fluorescent dyes,
it is possible to ascribe to each chromosome a unique spectral signature
or identifier tag (Fig. 1B). Only 5 fluorophores were needed to distinguish
the 24 different human chromosomes. A banding probe labeled with a sixth
fluorophore can be used to give banding profiles similar to Giemsa. Karyotyping
by color has been shown to detect chromosomal abnormalities that are difficult
or impossible to identify by Giemsa banding.
An
alternative use of multiple fluors is that demonstrated by Lignon and
coworkers. They developed a probe set for 4 common deletion regions: Prader-Willi
and Angelman, Williams, Smith-Magenis, and DiGeorge/velocardiofacial syndromes.
Ten of 46 cases were found to have one of these microdeletions.
Recent
advances have described the development of telomere- specific probes that
can identify previously undetectable rearrangements at the tips of chromosomes,
termed telomeric translocations or deletions. A number of retardation
disorders including the ATF-16 syndrome, cri-du-chat, Wolf-Hirschhorn,
and Miller-Dieker syndromes may result from telomere translocation or
subtelomeric deletions.
The
recognition that telomere translocations or deletions might be a frequent
cause of mental retardation led to a key study carried out by Flint and
colleagues that addressed the question of what percentage of mentally
retarded patients with apparently normal karyotypes will show cryptic
translocations and whether these translocations are frequent in the nonretarded
population. They used 3 dozen variable number tandem repeat (VNTR) probes
to examine 28 (of 48 possible) subtelomeric regions. They studied 99 patients
with varying degrees of idiopathic mental retardation and apparently normal
karyotypes. Three of the patients had telomere monosomy, 1 on chromosome
13 and 2 on chromosome 22. Two of the cases of monosomy resulted from
unbalanced translocations, and 1 resulted from an interstitial or terminal
deletion. Considering that only half the telomeres were surveyed and that
many of the alleles were noninformative, they estimated the percentage
of cryptic subtelomeric monosomy in retarded individuals to be 6% (95%
confidence limits are 1.2%17.6%). In a survey to determine the rate of
telomere monosomy of chromosome 13 and chromosome 22 telomeres in normal
individuals, no rearrangements were found in more than 160 nuclear families
with 1,000 individuals tested. Recently, by use of one additional hypervariable
polymorphic probe using the same patient population, they identified an
additional telomeric deletion, bringing the current estimate of frequency
of unbalanced telomeric rearrangements to slightly more than 7%. These
observations strongly suggest that cryptic translocations are a major
contributor to the mental retardation phenotype, yet this possibility
has not been rigorously examined.
The
recent development of M-FISH and multiplex telomere screening 1assays
provides a novel opportunity to definitively establish the role of cryptic
translocations and subtelomeric deletions in the etiology of mental retardation
and other childhood neuropsychiatric diseases. Are there discrete classes
of genomic rearrangements associated with certain subpopulations of affected
individuals? How many different types of cryptic translocations or deletions
are associated with neuropsychiatric disorders? Are they chromosomally
clustered or distributive? Although several hundred genes have been postulated
to contribute to the mental retardation phenotype, only a limited number
have been actually identified. The new molecular cytogenetic tools that
have emerged within the past few years permit the exploration of these
patient populations in a fashion previously unattainable.
