|Year : 2016 | Volume
| Issue : 2 | Page : 35-40
Karyotyping: Current perspectives in diagnosis of chromosomal disorders
Suresh Kandagal Veerabhadrappa1, Pramod Redder Chandrappa2, Seema Yadav Roodmal3, Sharan J Shetty4, Gunjiganur S Madhu Shankari2, Kumbar P Mohan Kumar2
1 Faculty of Dentistry, SEGi University, Petaling Jaya, Selangor, Malaysia
2 Department of Oral Pathology and Microbiology, College of Dental Sciences, Davangere, India
3 Department of Oral Pathology and Microbiology, AJ Institute of Dental Sciences, Mangalore, Karnataka, India
4 Department of Periodontology, Mahatma Gandhi Mission's Dental College and Hospital, Navi Mumbai, Maharashtra, India
|Date of Web Publication||9-May-2016|
Suresh Kandagal Veerabhadrappa
Faculty of Dentistry, SEGi University, No. 9, Jalan Teknologi, Taman Sains, Petaling Jaya, Kota Damansara, Selangor - 47810
Source of Support: None, Conflict of Interest: None
A majority of the genetic abnormalities are directly related to the chromosomal aberrations. Cytogenetics is the diagnostic study of the structure and properties of chromosomes and cell division, which employs various methods, one of them being "karyotyping." It refers to a procedure of photographic representation of a stained preparation in which the chromosomes are arranged in a standard manner. The development of newer techniques such as "karyotyping" has made it possible to visualize undetected chromosomal anomalies such as small portions of chromosomes and translocations of tiny parts of chromosomes to one another. Because such procedures also enabled each pair of chromosomes to be distinguished individually, it has helped to further our understanding of the chromosomal basis of certain important genetic disorders. This review article highlights the importance of "karyotyping" and its importance in the diagnosis of chromosomal disorders.
Keywords: Chromosomes, cytogenetics, genetic, translocations, karyotyping
|How to cite this article:|
Veerabhadrappa SK, Chandrappa PR, Roodmal SY, Shetty SJ, Madhu Shankari GS, Mohan Kumar KP. Karyotyping: Current perspectives in diagnosis of chromosomal disorders. Sifa Med J 2016;3:35-40
|How to cite this URL:|
Veerabhadrappa SK, Chandrappa PR, Roodmal SY, Shetty SJ, Madhu Shankari GS, Mohan Kumar KP. Karyotyping: Current perspectives in diagnosis of chromosomal disorders. Sifa Med J [serial online] 2016 [cited 2022 May 16];3:35-40. Available from: https://www.imjsu.org/text.asp?2016/3/2/35/182000
| Introduction|| |
Living beings include variegate species that are distinct from one another. In the same species, every member has his/her own individuality, all owing to the genetic constitution of an organism - the chromosomes, the genes, and the DNA.  The blueprint for the formation and maintenance of an organism is provided by the DNA, which is packaged into chromosomes. Chromosomes are the factors, which distinguish one species from one another and which enable transmission of genetic information from one generation to the next. Chromosomes are the vehicles, which facilitate the reproduction and maintenance of a species. ,
Since a number of genetic abnormalities can be directly related to the chromosomal pattern, the characterization of chromosomes is of considerable diagnostic importance. This can be done by "cytogenetics." It is a photographic representation of a stained preparation in which the chromosomes are arranged in a standard manner and "karyotype" refers to the constitution of chromosomes of an individual.  In 1956, Tijo and Levan, Ford and Hamerton found that the normal human somatic cell contains only 46 chromosomes, and that maleness is determined by the presence of a "Y" chromosome, regardless of the number of "X" chromosomes in each cell.  The methods they used, with certain modifications, are now being used in "cytogenetic" laboratories to analyze the "karyotypes." In 1960, fibroblasts were used first for "karyotyping." 
| Procedure for Karyotyping|| |
I. Chromosome preparation: Source of chromosomes - any tissue with nucleated cells undergoing division can be used for chromosomal study:
- Peripheral venous blood - most commonly, the lymphocytes.
