Cytogenetic approaches to studying chromosomes and their relationship to human disease have improved greatly over the past several decades. Modern cytogenetic approaches enable researchers to do the following, among other things:

  • Precisely label the chromosomal location of any gene using different colored dots
  • Examine cells from any type of tissue, even tumor cells
  • Identify cells that have lost or gained a specific chromosome, undergone a translocation event involving a specific set of chromosomes, or lost or gained a copy of a given gene or genes
  • Determine whether specific regions of chromosomes have been lost or gained without ever looking at the chromosomes under a microscope

Clearly, the field of cytogenetics has developed into a vital tool for studying and diagnosing human disease. But how did this field first emerge, and how did researchers develop the many different cytogenetic techniques that currently exist?

The Emergence of a New Field

The field of human cytogenetics was initiated in 1956, when the number of chromosomes in a diploid human cell was accurately determined to be 46 (Tjio & Levan, 1956). Since then, our knowledge of human cytogenetics and our ability to utilize cytogenetic data to understand and diagnose human disease has increased by leaps and bounds (Speicher & Carter, 2005; Trask, 2002).

As the field of human cytogenetics emerged, researchers began to develop methods to visualize chromosome structure and organization. Scientists quickly realized that not all chromosomes are created equal--specifically, they differ in their length and in the position of their centromere. Researchers also embarked on numerous studies to determine the relationship between human disease and chromosomes.

Early cytogenetic studies showed that an extra or missing copy of certain human chromosomes could lead to disease. For example, in 1959, an extra copy of chromosome 21 was shown to be associated with Down syndrome (also called trisomy 21) (Lejeune et al., 1959). In the same year, several abnormalities in sex chromosome number were linked to disease. In particular, Turner's syndrome was shown to be associated with the presence a single X chromosome and no Y chromosome (45,X) (Ford et al., 1959), whereas Klinefelter's syndrome was determined to be associated with the presence of two copies of the X chromosome and one copy of the Y chromosome (47,XXY) (Jacobs & Strong, 1959). Both Turner's syndrome and Klinefelter's syndrome affect sexual differentiation in affected invidivuals.

Using Cytogenetic Approaches to Map Genes

In addition to providing associations between chromosomal abnormalities and disease, cytogenetic approaches have also allowed researchers to map genes to particular chromosomes. For example, in 1968, Roger Donahue used new methods to study metaphase chromosomes in his own blood cells, and he noted that one of his copies of chromosome 1 had a region near the centromere that was loosely structured and uncoiled. Using his extended family pedigree and conducting biochemical tests to determine blood group markers, Donahue employed cytogenetic techniques to map the Duffy blood group locus to chromosome 1 (Donahue et al., 1968).

Shortly after the Duffy blood group locus was mapped, Maximo Drets and Margery Shaw established methods to stain metaphase chromosomes using a dye called Giemsa, which produces a signature banding pattern, called G-bands, for each of the 24 different human chromosomes (Drets & Shaw, 1971). G-banding patterns can be used to detect chromosomal translocations, deletions, and insertions, and they have made key advances in gene discovery possible. For instance, as previously mentioned, Rowley used G-banding patterns to determine that a translocation event involving chromosomes 9 and 22 was responsible for CML (Rowley, 1973). G-banding methods continue to be widely used today, though such approaches have certain drawbacks. For instance, G-banding requires metaphase chromosomes, which are easily obtained from blood samples but are more difficult to retrieve from solid tissue samples. Furthermore, metaphase chromosomes are highly condensed, which can lead to lower resolution in mapping.

Human-Mouse Somatic Cell Hybrids

Although cytogenetic approaches evolved over time such that chromosomes could be easily distinguished from each other, researchers also needed ways to study individual chromosomes in more detail. In an effort to meet this need, researchers used the Sendai virus to induce fusion between a human cell and a mouse cell, resulting in a human-mouse somatic cell hybrid that contained the complete mouse genome, as well as sparse numbers of human chromosomes (Ephrussi & Weiss, 1965; Harris & Watkins, 1965) . An extensive series of human-mouse hybrid cell lines that carried known combinations of human chromosomes was thus developed, and this series greatly facilitated the mapping of human genes to specific chromosomes prior to the advent of the Human Genome Project.

Using Flow Cytometry to Sort Chromosomes

Yet another advance in cytogenetic techniques involved the process known as flow cytometry, which was originally used to study distinct cell populations within a mixture of different cell types. With this technique, a fluorescent dye is used to specifically label the cell population of interest. Individual cells can then be examined one at a time as they are pulled through the flow cytometer and subjected to laser-diffracted light to determine cell size and shape. Fluorescently labeled cells can also be sorted into separate tubes, based on their size and the intensity of their fluorescence signal, using diffraction plates in a process called fluorescence-activated cell sorting (FACS).

Although flow cytometry and FACS were initially used to isolate populations of intact cells, researchers eventually adapted these techniques to isolate individual human chromosomes, as shown in Figure 1. Such techniques involve using mitotic cell suspensions and disrupting the cell membranes to release the condensed chromosomes that are labeled using two different types of fluorescent dyes (Carrano et al., 1979). The first dye, called Hoechst 33269, binds to A-T base pairs, and the second dye, called chromomycin A, binds to G-C base pairs.

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