Important letter
A letter was found in the genomic sequence, the replacement of which reduces the risk of autoimmune diseases
A research group of The V. A. Engelhardt Institute of Molecular Biology, the N. I. Vavilov Institute of General Genetics and MIPT have identified a letter in the genome that can participate in the mechanisms of development of autoimmune diseases, in particular, multiple sclerosis. In an article published in PLOS ONE, scientists examined how the replacement of this letter can affect the work of the IL2RA gene, which produces a protein important for the immune system.
When two DNA sequences differ by one letter, that is, by one nucleotide (A, T, G or C), this is called single nucleotide polymorphism, or SNP (English Single nucleotide polymorphism). A specific variant available to a particular person is called an "allele".
List of suspects
A list of SNPs that occur with different frequency in healthy people and in people with autoimmune diseases can be obtained from the data of GWAS – genome-wide association search. Genome–wide association search is a search for connections between different genome variants and, for example, diseases. Laboratories all over the world determine the genomes of a huge number of people, compare sick and healthy and look for characteristic mutations. Thanks to such studies, for some diseases it is already possible to assess the risk that a person will get sick simply by reading his DNA sequence. As a rule, several SNPs are associated with one trait, that is, single-letter substitutions in different parts of the genome. However, the list of suspicious alleles says nothing about the mechanisms of the disease. To open up the possibilities of accurate diagnosis and personalized medicine, it is necessary to conduct a detailed study of each case.
The replacement does not come alone: along with one letter, another is inherited. For example, in one place there is a letter A, and in another there is a letter T linked to it. Then, if instead of A there is a replaced G, then another letter will also stand instead of T. Single-letter substitutions are inherited by a group, and several characteristic alleles are observed in one person at once. However, in order to get to the bottom of the causes of the disease, it is necessary to understand who from this criminal group is the instigator of the disease, and who is rather a witness.
The affected gene
In order for the immune system to properly guard the body, its cells must transmit signals to each other. One cell releases a signaling molecule, and the other receives it with the help of a special receptor. For example, the interleukin-2 receptor (IL2R) is activated when an interleukin-2 molecule comes to it. Activation of this receptor gives the cells a command: to attack or, conversely, to slow down. Interestingly, the response to this signal is different for different types of cells. Therefore, a delicate balance is important here: if there are too many or too few receptors, it will lead to disease.
"It has been shown that when the signal transmitted by interleukin-2 is amplified or interrupted, an autoimmune reaction occurs in mice. In people with a number of autoimmune diseases, changes in the work of the corresponding receptors were also detected. Therefore, we focused on the IL2RA gene and on neighboring SNPs associated with autoimmune diseases," says Marina Afanasyeva, the first author of the study, an employee of the V. A. Engelhardt Institute of Molecular Biology.
In the non-coding part of the gene, scientists selected six substitutions in the genome associated with autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, Crohn's disease, ulcerative colitis and so on.
The protein is produced according to a "recipe" written in RNA. In turn, RNA "rewrites" with DNA – this is called transcription. Some sections of DNA are not included in the final "recipe", that is, they do not encode protein. However, these sites can enhance or suppress transcription, so that replacing one letter in them can affect the amount of protein.
Molecular Mediator
RNA polymerase synthesizes RNA based on DNA sequence. A promoter is a DNA site that serves as a launching pad for RNA polymerase to begin transcription.
For proper initiation of transcription, a protein complex must assemble.
There are different ways in which one letter can affect the fate of a gene. At the non-coding site, the effect can be exerted through proteins called transcription factors. Such proteins bind to certain parts of DNA and to other proteins. As a result, a large protein complex is assembled, which triggers transcription. Some factors stimulate transcription, and some inhibit it. It turns out that for the correct operation of the gene, not only those parts of it on which the protein is encoded are important, but also the areas on which transcription factors sit down. If the transcription factor does not "recognize" its sequence, it will not communicate with it and as a result, the wrong amount of protein will be obtained. However, not all substitutions have the same strong effect on compatibility with transcription factors. Consequently, the circle of suspects is narrowing.
Using bioinformatic analysis, the researchers predicted which SNPs are located at the sites of binding to transcription factors. Applying a few more criteria, they selected a replacement designated as rs12722489. Most people have the letter G in a certain area, but for some this letter has been replaced with A, and statistically replacement is less common in people with autoimmune diseases. Analysis of this DNA site showed that if there is no replacement, then the sequence has affinity for the transcription factors ERa and ERß (estrogen alpha and beta receptors). The change in the amount of protein under the influence of transcription factors may be the cause of disorders in the immune system.
Face-to-face
Scientists have tested how letter substitution can affect transcription using a test construct. It encoded the protein luciferase, which in the presence of luciferin firefly causes bioluminescence – a chemical reaction accompanied by the emission of light. This is a standard genetic engineering technique that allows you to determine the activity of transcription by luminosity. A copy of the site from which IL2RA transcription starts was inserted into the ring DNA. The firefly luciferase gene was inserted immediately after it, and a copy of the site with rs12722489 was inserted even further away. These rings were placed inside living cells to trigger intracellular reactions and determine the activity of the gene, which depends on the regulation of transcription, by the degree of luminosity. One part of the designs contained the A-variant, the other – the G-variant, the latter demonstrated a luminosity 1.5 times greater. This means that replacing rs12722489 weakens IL2RA transcription.
Ring DNA. The promoter is the site where transcription is triggered (shown in light green). Next comes the luciferase gene, and the area containing the substitution is indicated in red.
In the same experiment, the authors showed that in the absence of estrogen, the luminosity of the A- and G-alleles is the same. Three more experimental methods were used to prove that ERa does bind to the G-variant and does not bind to the A-variant. Thus, the authors of the study showed that, at least in cell cultures, the replacement of rs12722489 affects the production of the IL2RA protein. If the letter G is in the found position, the DNA site binds to the transcription factor ERa, which enhances the transcription of the IL2RA gene. And as a result of replacing with the letter A, less protein may be obtained, which may reduce the tendency to autoimmune diseases.
Of course, a complete diagnosis cannot be made by one letter, but the more we learn about the genetics of diseases, the more accurate the forecasts become, and treatment and prevention are more timely. Now only a small number of genes have been studied in such detail.
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11.05.2017