Chromosomes have been both understood and misunderstood for over the past century. As early as 1882, Walther Flemming was believed to have started the field of human cytogenetics by describing chromatin (chromosome) as the rod-shaped bodies that he microscopically saw in the nucleus of cells. He also coined the term mitosis as the division of cells. Both Theodor Boveri and Walter Sutton in 1903 independently hypothesized that units of heredity were carried on these (yet undefined) chromosomes. Knowing the importance of the human chromosome, the next logical step was to understand the true chromosome number in man. In 1912, Von Winiwater stated that he believed that women had 47 chromosomes and men had 48 chromosomes. And T. S. Painter, in 1923, showed that the “true chromosome” number was indeed 48. Given the crude equipment and methodologies that these investigators had at their disposal, it is laudatory that they were indeed so close.
The chromosome number in humans remained at 48 from 1923 to 1956; however, even during that time it became recognized that changes in chromosome number was probably responsible for phenotypic abnormalities. Several authors in the 1930s, including L. S. Penrose, believed that a change in chromosome number was the underlying cause of Down syndrome. In 1956, Tjio and Levan eloquently described the chromosome number in humans as 46 (Tjio and Levan, 1956). In their paper in Hereditas, they stated that “Before a renewed, careful control has been made of the chromosome number in spermatogonial mitoses of man we do not wish to generalize our present findings into a statement that the chromosome number of man is 2n = 46, but it is hard to avoid the conclusion that this would be the most natural explanation of our observations.” – a remarkable understatement for such a landmark finding. This finding did not occur in a vacuum as many advances in cytogenetics were used to make the chromosomes more readable. This included the use of colchicine as a mitotic inhibitor, the utilization of a fixative and, most notably, the development of a hypotonic solution, by T. C. Hsu in 1956, to help spread out chromosomes, making them more visible.
Up until this time, chromosome analysis was done using tissue preparations, such as testicular material from deceased prisoners that was used by Painter, or fetal lung fibroblasts as used by Tjio and Levan. Most of the work in 1956 consisted of cell culture establishment, colchicine pretreatment, hypotonic solution pretreatment, and squashing. However, the cell culture technology was an impediment to the better visualization of chromosomes, and as pointed out in 1932 by J. B. S. Haldane, “A technique for the counting of human chromosomes without involving the death of the person concerned is greatly to be desired”. In 1960, Nowell and Hungerford published their work on short-term cultures using lymphocyte cultures, which were preferable to long-term cultures. These were effective due to the discovery of phytohemagglutinin, extracted from the common navy bean, which allowed for the stimulation and growth of lymphocytes in culture. This work allowed for a glorious period of human cytogenetics in which a number of syndromes were shown to be chromosomal in origin including the cause of Down syndrome by Lejeune, Trisomy 13 by Patau, Trisomy 18 by Edwards, as well as Turner syndrome (Ford), and Klinefelter syndrome (Jacobs and Strong) (Lejeune et al., 1959; see also Article 11, Human cytogenetics and human chromosome abnormalities, Volume 1). However, at this point, all of the analysis and visualization was done using solidly stained chromosomes and their limited differentiation through the differences and size and centromere placement.
The next major breakthrough in the visualization of chromosomes came from the differential staining of chromosomes. In 1970, Caspersson and his colleagues published a landmark paper describing the identification of human chromosomes by DNA-binding fluorescent agents. Caspersson was not a cytogeneticist, but used his knowledge of chemistry to ultimately develop the first method of identifying each individual chromosome (Caspersson et al., 1970; Caspersson, 1989). This work utilized a fluorescent microscope and was difficult for many labs to utilize. Therefore, when Seabright (1972) published her paper on the rapid use or trypsin for producing a banding pattern that could be visualized by light microscopy, this technology was readily used by most laboratories to both visualize and characterize chromosomes. Many other banding technologies were developed and utilized to better understand chromosome structure and to define chromosome abnormalities. These include, but are not limited to, C-banding, G11-banding, NOR staining, R-banding, T-banding, and Cd-banding. While these were highly useful when developed, most have been superceded by a variety of DNA probes used with fluorescence in situ hybridization (FISH), as will be described below. The development of banding techniques again revitalized the field of cytogenetics and allowed for the visualization and characterizations of syndromes involving deletions (Cri-du-Chat; Wolf-Hirschhorn), duplications, reciprocal translocations (9/22 translocations), Robertsonian translocations, and inversions (recombinant 8 syndrome).
Since all of the individual chromosomes could now be visualized, it became obvious that at times two pairs of chromosomes showed different numbers of bands, which could not be accounted for the differential condensation of the chromosomes. If chromosomes were visualized in prometaphase rather than metaphase, it produced a higher resolution and allowed for the detection of abnormalities not seen in routine chromosomal analysis. Yunis first described this methodology in 1976 and continued to illustrate its usefulness in clinical cytogenetics (Yunis, 1976; Yunis et al., 1978; Yunis and Chandler, 1978). While the initial methodology focused on synchronizing the cell cycle in cultures using methotrexate, many different methodologies were used to either synchronize cultures (e.g., use of excess TdR) or to keep the chromosomes from condensing (e.g., the addition of ethidium bromide). Many submicroscopic deletions that are studied extensively today using FISH were initially described using high-resolution methodologies. These include the deletions seen in Prader-Willi and Angelman syndrome, the deletion in Di syndrome (see Article 17, Microdeletions, Volume 1), and the duplication in Beckwith-Wiedermann syndrome. This new methodology allowed for a higher resolution analysis of chromosomes, which most laboratories ultimately developed. New abnormalities and new syndromes were described, but cytogenetics mostly went into a period of senescence after the adaptation and utilization of this methodology.