- Skin (fibroblasts), bone marrow.
- For fetal chromosome patterns - amniotic fluid cells, chorionic villi.
- 5-10 mL of heparinized venous blood is the most commonly used source. The heparin prevents coagulation, which would interfere with the later separation of lymphocytes. ,,
Culture: The blood cells are grown in a suitable culture medium containing phytohemagglutinin which, acts as a mitogen, stimulating the T-lymphocytes to divide and agglutinate the red blood cells (RBCs). The commonly used medium has 5 mL culture medium, 1 mL fetal bovine serum, and 0.2 mL phytohemagglutinin. Cultures are incubated at 37°C for 48-72 h.
Arrest of division: Mitosis is then interrupted at metaphase with spindle inhibitors such as colchicine (0.01%). Chromosome number, size, and shape at metaphase are species-specific - in nondividing cells, the chromosomes are not visible even with the aid of histologic stains for DNA or electron microscopy. During mitosis and meiosis, the chromosomes condense and become visible in the light microscope. Therefore, almost all cytogenetic work is done at metaphase. , The culture is incubated for 45 min. The contents of the vial transferred to a tube and centrifuged at 800 rpm for 5 min. Supernatant is discarded and mixed thoroughly.
Suspension in hypotonic solution: Prewarmed hypotonic saline is added to culture. This causes the RBCs to lyse. The osmotic swelling of the lymphocytes results in spreading of the chromosomes. It is incubated at 37°C for 5 min, centrifuged at 800 rpm for 5 min, and the supernatant removed.
Fixation: A freshly prepared fixative (3 parts of methanol and 1 part of glacial acetic acid) is added. Two changes of fixatives are given at intervals of 45 min.
Slide preparation: The cells resuspended in fresh fixative and slide are prepared by gently placing a drop of cell suspension on previously cooled cleaned slide and dried followed by staining. ,
II. Chromosome staining-banding techniques:
Numerous methods are available for identifying chromosomes and preparing karyotypes for diagnosis purposes. Banding patterns became the barcodes with which "cytogeneticists" can easily identify chromosomes, detect subtle deletions, inversions, insertions, translocations, fragile sites and other more complex rearrangements, and refine breakpoints. The ability to analyze chromosomes is dependent on the length of the chromosomes and how well they are fixed, spread, and stained. When a large number of cells has to be examined for clinical purposes, automatic scanning light microscopy with computer control and analysis can greatly facilitate the identification of chromosomal abnormalities.
A. General techniques: Some human chromosomes may be distinguished on morphological grounds alone, for example, the length of arms and position of primary and secondary constrictions. Autoradiography can also be used, especially for "S" phase identification and identifying chromosomes 4, 5, 13, 14, 15, 17, and 18.
Procedure: Suitable tissue preparations with a nuclear emulsion are done in a dark room, after which they are stored in the dark for several weeks and then are photographically developed and fixed. Discrete silver grains can then be seen over the sites that emit radiation; their position indicates sites of incorporation of the radioisotope. Such a preparation is termed as autoradiograph or a radioautograph. ,
Aceto-orcein method: This was the original staining technique, which permits the study of chromosome morphology. It is prepared by adding 1-2 g orecin to 45 mL hot acetic acid.
