Brittle X chromosome syndrome

Molecular Biology in medicine: science gives a chance

Irina Grishchenko, "Biomolecule"

Half a century ago, molecular biology emerged and began to develop rapidly. Biologists and physicists (the usual union for the mid-twentieth century) discovered the most important cellular processes, invented the basic methods, without which the work of any biological laboratory is unthinkable today. Now we have a huge potential for solving all kinds of problems: clarifying aspects of the origin of life, studying the interactions of components in a living cell and complex biochemical cascades. We know and are able to do what seemed fantastic 60 years ago. And one of the tasks that scientists can already attempt to solve is the fight against hereditary human diseases. Some of them, such as phenylketonuria, are being successfully corrected, approaches to the treatment of many others have not yet been found. In this article we will talk about one of these diseases – the syndrome of the brittle X chromosome - and about the difficulties of studying it.

At the beginning of the 20th century, scientists noticed that mental retardation affects men more often. In 1934, the Irish physician James Martin and the English geneticist Julia Bell first described a family where mental retardation was inherited concatenated with gender. There were 11 oligophrenic men and two women with mild mental retardation in this family. The discovered familial form of the disease was called Martin-Bell syndrome. 35 years later, Herbert Labs, conducting a cytogenetic study, revealed in the karyotype of four feeble-minded men and three normal women (from three generations of the same family) a strange X chromosome, which he called a marker: near the end of the long shoulder she had a secondary constriction. Labs proposed to track the marker chromosome in male embryos in dysfunctional Martin-Bell syndrome families, since it can signal a high risk of the birth of oligophrenes (Fig. 1) [1]. This is how doctors gained the first prenatal marker of the syndrome, and their patients – the opportunity to make an informed decision about maintaining pregnancy. Marker constriction was localized on the Xq27.3 site.

Figure 1. Herbert Labs tells colleagues about the X-chromosome constriction in Martin-Bell syndrome. Here and below are the drawings of the author of the article.

Later, many researchers observed X chromosomes under a microscope not just with a constriction, but as if broken - with the tips of long shoulders "torn off". The place of the tightening / breakage began to be called a fragile site. Therefore, the disease has received another name – fragile X chromosome syndrome (fragile X syndrome).

Another interesting feature of this disease is the aggravation of the disease in each subsequent generation (genetic anticipation). This phenomenon was explained only in the last decade of the 20th century, after the discovery of a special type of mutations – the expansion of trinucleotide repeats.

If we classify diseases by pathogenetic mechanisms, then a fairly large group will consist of diseases caused by the expansion of repeats [2]. The essence of the mutation is as follows: in the human genome there are short sections (for example, triplets of nucleotides), normally repeated several times, but for some reason their number begins to grow sharply - tens and hundreds of times – and the total length of the "stuttering" (containing repeats) fragment can increase to several thousand nucleotide pairs (fig. 2).

Figure 2. Imagine that our genome is a very long text, and the ongoing expansion turns it into complete nonsense.

Later it turned out that expansion underlies the pathogenesis of not only the fragile X chromosome syndrome, but also myotonic dystrophy of types I and II, as well as a number of neurodegenerative human diseases - for example, amyotrophic lateral sclerosis and Huntington's disease. In total, about 30 diseases are known for which such a mutation is characteristic. Many of these pathologies are associated with an increase in the number of repetitions (CGG)n, (CAG)n, (GAA)n and others [3].

Causes and pathogenesis of brittle X-chromosome syndrome

The syndrome of the brittle X chromosome is perhaps the most common cause of hereditary mental retardation after Down syndrome. There are quite a lot of clinical manifestations of the syndrome and not all of them are observed and not always, but the main ones – a low level of intelligence and emotional development, coupled with a number of physical abnormalities – are most often present. These features are noticeable already in early childhood.

The cause of the disease lies in the increase in the number of repeats of the CGT triplet in the promoter region (the launch pad for the start of mRNA synthesis) of the FMR1 gene (Fig. 3). The product of this gene is the FMRP protein (fragile X mental retardation protein), which interacts with RNA and directs complex molecular cascades necessary for the normal formation of neurons, their synaptic plasticity [4]. In a healthy person, the number of repetitions varies from 5 to 54. When the number of repeats increases to 55-200, an allele called premutant occurs. In the population, it occurs quite often: in one of 200-250 people. Although the mRNA level of the gene turns out to be higher than normal, the FMRP content remains unchanged or even decreases slightly. Why this is happening is still unknown. It can be assumed that RNA interference is involved in this - the process of suppressing gene expression (some stage of the path from the nucleotide sequence to the final product, in this case – FMRP) using small RNAs [5].

Figure 3. Structure of the FMR1 gene and its expression scheme. 5’-NTO and 3’-NTO are 5’- and 3’-untranslated regions of the gene.

