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Vol. 13: Spring, 1996

Molecular Cytogenetics (FISH)

From the Editor and Authors:

This issue of Genetic Drift is the first in a series of two updates on the clinical application of genetic diagnostic technologies. The present issue opens with the basics of FISH (fluorescence in situ hybridization), from concept to application, and concludes with a discussion of regulatory issues and practical guidelines for use. The second issue in the series will deal with DNA diagnostic testing for single gene disorders. Our intent is to provide our primary care colleagues with practical working knowledge of these new tools - and we welcome your feedback. The Teratogen Hot Topic is Vitamin A intake during pregnancy. The issue concludes with information about the MSRGSN which produces this newsletter. Please contact the Editor if you would like the list of references for this issue.

This issue was spearheaded by Lisa J. Brothman (UT), with contributions from Loris McGavran, Ph.D. (CO), Kathy Richkind, Ph.D. (NM), Mary C. Lowery, Ph.D. (CO), Wendy Flejter, Ph.D. (UT), Lynda Fox, J.D. (CO), John Stone, Ph.D. (AZ), Art Brothman, Ph.D. (UT), and Lynn Martinez, (UT).

Carol Clericuzio, M.D. (NM),
Editor

Introduction to Molecular Genetics

Novel laboratory techniques are often the driving force behind new ideas in science. Certainly the use of cytogenetic studies for clinical testing literally exploded in the 1960s and 1970s because of three new techniques: 1) Peter Nowell discovered that the plant lectin phytohaemagglutinin could stimulate lymphocytes to grow in culture; 2) TC Hsu used hypotonic shock to prepare chromosomes so that they could be counted accurately; 3) T. Caspersson and Marina Seabright developed chromosome banding techniques that allow accurate pairing and identification of structural abnormalities. In the 1980s the techniques of molecular biology were applied to cytogenetic preparations.

We call this "hybrid" technology molecular cytogenetics, and it is transforming the way we study chromosomal changes in humans. It has improved the detection of, indeed in some cases defined, microdeletion syndromes. De novo derivative chromosomes in patients with chromosome imbalances can now be accurately characterized: the clinician now knows the identity of the partial monosomy and trisomy in these patients, and the molecular geneticist has accurate information from which to find genes relevant to the phenotype. The chromosomal origin of supernumerary markers can be identified and we now have a real potential of predicting phenotype for marker carriers, benign or adverse.

With molecular cytogenetic techniques, we can now detect some chromosomal abnormalities in nondividing cells with interphase nuclei. Standard cytogenetics requires actively dividing cells with metaphase nuclei. Some constitutional chromosomal abnormalities, and phenomena such as mosaicism and chimerism can be addressed without growing cells in culture or in cell types that do not adapt well to tissue culture.

Potentially, chromosomal aneuploidy studies (i.e. studies looking for abnormalities in chromosome number) can be done more quickly. Preserved, rather than fresh, tissue can be investigated for retrospective studies. We can ask questions of chromosome organization, its relationship to gene expression and tissue specificity in ways that were not possible a few years ago.

Does it replace standard cytogenetics? No, molecular cytogenetic techniques focus on specific chromosomes, chromosome regions, and unique DNA sequences or genes. Standard cytogenetic studies, including high resolution analysis, allow us to survey the whole genome for abnormalities of chromosome number or structure. But as complementary studies or for certain targeted abnormalities, molecular cytogenetic techniques expand our capabilities for making more accurate and refined cytogenetic diagnoses, both for constitutional abnormalities and acquired chromosomal changes in cancer cells.

Basic techniques

The most commonly used molecular cytogenetic technique is fluorescence in situ hybridization, or FISH. FISH is usually applied to standard cytogenetic preparations on microscope slides, but it can be used on slides of formalin-fixed tissue, blood or bone marrow smears, and directly fixed cells or other nuclear isolates. The basic principle of the method is that single-stranded DNA will bind or anneal to its complementary DNA sequence. Thus, a DNA probe for a specific chromosomal region will recognize and hybridize to its complementary sequence on a metaphase chromosome or within an interphase nucleus. Both have to be in single-strand conformation, therefore the DNA probe and the target DNA must be denatured, usually by heating them in a formamide-containing solution.