Even after the introduction of high-resolution methodologies, it had become more apparent that there was a limitation to the resolution of chromosomes microscopically. In order to produce more resolution, many investigators tried to utilize a technique pioneered by Pardue and Gall in 1969, in which they utilized radioactive DNA for direct hybridization to chromosomes (Pardue and Gall, 1969). Their work was very successful as they utilized repetitive DNA for this hybridization. Other workers had varying success, and the technology was used in many instances for the chromosomal localization of known genes. However, this work was very time-consuming and tedious and was not ready to be used routinely by clinical laboratories. Simultaneously, both Pinkel and his colleagues and Ward and his coworkers were developing similar nonradioactive fluorescence technology. Pinkel et al.’s initial paper in 1986 opened the doors for the use of FISH for the study of chromosomes, although it took many years for laboratories to incorporate this technology into their routine work (Pinkel et al., 1986). Since its initial use, FISH has changed the face of cytogenetics, invigorated the field, and no laboratory can be considered complete without using the technology routinely (see Article 22, FISH, Volume 1).
Briefly speaking, FISH utilizes a DNA probe that has been labeled to hybridize to denature DNA on a slide. The initial probes were indirectly labeled with a hapten and then detected fluorescently. However, with advances in technology, almost all FISH is currently done using direct fluorescent labels. FISH can be utilized to study metaphase chromosomes or DNA in interphase cells whether from cultures or paraffin sections. As long as the DNA, that is to be studied, is not degraded, it can be analyzed using FISH. This technology has a phenomenal range in that not only can metaphase and interphase cells be studied but essentially any tissue with a nucleus can also be analyzed. Constitutionally, FISH is routinely used in interphase cells to rapidly detect aneuploidy and in metaphase cells to detect subtle deletions. The microdeletions (e.g., Prader-Willi syndrome), described above, that could previously only be detected with good high-resolution banding can now easily be detected with appropriate FISH probes. FISH has also been extremely useful in the delineation of structural abnormalities such as marker chromosomes, duplications, or derivative chromosomes by the simple use of or combination of alpha-satellite probes and/or chromosomes paints. More recently, a set of chromosome-specific subtelomere probes have been developed and used to detect cryptic rearrangements that have been missed with routine banding. Many of these rearrangements (either simple deletions or derivative chromosomes) have been too small to detect with routine banding, but contain enough loss or gain of genetic material to lead to phenotypic anomalies. It is estimated that between 2 and 6% of individuals with mental retardation/dysmorphic features without a recognizable G-banded anomaly may have a cryptic rearrangement. Thus, it is apparent that our resolution (and visualization) of chromosomes have been increased 20-fold from about 3 Mb with the analysis of banded chromosomes to approximately 150 kb with directed FISH analysis looking for specific DNA alterations.
This increased resolution is also important and easily seen with somatic changes in chromosomes associated with cancer (see Article 14, Acquired chromosome abnormalities: the cytogenetics of cancer, Volume 1). For these specimens, the development of probes has mainly involved resources for the detection of specific rearrangements associated with a variety of leukemias and lymphomas (see Article 24, Cytogenetic analysis of lymphomas, Volume 1). Again, as for constitutional abnormalities, these probes can be utilized in both metaphase and interphase preparations. Thus, while a bcr-abl fusion in chronic myelogenous leukemia can easily be detected routinely in metaphase G-banded chromosomes, FISH can be utilized in interphase cells (when appropriate metaphase preparations are not available) to detect the fusion. Similarly, probes for the TEL-AML1 fusion are routinely used to detect this change as the underlying 12/21 translocation cannot usually be detected analyzing the banded chromosomes. These are just two examples showing how FISH has changed, how chromosomes are studied, and what can be detected. More specific examples of its use can be seen throughout this topic.
Most of the FISH analysis described above and throughout the rest of the chapters in this topic has been possible because of the advances in technologies and the development of a series of commercial probes for these FISH studies. Most work in clinical laboratories, whether doing subtelomeric analysis or studying specific translocations in cancer, is done with prepared commercial probes. However, the study of the Human Genome has been a veritable boom for cytogeneticists. The resources produced in the elucidation of the genome allows for the study of any region of chromosomes. As almost the entire genome has been sequenced, there are BAC probes readily available, for any location in the genome, to delineate any structural change or better describe a chromosome anomaly. These probes have now easily been used to show cryptic deletions and the complexity of changes in chromosomes and have immensely improved our detection of chromosome changes. Again, however, most of these analyses need to be used as a directed study of abnormalities in that specific areas can be studied as desired. However, it is the development of these resources that has led to the next milestone in cytogenetics, the utilization of comparative genomic hybridization with BAC arrays to detect and study chromosome anomalies. Comparative genomic hybridization has been utilized for many years (see Article 24, Cytogenetic analysis of lymphomas, Volume 1). In its inception, it is a technology where you take DNA from a specimen you are interested in studying, label that DNA, and then hybridize to normal metaphase chromosomes. Utilizing this technology, you could determine if there were any excess or deficiencies in the DNA sample studied. While this is a good technique in most hands, its resolution was limited to about 10 Mb, not necessarily adding any resolution to routine banding. However, with the delineation of the genome, the DNA of interest can be labeled and hybridized against a BAC array. This array can contain BACs that are 1 MB apart, allowing for a resolution of this magnitude, not just for an area of interest but for the entire human karyotype (Fiegler et al., 2003; Greshock et al., 2004; Bignell et al., 2004; Ishkanian et al., 2004). While this work is still in the research stage, the potential it can provide is enormous and soon will allow visualization of the chromosomes past the bands and to the DNA backbone. These advances will not only allow us to have greater visualization of chromosomes but also allow for a better understanding of chromosome abnormalities over the next 100 years.
