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Molecular Cytogenetics (FISH) Vol. 13: Spring, 1996 |
Syndromes such as Prader-Willi syndrome or Smith-Magenis syndrome were first characterized as chromosome deletion syndromes by high resolution cytogenetic analysis of prometaphase chromosomes. These deletions are small and often difficult to detect, or in the case of Williams syndrome, undetectable on prometaphase chromosomes. The term microdeletion syndromes is commonly applied to these.
Cosmid probes have been developed that recognize unique DNA sequences within the critical region of chromosomal deletion for several of the microdeletion syndromes. These probes, and their commercial availability, has made the laboratory diagnosis of these syndromes more accurate. In some cases, such as Velocardiofacial/DiGeorge syndrome, the prevalence of the deletion 22q11.2 both de novo and familial, in the population had not been appreciated.
In many labs, FISH is now the test of choice to document these deletions because it is more accurate, and arguably faster to perform. The tests are performed on metaphase chromosomes. Usually the microdeletion cosmid (locus-specific) and a second cosmid (chromosome-specific) are hybridized together to insure identification of the chromosome and to provide an internal control.
Combinations of FISH probes and standard cytogenetics can characterize structural rearrangements and marker chromosomes. Take the example of a patient identified in the newborn period who has multiple congenital anomalies consistent with possible trisomy of the long (q) arm of #10. Standard cytogenetic studies demonstrate a structural abnormality of 10q: it is twice the length of the normal homologue. A chromosome #10 whole chromosome paint hybridizes to the normal #10, and to most, but not all of the abnormal #10. This helps to confirm a duplication of chromosome 10 material, and the G-banding pattern is consistent with 10q.
What about the rest of the chromatin that does not hybridize with the paint? Again, by the banding pattern, there is a suggestion that part of chromosome 18 is present. An alpha satellite probe for #18 centromere hybridizes to the abnormal #10. We have a dicentric! Chromosome 18 paint confirms that there are unique, #18-specific sequences present, and the banding pattern suggests 18p and proximal 18q. This child is in fact trisomic for 10q, but also for part of chromosome 18.
This kind of diagnosis would not have been confirmed prior to the advent of clinical molecular cytogenetics. FISH is also teaching us to expect the unexpected: abnormalities are often more complex than they first appear.
The autosomal trisomies 21, 13, and 18, together with sex chromosomal aneuploidies, are responsible for well over 90% of the chromosomal disorders found in human conceptuses. They are easily diagnosed by standard metaphase cytogenetic studies of chorionic villus samples (CVS), amniotic fluid cells, and peripheral or cord blood lymphocytes, or other tissue that can be grown in tissue culture.
Chromosome-specific cosmids or alpha satellite probes can be used in FISH studies to document chromosome gain or loss in cells that are not dividing. Interphase cytogenetics using FISH is a rapidly growing field. One can enumerate the number of fluorescent signals present in interphase nuclei, for instance using an alpha satellite probe for the centromere of chromosome 18, or a unique sequence cosmid for a locus on chromosome 21.
Some laboratories offer interphase FISH aneuploidy studies of prenatal samples and for newborns suspected of having an autosomal trisomy.
Currently, these studies are used for screening, not as a stand-alone diagnostic test. It is the opinion of most geneticists that interphase FISH studies must be performed in conjunction with standard cytogenetic testing for clinical diagnosis. The results must be interpreted with caution, and appropriate controls must be developed.
We cannot always extrapolate from signal number to the type of chromosome abnormality: three #18 alpha satellite signals in 80 of 100 nuclei may signify Trisomy 18, but we would see the same pattern with an extra isochromosome 18p. We can say that the signal pattern is consistent with a chromosomal abnormality of #18. In this example, have we documented mosaicism? What about those other 20 cells? Most likely, hybridization was not complete in these cells, and not all cells are informative. We must beware of overinterpretation.
Interphase FISH can be applied to noncultured cells and fixed tissue. An interesting use is the detection of tissue limited mosaicism of an aneuploid cell line, for instance in placental tissue or in buccal mucosa. Again, the technology is driving a new appreciation of the extent of chromosomal mosaicism to be found in humans.
The study of the acquired chromosomal abnormalities in cancer cells continues to expand. Cancer cells are often difficult to grow in culture and FISH techniques can augment standard cytogenetic testing. Numeric changes such as trisomy 8 in myeloid disorders can be detected by interphase FISH. Certain translocations, such as the bcr/abl rearrangement in chronic myelogenous leukemia and acute lymphoblastic leukemia can be detected by FISH on both metaphase preparations and interphase nuclei.
FISH studies are being used to look for early relapse and residual disease in nondividing cells. In allogeneic bone marrow transplant patients who received opposite sex donor cells, the success of engraftment can be monitored by FISH chimerism studies. Differentially labeled X- and Y-specific probes can be used to detect the proportions of XX to XY cells in bone marrow or peripheral blood nuclei in a dual color FISH procedure. By combining immunocytochemical detection of cancer cells and FISH techniques, chromosomal abnormalities and cell type can be simultaneously studied.
FISH is used to map genes to specific chromosomes and chromosomal regions. The order of genes or gene sequences within a chromosome can be established by labeling the probes with different fluorochromes and detecting their hybridization color pattern. In a new technique called fiber FISH, chromosome-specific chromatin fibers can be spread on a slide and then hybridized with locus-specific probes to allow fine resolution mapping of DNA sequences.
Comparative genomic hybridization (CGH) is a molecular cytogenetic technique that combines FISH and standard cytogenetics to look for genome-wide DNA amplification and deletions in tumor cells or cells of a patient with a possible chromosomal disorder. Its value is that unlike standard cytogenetics, cell culture is not necessary. Like standard cytogenetics, chromosome gains and losses, and amplification or deletion of chromosome regions can be detected without having to selectively FISH for a specific chromosome or sequence. Its use is currently in the research lab. Issues of sensitivity and reliability are being addressed. However, as with other molecular cytogenetic techniques, one year's experiment is next year's clinical test.
Contributed by Loris McGavran, Ph.D. (CO)
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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