Technique: Add a few drops of stain to the prepared slide, lower coverslip, and apply gentle firm pressure with filter paper or glass rod. Remove excess stain by applying filter paper to the edge of coverslip. Chromosomes stain deep purple. This method is indelible and does not permit destaining and use of subsequent staining methods for banding. It is replaced by the banding techniques. ,
B. Banding techniques: First-banding technique was introduced by Caspersson (1969); it includes G (Giemsa) banding, Q (Quinacrine) banding, R (Reverse) banding, C (centromeric heterochromatin) banding, T (Telomeric) banding, and nucleolar organizing regions (NORs), high resolution (fine) banding. ,,
1) G (GIEMSA) BANDING: It is the most common method, which produces permanent slides that can be studied under a standard light microscope. It produces the same banding pattern as quinacrine with even greater resolution and does not necessitate the use of fluorescence microscopy. It can be used to pair and identify each of the human chromosomes accurately. ,
Prior to staining, the fixed chromosomes are treated with agents capable of denaturing chromosomal proteins such as proteolytic enzymes (trypsin, most commonly), salts, heat, detergents and urea. G-banding is most consistently produced by pretreatment of chromosomes with trypsin before staining with Giemsa. ,
Laboratory procedure: Slides for G-banding should be 1 day at room temperature or overnight at 50-60°C for optimal results.
- Incubate the slide for 20-40 s in 0.025% solution of trypsin in distilled water.
- Rinse thoroughly with phosphate-buffered saline (PBS) or distilled water.
- Stain in 4% buffered Giemsa solution for 5-10 min.
- Rinse slides in distilled water and air-dry.
- When the chromosomes are stained with Giemsa, a DNA-binding dye, after such treatment, G-bands can be seen with a light microscope.
- The G-bands constitute 300-400 alternate dark and light bands, which are characteristic for each chromosome and reflect differential chromosomal condensation. ,,,
Digital photograph of the entire metaphase spread is taken and both homologs of each chromosome pair are placed side by side in the numerical order for careful band-by-band analysis, which permits identification of relatively subtle changes in banding patterns caused by structural chromosome abnormalities  [Table 1].
R-banding: A pattern that is opposite of G- or Q-banding can be produced by various means and is referred to as reverse (R-) banding. Fluorescent R-banding patterns are produced by dyes with GC base-pair affinity such as chromomycin A3, olivomycin, and mithramycin. It is produced by subjecting slides to high temperatures followed by staining with Giemsa or acridine orange. R-bands have the advantage of staining the gene-rich chromatin, thus enhancing the ability to visualize small structural rearrangements in the parts of the genome that are most likely to result in phenotypic abnormalities.
C-Banding: C-bands localize the heterochromatic regions of chromosomes. Pardue and Gall (1970) first reported C-bands when they discovered that the centromeric region of mouse chromosomes is rich in repetitive DNA sequences and stains dark with Giemsa. C-banding is also useful to show chromosomes with multiple centromeres, to study the origin of diploid molar pregnancies and true hermaphroditism, and to distinguish between donor and recipient cells in bone marrow transplantation. 
T-banding: This method involves staining the telomeric (end) regions of the chromosomes. Slides are treated with phosphate buffer or Earle's balanced salt solution and then stained using mixed Giemsa solution to produce the t-bands.
CT-banding: In this method, slides are treated with barium hydroxide to stain both the centromeric heterochromatin as well as the telomere of chromosomes to produce the CT-bands.
Nucleolar organizing region (NOR) banding: This technique stains NOR located in the satellite stalks of acrocentric chromosomes and house genes for ribosomal RNA. NOR-bands represent structural nonhistone proteins that are specifically linked to NOR and bind to ammoniacal silver. It is useful in clinical practice to study certain chromosome polymorphisms such as double satellites and also helpful to identify satellite stalks that are occasionally seen on nonacrocentric chromosomes.
| High-Resolution Banding: High|| |
Resolution cytogenetics provides precision in the delineation of chromosomal breakpoints.
and assignment of gene loci, greater than with earlier banding techniques. This is achieved by synchronizing the lymphocyte cultures and obtaining more number of cells in prometaphase or even prophase (increasing resolution from 500 to over 1,000 bands in a haploid genome).
| Molecular Cytogenetics|| |
Fluorescent in situ hybridization (FISH): This technique allows the visualization of chromosomal location and nuclear location of specific DNA sequences and permits the detection of specific nucleic acid sequences in morphologically preserved chromosomes. It can be performed on either metaphase or interphase cells and involves denaturing genomic DNA by using heat and formamide. ,,
| Karyotype Analysis|| |
After banding/staining, the chromosome component of single cells is selected on the basis of representative morphology and chromosome number and photographed under a light microscope. Photographic representation of each individual chromosome is then cut out from such a photomicrograph and arranged so as to construct a "karyotype." , Analysis is done either by looking down the microscope under oil immersion or on a photograph and involves the following steps.
