CYTOGENETICS: HISTORY, WHAT IT STUDIES, TECHNIQUES, APPLICATIONS - BIOLOGY - 2025

The cytogenetic is the study of the morphology, structure and function of chromosomes, including their changes during somatic cell division, or mitosis, and during reproductive cell division, or meiosis.

Cytology also studies the factors that cause chromosomal changes, including pathological ones, which appear from one generation to another, and evolutionary ones, which act over many generations.

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History

The memorable years and events in the history of cytogenetics are as follows:

- In 1842, Karl Wilhelm von Nägeli observed “transient stem cells”, later called chromosomes.

- In 1875, Eduard Strasburger identified chromosomes in plants. In 1979, Walther Flemming did it in animals. Flemming coined the terms chromatin, prophase, metaphase, anaphase, and telophase.

- In 1888, W. Waldeyer coined the term chromosome.

- In 1893, Oscar Hertwig published the first text on cytogenetics.

- In 1902, Theodor Boveri and Walter Sutton discovered homologous chromosomes.

- In 1905, Nettie Stevens identified the Y chromosome.

- In 1937, Albert Blakeslee and AG Avery stopped the metaphase with colchicine, greatly facilitating the observation of chromosomes.

- In 1968, Torbjörn Caspersson et al. Described the Q bands. In 1971, Bernard Dutrillaux and Jerome Lejeune described the R bands.

- In 1971, C bands were discussed at a conference on human chromosome nomenclature.

- In 1975, C. Goodpasture and SE Bloom described Ag-NOR staining.

- In 1979, Jorge Yunis described the high resolution methods for G bands.

- In 1986–1988, Daniel Pinkel and Joe Gray developed the FISH (fluorescent in situ hybridization) technique.

- In 1989, Hermann ‐ Josef Lüdecke microdissected chromosomes.

- In 1996, Evelyn Schröck and Thomas Ried described multichromatic spectral karyotypic typing.

Discoveries in humans

In 1914, Theodor Boveri suggested that cancer could be due to chromosomal changes. In 1958, Charles E. Ford observed chromosomal abnormalities during leukemia.

In 1922, Theophilus Painter published that humans have 48 chromosomes. It took until 1956 for Jo Hin Tjio and Albert Levan to establish that they actually have 46 chromosomes.

In 1932, PJ Waardenburg suggested, without proving it, that Down syndrome could be the result of a chromosomal aberration. In 1959, Jerome Lejeune demonstrated the presence of an extra somatic chromosome in patients with Down syndrome.

Also in 1959, Charles E. Ford reported that women with Turner syndrome lack one of the two X chromosomes, while Patricia Jacobs and John Strong discovered the presence of an extra X chromosome in men with Klinefelter syndrome.

In 1960, JA Böök and Berta Santesson described triploidy, Klaus Patau described trisomy 13, and John Edwards described trisomy 18.

In 1969, Herbert Lubs First discovered Fragile X syndrome. That same year, amniocentesis began to be used for cytogenetic diagnosis.

Field of study

Cytogeneticists study the chromosomal evolution of living things, using karyotypes to do phylogenetic analysis and solve taxonomic problems.

In addition, they investigate epidemiological aspects of human chromosomal aberrations and the environmental factors that produce them, diagnose and treat patients affected by chromosomal abnormalities, and develop molecular approaches to decipher the structure, function, and evolution of chromosomes.

Chromosome morphology

Each chromosome is made up of two chromatids, held together by a constriction called the centromere. The sections of chromosome that start from the centromere are called arms.

Chromosomes are called metacentric when they have the centromere in their middle; submetacentric if they have it slightly away from the middle, so that the opposite arms are not of equal length; acrocentric if the centromere is close to one of the extremes; and telocentric if the centromere is just at one end of the chromosome.

Techniques: sample processing

The steps to take to process the samples are as follows.

Obtaining the sample

Acquisition of the required tissue, storing it in the medium and in suitable vials.

Culture

With the exception of samples for FISH analysis, a culture period of between one day and several weeks is required before harvest.

Harvested

It is the obtaining of cells in metaphase.

Stopping mitosis

Standard cytogenetic analysis requires halting mitosis so that cells remain in metaphase, using colchicine or Colcemid®.

Hypotonic treatment

It increases the volume of cells, which allows chromosomes to extend.

Fixation

3: 1 methanol – acetic acid is used to remove water from cells, hardening the membranes and chromatin for staining.

Sheet preparation

The fixed cells are spread on microscope slides, after which they are dried.

Chromosome staining

There are several staining methods to recognize differences between chromosomes. The most common is the G.