With a slightly more pronounced expansion of CGT repeats, special intracellular inclusions consisting of FMR1 mRNA and RNA-binding proteins can be detected in patients. This is evidence that mRNA becomes toxic to the cell [6]. Interestingly, the "normal" mRNA does not have a toxic effect even at very high concentrations. Most women carriers of premutation, unlike men, have no external manifestations of pathology. This is the merit of the second X chromosome, which compensates for the defect in a greater or lesser proportion of cells. Moreover, there is evidence of preferential inactivation ("shutdown") of the defective chromosome. But often such women are characterized by emotional problems, depression and phobias.

Inactivation of one of the X chromosomes is a vital process of dose compensation of genes, which prevents the doubling of the expression of all X chromosome genes in females compared to males. That is, in each cell of an individual of any sex, despite the diploid set of chromosomes, only one X chromosome is active - inherited either from the father or from the mother. The fascinating details of the "shutdown" of the sex chromosomes in humans and worms are described in the articles "The mysterious journey of non-coding Xist RNA on the X chromosome" [8] and "Stories from the life of the X chromosome of the hermaphrodite roundworm" [9]. – Ed

And, of course, even in the absence of external signs of the disease, the premutant allele is transmitted to offspring. At the same time, there is an "amplification" of repetition – with each ovogenesis more and more, up to several thousand "copies". And this leads to the fact that the premutant allele turns into the most mutant one [2, 7]. In this case, we are already talking about the syndrome of the brittle X chromosome. Its frequency in the population is about 1:3600-6000. That's quite a lot! With such a significant increase in the number of CHG repeats, epigenetic changes occur: attachment of methyl groups to the cytosine of CHG triplets in the region of the FMR1 promoter and modification of DNA–related proteins - histones. All this leads to a local change in the density of DNA stacking – the formation of condensed, inactive chromatin, called heterochromatin. The expression of genes located in such a zone is suppressed. Therefore, in the case of Martin-Bell syndrome, the production of FMRP protein is sharply reduced. Moreover, chromatin modifications cause a visual "fragility" of the chromosome in the Xq27 region – the same one that scientists observed in the middle of the 20th century. However, in fairness, it should be noted that in a couple of percent of patients, the syndrome is caused not by the expansion of CGT repeats, but by other mutations of the FMR1 gene.

So, apparently, the pathogenetic mechanisms of the fragile X chromosome syndrome and other "expansive" diseases are common: all of them are characterized by some critical number of triplets, in which the gene still functions normally. The reasons for the expansion itself are not completely clear. To date, many hypotheses and models have been proposed that try to explain it, for example, violations during replication, problems with repair systems, etc. However, so far none of them has found experimental confirmation.

Why is it difficult to diagnose expansion and how is this problem solved?

As already mentioned, the syndrome of the brittle X chromosome is far from the only disease manifested by mental retardation. But the accumulated knowledge helped to develop a fairly detailed method of diagnosing this particular syndrome. It is possible to detect even premutation in people with a normal phenotype (with a normal IQ level and without developmental abnormalities) [10]. This is very important because female carriers have a high risk of having children with a pronounced syndrome. However, this technique is not without drawbacks and, unfortunately, is not widely used, so the development of methods of molecular diagnostics is still given a special place.

Initially, a study of the patient's chromosome set was performed – karyotyping – and when damage was detected in the Xq27.3 site, a diagnosis was made. This is still the first thing that geneticists do today - at least in Russia. The problem with karyotyping is that this method is not sensitive enough, which means it is not too reliable. Therefore, more and more modern methods are being used for diagnosis. There are test systems for DNA diagnostics based on key methods of molecular biology: PCR (Fig. 4), Southern blot, immunoprecipitation, etc. They allow us to estimate the amount of FMRP protein and its mRNA, determine the number of CGT repeats and the level of cytosine methylation in the promoter of the FMR1 gene. This, in turn, helps to better understand the pathophysiology of the syndrome, because it is possible to correlate the results of the analysis with the phenotype of patients and carriers of premutation.

Figure 4. PCR – polymerase chain reaction, one of the standard methods of molecular biology used in diagnostics. The main components without which the reaction will not go are shown. A DNA matrix is a DNA molecule, a section of which needs to be multiplied (amplified) many times. Primers – oligonucleotides complementary to the ends (on different chains) of the DNA matrix site of interest, as if limiting it, perform the function of a seed for an enzyme that copies DNA (DNA polymerase). dNTP – deoxyribonucleoside triphosphates are the building material for a new DNA molecule. The buffer is a solution of salts that provides the necessary conditions (pH, ionic strength); it necessarily contains a magnesium salt, because DNA polymerase works only in the presence of Mg2+ ions. If all the components are mixed, placed in a device called an amplifier (cycler) and run the desired program of cyclic increase-decrease in temperature, thousands of copies of the site of interest are synthesized on the matrix of the original single DNA molecules, which will eventually be easy to study. If, due to some mutations, sequences that are normally complementary to primers change, or the distance between them radically increases, there will simply be no PCR product.

