Scientific Fights, or How Geneticists Have Made Use of Molecular Competition?

Do battles take place in nature? Of course! Plants fight for access to the sun, wild animals wrestle with carcasses in a struggle for the biggest bite, and parasites are advantageously equipped to exploit their hosts, who in turn attempt to withstand the parasites. Hosts, on the other hand, have cells – like eosinophils in humans – that specialise in killing parasites. These are just a few examples of scientific battles in which one loses in order for the other to win. Scientific battles can also be successfully used in order to diagnose and treat diseases. Some very interesting battles, which I am going to talk about, are fought in genetics.

Research scientists are continually discovering new compounds and their surprising properties. Compounds can be indicators, drugs or very powerful inhibitors. The combination of these competitive scientific “battles” with special molecular features has led to the development of an extremely accurate and very useful genetic test, called “array Comparative Genomic Hybridization” (aCGH) [1].

The field of genetics unapologetically demands precision. The intricacy of genetic processes and their complexity can be best depicted by the very structure of DNA, the most important acid in human body. It is a double helix, formed by four nitrogenous bases: adenine, thymine, cytosine and guanine. The functions of all genes are entirely dependent on the order of these bases, and so in order to locate and identify genes researchers and diagnosticians need to know their base order.

But what if, due to a mutation, imbalances occur within chromosomes? What if a fragment of a chromosome breaks off and goes missing? Or two fragments of chromosomes switch places? The aCGH genetic test can be imagined as a great field (microarray plate) in which very high towers (fragments of single stranded human DNA) built entirely of four types of blocks (bases) are set side-by side [2]. New towers labelled different colours: red, from the patient; and blue, control human DNA; then rain over the field. These bind to the original towers in the field to form double stranded DNA, as adenine forms bonds with thymine and cytosine pairs with guanine.

When using this test geneticists do not know whether the patient has deletions or duplications [3]. This is where the blue towers are required, as a reference sample, where the base order is known. When red and blue stained oligonucleotides are placed together in a solution on a plate, they compete for colourless oligonucleotides from the microarray. If the amounts of DNA from the patient and control are the same, the colour visible on the computer scan of the array would be a combination of the colours of dyes used – green [4].

Figure 1. The principle of aCGH test. [Source: Baylor College of Medicine]

Figure 1. The principle of aCGH test. [Source: Baylor College of Medicine]

But how can aCGH tests help the researchers find sub-microscopic changes? Imagine that the patient has a deletion – a missing chromosome fragment. In the control DNA these oligonucleotides are present, but they are absent from the patient’s sample. Control DNA fragments will bind to their corresponding oligonucleotides on the array, but they will not have a counterpart in the form of fragments of DNA from the patient [5, 6]. Therefore, the colour of the control DNA (blue) at a given point of the matrix will dominate. Thanks to a super sensitive (and very expensive) scanner, it is possible to detect this slight inequality in the colour. It tells the researchers at which point the patient’s chromosome is missing genes. On the other hand, if a patient has duplication, the control DNA will be less represented. There will simply be more oligonucleotides from the patient due to this duplication. Therefore, they will be more likely to bind to the oligonucleotides on the array [7]. Just as before, this will cause unevenness in the matrix colour at this point. This time the dominating colour will be the colour of the patient’s DNA (red). Just as before, it can be read and analysed by the scanner, giving the researcher an insight into where there is errors within the DNA.

Figure 2. A sample result of patient’s aCGH test. Areas marked in red are deletions; areas marked in green are duplications. [Source: author’s own research from the Institute of Mother and Child in Warsaw]

Figure 2. A sample result of patient’s aCGH test. Areas marked in red are deletions; areas marked in green are duplications. [Source: author’s own research from the Institute of Mother and Child in Warsaw]

The aCGH test was developed because of the need for a more precise tool for genetic analysis, compared to the tests available at the time, at the end of the twentieth century. The aCGH test gave more exact localisation of changes occurring within the karyotype, compared to microscopic techniques of chromosome analysis. With the use of this test the causes of numerous diseases can be diagnosed, such as congenital epilepsy, Prader–Willi syndrome, Angelman syndrome (AS) and many more, including an individual’s risk of cancer. Moreover, aCGH test can also be combined with other techniques, like fluorescent in situ hybridization. This method allows one to observe the imbalances marked with molecular probes at the metaphase chromosomes, which is useful as a verification of accuracy of the microarray itself. In this way genetic based neurological problems, such as epilepsy, mental retardation and other issues can be successfully diagnosed, which is often a big relief for the patients or their family.

