6.1: Karyotypes and cytogenetics

Karyotypes

The entire chromosome set of a species is known as a karyotype, which can be thought of as a global map of the nuclear genome. Karyotyping is the process by which the condensed chromosomes of an organism are stained and photographed using light microscopy. Karyotyping can be used to determine the chromosome complement of an individual, including the number of chromosomes and any abnormalities.

Karyotypes describe the chromosome count of an organism and what these chromosomes look like under a light microscope. Attention is paid to their length, the position of the centromeres, banding pattern, any differences between the sex chromosomes, and any other physical characteristics. The preparation and study of karyotypes are part of the larger field of cytogenetics. The field of cytogenetics involves the study of inheritance in relation to the structure and function of chromosomes. Thus, karyotyping is a fundamental process within this field.

The study of whole sets of chromosomes is sometimes known as karyology. The chromosomes are depicted (by rearranging a photomicrograph) in a standard format known as a karyogram or idiogram: in pairs, ordered by size and position of centromere for chromosomes of the same size, as shown in Figure \(\PageIndex{3}\).

Figure \(\PageIndex{3}\): Human Karyogram. The National Cancer Institute

The basic number of chromosomes in the somatic cells of an individual or a species is called the somatic number and is designated 2n. In the germ line (the sex cells) the chromosome number is n (humans: n = 23). Thus, in humans 2n = 46. In normal diploid organisms, autosomal chromosomes are present in two copies. There may, or may not, be sex chromosomes. Polyploid cells have multiple copies of chromosomes and haploid cells, usually, gametes, have single copies. Karyotypes can be used for many purposes; such as to study chromosomal aberrations, cellular function, taxonomic relationships, medicine and to gather information about past evolutionary events (karyosystematics).

During the chromosomal staining processes used to produce a karyotype, the staining intensity along the chromosome can vary due to localized sequence and structural differences. These banding patterns are an inherent characteristic of a chromosome and can be utilized as a diagnostic tool. Typically, karyotypes are prepared from cells that are actively undergoing mitosis. The mitotic progression is blocked in prometaphase or metaphase when chromosomes exist in their most condensed state. The cells are lysed, but the nuclei are retained intact and are subsequently treated with a chemical fixing agent. Once fixed, a number of different types of stains can be used to visualize the chromosomes.

One of the first types of chromosomal staining procedures developed was known as Q-banding, which was developed in 1970 by Torbjorn Caspersson. This technique uses the DNA-alkylating dye, quinacrine, which forms a covalent link with the DNA. Researchers noted that the staining patterns resulting from this technique were consistent and repeatable, demonstrating that banding patterns can be used to identify and characterize individual chromosomes. Giemsa dye, as shown in Figure 24.3, is more commonly used today, as it can be used with bright field microscopy and produces high detail banding patterns. A specific technique, called G-banding uses Giemsa staining following the treatment of mitotic chromosomes with the protease, trypsin. Pre-treating the sample with trypsin before staining causes the partial breakdown of chromosomal proteins leading to chromosomal relaxation. This allows more thorough staining of the chromosomes when treated with Giemsa dye. When the chromosomal region is more tightly packed into heterochromatin, it tends to stain more darkly with the Giemsa dye, than the more lightly packaged euchromatic regions. Heterochromatic regions tend to have higher A-T content and don't contain as many gene regions as euchromatic regions. Euchromatic regions stain more lightly with G-banding. Other types of staining with Giemsa, include R-banding or Reverse-banding, which involves heating the DNA before staining. This is thought to cause the melting of A-T-rich regions, reducing the Giemsa staining, when compared to G-C-rich, gene-containing regions of the chromosomes.

 

Figure \(\PageIndex{4}\): Female Down Syndrome Karyotype Demonstrating Trisomy 21. (Karyotype prepared by Fatma Soylemez). Image from: Soylemez, F. (2014) Chapter in Health Problems in Down Syndrome.

When visualizing a karyotype, the chromosomal images are aligned so that heterologous chromosomes are paired together and positioned such that the p-arm (short arm) is on top and the q-arm (long arm) points downward. Karyotypes can be used to quickly identify gross chromatic abnormalities that are larger than a few megabases in difference. This includes abnormalities such as aneuploidy (the addition or absence of an entire chromosome), or translocations (the transfer of part of a chromosome to a neighboring chromosome), as shown in Figure \(\PageIndex{4}\) and Figure \(\PageIndex{5}\).

Figure \(\PageIndex{5}\): Karyotype of a Translocation (A) G-banding karyotype of a patient with t(15;22) translocation. Chromosome 22 monosomy with an unbalanced translocation is observed. The red arrows indicate the abnormal chromosomes. X indicated total monosomy. Original magnification, x110. (B) Ideogram and partial G-banding karyotype showing a more defined image of the translocation of the q-arm of chromosome 22 to chromosome 15. Image from: Gug, C., et al (2018) Exp & Ther Med 16(4) 6609

The telomeric regions of chromosomes can also be identified using fluorescent staining techniques, as shown in Figure \(\PageIndex{6}\). The structure of telomeric chromosomal regions is described in section 24.3.

Figure \(\PageIndex{6}\): Telomere Staining. Image from: U.S. Department of Energy Human Genome Program

More recently, techniques such as chromosome painting, use fluorescently labeled probes to hybridize with specific chromosomes or even specific gene regions of a chromosome. Karyotypes originating from this technique are called spectral karyotypes. This technique can be especially useful in identifying translocations that have occurred in human cells, as shown in Figure \(\PageIndex{7}\).

Figure \(\PageIndex{7}\): Spectral karyotyping (SKY) analysis of a fibroblast cell line derived from lung tissue (WI-38T) Different strains of WI-38T were developed and two showed high proliferation rates typical of the premalignant state. (A) shows SKY analysis for WI-38T and the two high proliferation strains. Both high proliferation strains show translocations of chromosome 17 to the X chromosome. (B) shows specific staining of WI-38T(HP-1) for the 17q25 region of chromosome 17 (pink) which is visible on both copies of chromosome 17 and also on one of the X chromosomes, indicating the translocation. (C) shows a graphic image of the translocation. Image from: Buganim, Y., et al (2010) PLoS ONE 5(3) e9657