Chromosome Charting Takes a Giant Step

As the Human Genome Project continues, researchers the world over are analyzing our genetic instructions, gene by gene. But the human genome can also be viewed from another level, the rodshaped chromosomes that carry the genes.

In the past few months, viewing chromosomes has taken a giant leap forward, thanks to fluorescence microscopy. Two groups of researchersñDavid C. Wardís group at the Yale University School of Medicine and Thomas Riedís team at the National Institutes of Health, in collaboration with Applied Spectral Imaging of Migdal Haíemek, Israel, and Carlsbad, Calif.ñhave used fluorescence in situ hybridization, or FISH, to distinctly label each of the 24 types of human chromosomes.

Applying FISH to human chromosomes elevates chromosome charting, or karyotyping, from an art to more of a science, because FISH uses fluorescent dyes tagged to DNA sequences to detect specific genes on chromosomes. Some earlier approaches to visualizing chromosomes were generalized in comparison.

The idea for FISH grew out of the work of two staff scientists at Lawrence Livermore National Laboratory in 1985. Joseph W. Gray and Daniel Pinkel were using a fluorescence-activated cell sorter to collect large numbers of a single type of chromosome and realized that the probes they were using to identify chromosomes could also be used in clinical applications. A patent was filed in early 1986 for a method of blocking nonfluorescent chromosome regions, which was key to the technology. Soon, Imagenetics Corp. in Naperville, Ill., developed clinical kits for research use that were marketed by Life Technologies Inc. of Bethesda, Md., under the name "whole chromosome paints." Today, Vysis Inc. of Downers Grove, Ill., markets research-use-only kits based on the Livermore probes to detect certain chromosomal anomalies.

In the early 1990s, a few companies developed diagnostic kits to scan for extra chromosomes 13, 18, 21, X or Y in fetal cells to diagnose prenatally the most common chromosomal problems. FISH could also reveal, literally at a glance, when two different chromosomes exchange parts, a type of abnormality called a translocation. But the holy grail remained simultaneous FISH probing of all 46 chromosomes.

Like any new kid on the block, FlSHing the human genome faces obstacles: both FDA approval and acceptance by laboratories accustomed to the existing technology. For example, prenatal FISH tests must still be backed up with FDA-sanctioned tests.

To apply FISH probes to all the chromosomes, the Yale researchers used five fluorescent dyes: fluorescein and four cyanine dyes. In one experiment, they tagged 24 chromosome-specific DNA probes with different combinations of these fluorophores. For example, they marked chromosome 21 with fluorescein, Cy3.5 and Cy5; chromosome 6 with fluorescein and Cy3.5; and the X chromosome with Cy3 and Cy3.5. They have since added a sixth fluor, increasing discrimination. Ward terms this combinatorial approach M-FISH, for multiplex-FISH.

The key to the techniqueís ability to clearly distinguish between the chromosome marking fluors is in analyzing the excitation and emission spectra to select laser excitation sources and filter sets that avoid overlapping wavelengths. Software assigns spectral signatures for each probed chromosome, translates these into gray values, then "paints" each chromosome with a pseudocolor.

But thatís just the beginning, said Ward, who foresees combining M-FISH with old-fashioned banding. "The computer can identify each chromosome according to its profile. Then it can make them black and white and lay on top of that a banding pattern," he explained.

The approach clearly depicts chromosome abnormalities. "For example, if there is a translocation," Ward adds, "the computer draws a line across the breakpoint. Above the breakpoint it corresponds to chromosome 8 and below to chromosome 10." The Yale technique produces a chromosome chartñfrom properly prepared cellsñin about five minutes. Applied Spectral Imagingís approach uses an interferometer. It takes only one minute but requires more equipment and is costlier. "It is a microscope system that gives spectra for each pixel element in a CCD image. A computer program looks at the interference patterns and gets the wavelength of the light at that pixel. Two fluors at a particular element of a picture give a spectrum of components that made up the signal," says Ward.

The company calls its approach SKY, for "spectral karyotyping." The interferometer system, called the SD-200 Spectral Bio-Imaging System, costs nearly $90,000, compared with the $2000 to $3000 that Ward estimates the Yale system will cost when it is commercialized.

Prenatal testing is an obvious potential market for M-FISH, SKY or whatever it is eventually called, but it will be an uphill battle against well-entrenched technology. A more immediate market may be in cancer diagnosis, prognosis and treatment monitoring.

Fewer than 10 percent of the 1.2 million cases of cancer diagnosed each year are scrutinized for chromosome abnormalities or specific cancer genes. That will change as the Human Genome Project identifies the genetic underpinnings of many malignancies. Leukemias and lymphomas are the cancers most often linked to abnormal chromosomes. In fact, one of the earliest landmarks in cytogenetics, in 1960, was the association of a translocation of a piece of chromosome 9 to chromosome 22 with chronic myelogenous leukemia (CML)

Today, several chromosomal clues to cancer type and progression are recognized, if not completely understood. For example, the outcome of CML is more favorable if the patientís cells show extra chromosomes 4 and 10 than if they have extra chromosome 8s.

FISH promises to add to the list of chromosome clues to cancer that conventional chromosome banding has already compiled. For example, a cell line of a type of head and neck cancer eluded conventional techniques because cells would typically have anywhere from 48 to 62 chromosomes. M-FISH identified a prevalent karyotype of 49 chromosomes, with the genome riddled with translocations. And FISH revealed a previously unknown translocation associated with childhood B-cell acute lymphoblastic leukemia. Because the translocation occurs between two chromosomes of similar size and with similar banding, conventional banding had failed to pick it up.

FISH has a bright future in cancer management. Dr. Thomas Look, chairman of experimental oncology at St. Jude Childrenís Research Hospital in Memphis, Tenn., calls FISH "one of the best ways to detect genetic abnormalities in childhood cancers that are important for predicting how the patient will respond." We will be hearing more about it.

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