- The number of chromosomes is determined: Usually the total chromosome count is determined in 15-20 cells but if mosaicism is suspected, then 30 or more cell counts are undertaken.
- Chromosomes are assembled as homologous pairs: The homologous chromosomes pairs are arranged in a decreasing order of size to construct a "karyotype."
- Detailed analysis of the banding pattern of individual chromosomes: This is performed on both members of each pair of homologs in approximately three-five metaphase spreads. ,
The banding pattern of each chromosome is specific and shown in the form of a stylized ideal karyotype known as an idiogram.
- If the total count is 46, Group G is identified to diagnose the sex of the individual.
- If the count is more or less, the precise numerical abnormality is identified. Then, any structural abnormality present is identified.
- The chromosomes are usually identified in groups starting with Group G, and then Group D, Group F, Group E, Group A, Group B, and finally Group C.
- Identification of individual chromosomes is possible on the basis of total length of the chromosome, arm ratio, position of secondary constrictions and nucleolar organizers (satellites), subdivision of chromosome into euchromatic and heterochromatic regions, and characteristic banding patterns. ,
The "karyotypes" of different groups and species are compared, and similarities in karyotypes are presumed to represent evolutionary relationships and suggest primitive or advanced feature of an organism. ,,
| Normal Human Karyotype|| |
A human somatic cell contains 46 chromosomes, made up of 22 pairs of autosomes and a single pair of sex chromosomes, XX in the female, XY in the male [Figure 1].
| Classification of Chromosomes for 'Karyotyping'|| |
Chromosomes are arranged into seven groups based on size and centromere location. The centromeres can be found in the middle of the chromosome (median), near one end (acrocentric), or in between these first two (submedian) [Figure 2].
Group A: Chromosomes 1-3 are the largest with median centromere.
Group B: Chromosomes 4-5 are large with submedian centromere.
Group C: Chromosomes 6-12 are medium-sized with submedian centromere.
Group D: Chromosomes 13-15 are medium-sized with acrocentric centromere.
Group E: Chromosomes 16-18 are short with median or submedian centromere.
Group F: Chromosomes 19-20 are short with median centromere.
Group G: Chromosomes 21-22 are very short with acrocentric centromere.
Chromosome X is similar to group C.
Chromosome Y is similar to group G
Applications of "karyotyping" includes: ,
- Cytogenetic aspect of neonatally discovered congenital abnormalities and prenatal diagnosis.
- Mental retardation and neuropsychiatric disorders.
- Reproductive failure, endocrinology, gynecology, urology.
- Malignancy: Hematology and oncology.
- Mutagenesis: Environmental medicine and industrial medicine. ,
- "Karyotyping" in chromosomal abnormalities: Approximately 60% of all spontaneous abortions and 1 out of 200 newborns have some form of chromosomal abnormality, which can be detected by "karyotyping" pre/postnatally.
- Structural Abnormalities: Structural chromosomal rearrangements result from chromosome breakage, with/without subsequent reunion in a different configuration. They can be balanced generally harmless with few effects and unbalanced serious clinical effects. Commonly identified structural abnormalities include translocation, deletion, inversion, ring chromosomes, isochromosomes, mosaicism, and chimerism [Table 2].