PCR is the main diagnostic method. It allows you to develop an area containing (CGG)n. After conducting such an analysis, it is possible to determine the exact size of this area, and hence the number of repeats, and in this way to detect premutant or mutant alleles in patients. But I must say that it is not easy to achieve this. Researchers face a number of difficulties when amplifying these fragments. DNA, which will act as a matrix for the synthesis of new molecules, has such a characteristic as the HZ composition, reflecting how rich the matrix is in guanine-cytosine pairs (a rich matrix contains about 60% of HZ pairs). If the percentage of HZ pairs is high, then the molecule will be refractory, and at some stages of PCR it will be necessary to carry out a longer denaturation. (CGG)The n-region is 100% composed of HZ pairs, and it is clear that this is a very difficult matrix.

Everything is even more complicated by the fact that such a sequence effortlessly forms various secondary structures that are very stable thermodynamically: all kinds of hairpins, G-quadruplexes (four chains interconnected by guanine and supported by a monovalent cation, for example K+), i-motifs (structures consisting of four DNA chains rich in cytosine, stable in an acidic environment) [11]. The study of such structures is a very beautiful and intriguing task for biochemists and biophysicists, but it is a serious obstacle to establishing the size (CGG) of n–regions. Well, on top of everything else, primers (oligonucleotide seeds for DNA polymerase) can form dimers with such sequences, and the mixture of molecules turns into one thermostable, unraveling tangle! It is clear that you can't work with such a matrix just like that. But! For several years, scientists have been actively coming up with more and more new modifications of conventional PCR, significantly improving the result.

Since the HZ-rich matrix needs a longer and high-temperature denaturation, previously they tried to warm up the matrix additionally, before PCR. However, as you might guess, this did not solve the problem. Back in the late 90s, it was found out that DNA synthesis is interrupted in extended sections of CGG repeats in the presence of K+, and a little later they realized that the very same quadruplexes are to blame [12]. Since KCl is included in the most common buffer for PCR, the most obvious solution was to exclude it from the buffer; this gave certain results, but I wanted more. Therefore, they began to actively come up with alternative buffers.

Nowadays, PCR is often performed with the addition of pure Tris-HCl as a buffer. Tris is a standard component for the production of nucleic acid solutions: it is cheap, and its buffering properties are high at pH 7-9 – values that are physiological for living organisms. Magnesium chloride is necessarily added to Tris in concentrations that do not inhibit DNA polymerase and therefore do not reduce the yield of a specific product. Very often, the mixture is "improved" by various substances that change the properties of the entire complex PCR system: DMSO, betaine, formamide – they stabilize denatured DNA, help reduce the melting point. Some use modified dNTPs, in particular 7-dease-dhtps, and note its effectiveness (Fig. 5); this modified nucleotide prevents the formation of complex duplexes.

Figure 5. An improved mixture for PCR is the first thing that is needed for amplification (CGG) of the n–region. Drawing by the author of the article.

In addition to the components of the mixture, there are interesting variants of temperature cycles. The simplest option, often used for amplification of not the most "impenetrable" sequences, is PCR with a hot start (hot–start PCR). The difference between this modification and standard PCR is the use of special antibodies that prevent polymerase activation until the desired temperature is reached, which avoids non-specific synthesis. To work with extremely HZ-rich matrices (>83%), a PCR variant called Slowdown (Touchdown modification) is proposed: slow heating and cooling speeds, a stepwise decrease in the annealing temperature after a certain number of cycles, the addition of 7-dease-dHTF – all this leads to an increase in the yield of the target PCR product.

However, it cannot be said that the problem of obtaining the amounts of HZ-rich fragments necessary for further analysis (such as the FMR1 promoter region) has been completely solved: articles on this topic appear frequently, but the published results contradict each other; commercial companies compete in the development of "magic" kits, but they can cost so much that they are not in even successful foreign laboratories can afford it.

Determining the size (CGT) of the n-region is the very first and very important stage in the study of the fragile X chromosome syndrome, which, however, still needs to be optimized. If we learn to count repetitions quickly and efficiently, then diagnostics will become simple and relatively cheap. It will be possible to conduct it en masse, which means to distinguish the syndrome of the brittle X chromosome from many other diseases accompanied by mental retardation, which is extremely important for the selection of therapeutic approaches.

Literature

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Portal "Eternal youth" http://vechnayamolodost.ru  26.10.2016