The probe is hybridized to the target DNA under conditions that allow the DNA to reanneal in double-strand form. Added to the hybridization mixture is an excess of repetitive sequence DNA to block non-specific binding of the probe to the target. After hybridization is complete (often after 2-18 hr at 37 C), the slides are washed in formamide-saline citrate solutions to remove excess or non-specifically bound probe. To detect the location of the probe on the target DNA, the probe DNA can be directly labeled with a fluorescent tag. It can also be chemically modified by the addition of hapten molecules (biotin or digoxigenin) that can then be indirectly fluorescently labeled with immunocytochemical techniques. The target DNA is counterstained with another fluorochrome of a complementary color.

The probe DNA can be observed on its target by using a fluorescent microscope with filters specific for the fluorochrome label and the counterstain. Special filters have been developed to allow simultaneous visualization of several fluorochromes. Digital cameras designed to detect low light level emissions and computer imaging are used to increase the sensitivity of probe detection. Because fluorescent dyes are subject to photobleaching (fading), the preparations are not permanent and must be stored away from light. Use of an antifade solution (phenylenediamine) has improved the capacity to observe and document fluorescently labeled samples.

Three different types of probes are typically used in clinical FISH studies:

Probes that bind to specific chromosome structures typically recognize repetitive DNA sequences, such as within centromeres (alpha satellite DNA ) or telomeric sequences. Within the repeat sequence are nucleotide patterns that are unique for specific chromosomes. There are now available centromeric probes that can identify most of the individual chromosome homologues. Similarly, there are probes that specifically recognize the telomeres (chromosome ends) of the long and short arms of many of the homologues, and more are rapidly being developed. Most alpha satellite centromeric probes give a large, bright signal and are useful for both chromosome identification in metaphase preparations and chromosome enumeration in interphase nuclei. Chromosome-specific telomere probes can be used for the above, for detection of cryptic translocations, and to define interstitial and terminal deletions.
Unique sequence probes hybridize to single copy DNA sequences in a specific chromosomal region or gene. In clinical cytogenetics these probes are usually referred to as cosmids, named for their cloning vector. These are the probes used to identify the chromosomal critical region or gene associated with microdeletion syndromes. On metaphase chromosomes, they hybridize to each chromatid, usually giving two small, discrete signals per chromosome.
Whole chromosome paints are cocktails of unique sequence probes that recognize the unique sequences spanning the length of a particular chromosome. At metaphase, both chromosome homologues are "painted" or fluoresce brightly. Among other applications, paint probes are used to define the chromosomal origin of derivative segments on translocation chromosomes or supernumerary markers.

A new nomenclature to describe FISH findings has been introduced by the standing committee for the International System on Cytogenetic Nomenclature (ISCN 1995). It is designed to be compatible with standard cytogenetic nomenclature and to provide precise information about FISH results. Many laboratories will be incorporating the new nomenclature into their reports, together with text to explain their findings.

The Genetic Drift Newsletter is not copyrighted. Readers are free to duplicate all or parts of its contents. The Genetic Drift Newsletter is published semiannually by the Mountain States Genetics Network for associates & those interested in Human Genetics. In accordance with accepted publication standards, we request acknowledgement in print of any article reproduced in another publication. The views expressed in the newsletter do not necessarily reflect local, state, or federal policy. For additional information, contact Carol Clericuzio, M.D., Editor, Department of Pediatrics, The University of New Mexico, Albuquerque, NM, 87131

Table Of Contents: Molecular Cytogenetics (FISH)
Introduction & Basic Techniques
Applications of FISH Technology
FISH Applications in Cancer Cytogenetics
FISH in Microdeletion Syndromes
FISHing in Unknown Waters
Regulatory Issues and FISH

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