Syndromes associated with chromosome abnormalities [Table 3].
| Conclusion|| |
The development of newer techniques such as "karyotyping" has made it possible to visualize undetected chromosomal anomalies such as small portions of chromosomes and translocations of tiny parts of chromosomes to one another. Because such procedures also enabled each pair of chromosomes to be distinguished individually, it has helped to further our understanding of chromosomal basis of certain important genetic disorders.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Tobias ES, Connor M, Ferguson-Smith M. Essential Medical Genetics. 6 th
ed. Wiley Blackwell Science; 1997. p. 57-69.
Ciccone R, Giorda R, Gregato G, Guerrini R, Giglio S, Carrozzo R, et al
. Reciprocal translocations: A trap for cytogenetists? Hum Genet 2005;117:571-82.
de Ravel TJ, Balikova I, Thienpont B, Hannes F, Maas N, Fryns JP, et al
. Molecular karyotyping of patients with MCA/MR: The blurred boundary between normal and pathogenic variation. Cytogenet Genome Res 2006;115:225-30.
Williams PL. Gray′s Anatomy. 38 th
ed. London: Churchill Livingstone; p. 193-211.
de Vries BB, Pfundt R, Leisink M, Koolen DA, Vissers LE, Janssen IM, et al
. Diagnostic genome profiling in mental retardation. Am J Hum Genet 2005;77:606-16.
Friedman JM, Baross A, Delaney AD, Ally A, Arbour L, Armstrong L, et al
. Oligonucleotide microarray analysis of genomic imbalance in children with mental retardation. Am J Hum Genet 2006;79:500-13.
Mao X, Lillington D, Child F, Russell-Jones R, Young B, Whittaker S. Comparative genomic hybridization analysis of primary cutaneous B-cell lymphomas: Identification of common genomic alterations in disease pathogenesis. Genes Chromosomes Cancer 2002;35: 144-55.
Mueller RF, Ian D. Young: Emery′s Elements of Medical Genetics. 11 th
ed. London: Churchill Livingstone; 2001. p. 84-101.
Kumar V, Cotran RS, Robbins SL. Robbin′s Basic Pathology. 7 th
ed. Philadelphia: WB Saunders; 2003. p. 120-72.
Nora JJ, Fraser FC. Medical Genetics - Principles and Practice. 2 nd
ed. Philadelphia: Lea and Febiger Publications; 1981. p. 145-80.
Rubin E, Farber JL. Pathology. Philadelphia: JB Lippincott Company; 1988. p. 332-52.
Walter JB, Talbot IC. Walter and Israel′s General Pathology. 7 th
ed. New York: Churchill Livingstone; 1996. p. 232-51.
Schoumans J, Ruivenkamp C, Holmberg E, Kyllerman M, Anderlid BM, Nordenskjöld M. Detection of chromosomal imbalances in children with idiopathic mental retardation by array based comparative genomic hybridisation (array-CGH). J Med Genet 2005;42:699-705.
Tompa R, McCallum CM, Delrow J, Henikoff JG, van Steensel B, Henikoff S. Genome-wide profiling of DNA methylation reveals transposon targets of CHROMOMETHYLASE3. Curr Biol 2002;12:65-8.
Pinkel D, Albertson DG. Comparative genomic hybridization. Annu Rev Genomics Hum Genet 2005;6:331-54.
Cheung SW, Shaw CA, Yu W, Li J, Ou Z, Patel A, et al
. Development and validation of a CGH microarray for clinical cytogenetic diagnosis. Genet Med 2005;7:422-32.
Shaffer LG, Kashork CD, Saleki R, Rorem E, Sundin K, Ballif BC, et al
. Targeted genomic microarray analysis for identification of chromosome abnormalities in 1500 consecutive clinical cases. J Pediatr 2006;149:98-102.
Schaeffer AJ, Chung J, Heretis K, Wong A, Ledbetter DH, Lese Martin C. Comparative genomic hybridization-array analysis enhances the detection of aneuploidies and submicroscopic imbalances in spontaneous miscarriages. Am J Hum Genet 2004; 74:1168-74.
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3]