Advances in Molecular Cytogenetic Techniques: Fishing for Answers - Wave of the Future
by Beth A. Pletcher, MD, May 1999

Over the past decade, tremendous advances have been made in the fields of molecular and cytogenetic diagnosis. As the Human Genome Project nears completion, with the expected comprehensive genetic map of man to be elucidated by 2003, the power for detection of human genetic diseases increases exponentially. Along with this intricate map goes the ability to identify both small deletions of genetic material previously too tiny to be detected (as in the past) through routine microscopy and single point mutations (genetic typos if you will) which can result in genetic disease or dysfunction. One of the molecular cytogenetic tools that will become clinically important in the future is the so called FISH (fluorescence in-situ hybridization) technology which, even in its current infancy, provides great insight into a number of genetic disorders.

Use of FISH requires advance knowledge of a very specific sequence or sequences in the area under study. Simply put, FISH utilizes a sequence of DNA (a unique genetic phrase) labeled with one of a variety of fluorescent "tags" to highlight or point out the genetic region of interest. The FISH probe, during the process known as hybridization, finds and attaches to its identical twin genetic sequence; not unlike a molecular search engine. Depending on the size and specificity of a given probe sequence, a variety of signals can be visualized. DNA probes can be developed that are specific to a certain chromosome or even a single gene as long as one knows the exact spelling of the sequence and that sequence or phrase doesn't occur in other areas of the genome.

The first widespread clinical application of FISH in prenatal patients enabled cytogeneticists to identify fetuses with common chromosomal trisomies or sex chromosome aneuploidies in a rapid assay. These probes can be applied to either dividing cells or cells at rest, unlike routine cytogenetic studies that require mitotic cells for analysis, and gives preliminary cytogenetic results in a day or two rather than one to two weeks. However, FISH in this context is still limited to identifying common chromosomal errors resulting from non-disjunction during meiosis and as currently applied will not detect other less common and yet potentially devastating cytogenetic abnormalities.

Further development of FISH technology is now enabling scientists and clinical cytogeneticists to do whole chromosome painting. Probes are now available that will label or paint entire chromosomes and because of the variety of fluorescent colors currently in-hand, all twenty-three chromosomes can be painted different colors. This is especially helpful when studying complex cytogenetic rearrangements such as those that occur in malignancies. Someday this painting strategy may also be used for evaluation of children with multiple congenital anomalies or other features of a cytogenetic disorder where routine cytogenetic analysis fails to uncover a deletion or rearrangement.

FISH is also being used in a clinical genetic setting to confirm diagnoses for a number of conditions that are caused by small deletions of genetic material (also called contiguous gene syndromes). Although FISH deletions may not be found in all affected individuals, it can augment clinical diagnosis and is being used routinely to evaluate individuals with features of: DiGeorge, velo-cardio-facial, Prader-Willi, Angelman, Williams, Miller-Dieker and Smith-Magenis syndromes. It is also quite useful for detecting submicrosopic deletions on the ends of chromosomes when a patient has clinical features of a chromosomal deletion but there are no findings noted with routine metaphase or prophase cytogenetic studies.

Perhaps one of the most promising uses of this technology in the future will be in the field of cancer cytogenetics. Already use of FISH to detect Her-2/neu gene amplification in breast tumor tissue has enabled oncologists to provide better prognostic information to patients. Many more probes have been developed to detect subtle and not so subtle cytogenetic changes in paraffin embedded tissues that assist clinicians in therapeutic decision making as well as assessment of tumor response to treatment. With the unraveling of the entire genetic code, the presence or absence of specific tumor markers, cytogenetic deletions or gene amplification will more directly impact the individualization of treatment strategies. In the future, FISH will enable clinicians to elucidate variable characteristics of any given tumor and address these subtle differences in a direct, therapeutically sound way. Because of the exquisite sensitivity of the FISH technology in the area of cancer genetics, detection of minimal residual disease and discovery of early relapses are now possible.