Microscopic analysis

Allows you to choose suitable cells to observe and photograph chromosomes.

Preparation of karyograms

Based on photographs of cells in metaphase, images of the set of chromosomes of a representative cell are composed for later study.

Chromosome bands

There are four types of chromosomal bands: heterochromatic bands; euchromatic bands, nucleolus organizing regions (NORs); kinetochores.

Heterochromatic bands appear as discrete blocks. They correspond to heterochromatin, which contains highly repetitive DNA sequences that represent conventional genes and are not decondensed at the interface.

Euchromatic bands consist of a series of alternating segments that are or are not affected by staining. These bands differ in size, forming distinctive patterns characteristic of each pair of chromosomes of a species, which makes them very useful for identifying chromosomal translocations and rearrangements.

NORs are those segments of the chromosomes that contain hundreds or thousands of ribosomal RNA genes. They are commonly visualized as constrictions.

Kinetochores are the binding sites of the microtubule spindle to chromosomes.

Chromosomal band staining

Chromosome banding consists of staining techniques that reveal patterns of longitudinal differentiation (light and dark regions) that could not be seen otherwise. These patterns make it possible to compare different species and study evolutionary and pathological changes at the chromosome level.

Chromosome banding methods are divided into those that use absorption staining, typically Giemsa pigments, and those that use fluorescence. Absorption staining methods require a preliminary physico-chemical treatment, as described in "Sample Processing."

Some types of banding allow the evidence of patterns of restricted regions of chromosomes related to functional properties. Others allow the visualization of differences between homologous chromosomes that make it possible to identify segments.

C bands

The C-band stains most heterochromatic bands, making it the universal technique to show the presence of heterochromatin in chromosomes. Other methods stain only part of the total heterochromatin, making them more useful than C-banding to differentiate between types of heterochromatin.

Q bands

Q-banding is the oldest staining technique. It owes its name to the use of quinacrine. It is effective regardless of the chromosome preparation method. It is an alternative method to G banding. It is rarely used, but its reliability makes it useful when the material is scarce or difficult to banding.

G bands

The G-band, based on the use of Giemsa and trypsin, is the most used today. It allows the detection of translocations, inversions, deletions and duplications. It is the most used method for the characterization of karyotypes in vertebrates, showing differences between chromosomes that cannot be distinguished based only on their morphology.

R bands

The R banding produces an inverse staining pattern with respect to the G banding (light R bands equal dark G bands and vice versa). The R band is particularly useful for highlighting the ends of chromosomes, which are slightly stained when the G band is used.

T bands

The T-band is a variant of the R-band in which there is no staining of most of the interstitial bands of the chromosomes, so that the terminal regions of the chromosomes are intensely stained.

Ag-NOR bands

Ag-NOR banding is used to locate NORs by silver staining. In Ag-NOR banding, inactive NOR genes may not be stained. Therefore, this banding is used to study changes in the activity of ribosomal genes during gametogenesis and embryonic development.

Fluorescent in situ hybridization (FISH)

FISH banding allows chromosomes to be visualized using fluorescently labeled probes. FISH technology allows karyotypic analysis of cells that are not dividing.

FISH banding allows the detection of specific DNA sequences in chromosomes, cells and tissues. Therefore, it can be used to detect chromosomal abnormalities that involve small segments of DNA.

FISH banding paved the way for two more sophisticated related techniques, known as spectral karyotyping (SKY) and multi-color FISH (M-FISH).

In SKY and M-FISH, fluorescent dyes are used, which together produce combinations of colors, one for each chromosome. These techniques have been very useful in detecting complex chromosomal aberrations, such as those seen in certain tumors and in acute lymphoblastic leukemia.

Medical applications

- Cytogenetics of cancer. Chromosomal aberrations and aneuploidy are common in tumors. Chromosomal translocations can have carcinogenic effects through the production of fusion proteins. Cytogenetics is used to monitor the progress of cancer treatments.

- Fragile sites and chromosome fracture. Fragile chromosome sites can lead to pathologies such as Fragile X syndrome. Exposure to cytotoxic agents can cause chromosome fracture. Carriers of certain autosomal mutations lack the ability to repair DNA damaged during chromosome fracture.

- Numerical abnormalities of chromosomes. The chromosome count can diagnose trisomies, such as the one that causes Down, Edwards and Patau syndromes. It also allows the diagnosis of Turner and Klinefelter syndromes.

- In chronic myelogenous leukemia, the white blood cells have a “Philadelphia chromosome”. This abnormal chromosome is the result of the translocation of chromosomes 9 and 22.

References

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