However, there are certain societal aspects that are still of much concern. As the method is used in prenatal diagnosing, it can be the source of information about the foetus sex, potential diseases and even a basis to try to predict baby’s character features. This can – and does – raise some objections to the ethical side of using aCGH for that purpose. Moreover – the same think can happen with any adult person diagnosed with the use of microarrays; a possibility of developing Parkinson’s disease or Alzheimer’s disease can be detected, sentencing the patient to a life under a cloud of fear.

As with every molecular method, aCGH too has limitations. It is not possible to detect structural chromosomal aberrations, without copy number changes, such as mosaicism, balanced chromosomal translocations, and inversions. Additionally, chromosomal regions with short repetitive DNA sequences are highly variable between individuals. They can interfere with CGH analysis, which is why repetitive DNA regions like centromeres and telomeres need to be blocked and omitted from screening. This is why there are still many improvements that need to be made on the technique to make it even more precise.

The aCGH test uses natural tendency of molecules to compete for the binding slots. Because of these properties, researchers have a chance to diagnose patients, look into their genome and discover the causes of irregularities [8]. Clever natural mechanisms can therefore also be adopted by researchers to obtain tangible benefits which allow us to learn more about ourselves. Thus, the struggle for supremacy does not have to have such negative connotations. Those who understand this best are the geneticists, struggling every day with the mysteries encoded in the order of these four inconspicuous nitrogen blocks on which the majority of the living world is built.

References:

[1] Wicker, N., A. Carles, I. G. Mills, M. Wolf, A. Veerakumarasivam, H. Edgren, F. Boileau, B. Wasylyk, J. A. Schalken, D. E. Neal, O. Kallioniemi, and O. Poch. 2007. A new look towards BAC-based array CGH through a comprehensive comparison with oligo-based array CGH. BMC Genomics 8:84.

[2] Shaw-Smith, C., R. Redon, L. Rickman, M. Rio, L. Willatt, H. Fiegler, H. Firth, D. Sanlaville, R. Winter, L. Colleaux, M. Bobrow, and N. P. Carter. 2004. Microarray based comparative genomic hybridisation (array-CGH) detects submicroscopic chromosomal deletions and duplications in patients with learning disability/mental retardation and dysmorphic features. J Med Genet 41 (4):241-8.

[3] Levsky, J. M., and R. H. Singer. 2003. Fluorescence in situ hybridization: past, present and future. J Cell Sci 116 (Pt 14):2833-8.

[4] de Ravel, T. J., K. Devriendt, J. P. Fryns, and J. R. Vermeesch. 2007. What’s new in karyotyping? The move towards array comparative genomic hybridisation (CGH). Eur J Pediatr 166 (7):637-43.

[5] DeVries, S., J. W. Gray, D. Pinkel, F. M. Waldman, and D. Sudar. 2001. Comparative genomic hybridization. Curr Protoc Hum Genet Chapter 4:Unit4.6.

[6] Béri-Dexheimer, M., C. Bonnet, P. Chambon, K. Brochet, M. J. Grégoire, and P. Jonveaux. 2007. Microarray-based comparative genomic hybridization in the study of constitutional chromosomal abnormalities. Pathol Biol (Paris) 55 (1):13-8.

[7] Shaffer, L. G., and B. A. Bejjani. 2006. Medical applications of array CGH and the transformation of clinical cytogenetics. Cytogenet Genome Res 115 (3-4):303-9.

[8] Miller, D. T., Y. Shen, and B. L. Wu. 2012. Oligonucleotide microarrays for clinical diagnosis of copy number variation and zygosity status. Curr Protoc Hum Genet Chapter 8:Unit8.12.

Image credits:

[1] Image credit (public domain): Baylor College of Medicine ‘Agilent aCGH’ , Baylor College of Medicine website, Last modified: May 29, 2013.

Dalia Gala is an undergraduate student at University of Glasgow, majoring in Molecular and Cellular Biology. She is interested in cells and travelling, which is why she spent last summer in Oslo University Hospital in Norway, working on cells in the weekdays and exploring Norway in the weekends.

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