The molecular basis of Down syndrome

It was relatively easy to adopt the concept that a defective gene could cause disturbances in the body's biological function, and even diseases. But what happens when there are too many normal copies of a certain gene in the genetic load?

In the days when genetics took its first molecular steps, it was relatively easy to adopt a concept according to which a defective gene could cause disturbances in the body's biological function, and even diseases. But what happens when there are too many normal copies of a certain gene in the genetic load? Can an excess of normal genes cause negative effects? The answer to this question was not clear, since too many copies of the same gene do not necessarily result in the production of an excess amount of the protein encoded in the gene.

And in the end, the proteins are the real players in the field of the living cells. The genes "only" encode the information necessary for their construction. In addition to the lack of clarity on this question, the geneticists had before their eyes many examples from the world of plants, where multiple copies of normal genes is a widespread phenomenon (and important in development), which does not cause any disorders.

This is the context in which the pioneering work of Prof. Yoram Groner, who founded the department of molecular genetics at the Weizmann Institute of Science (as a merger of the department of genetics with the department of virology), and later served as the vice president of the institute, should be seen. Prof. Gruner recently won the AMT Prize in Life Sciences - Genetics for 2008. The judges' reasoning stated, among other things: "For his ground-breaking research in the molecular biology of Down's syndrome which proved in practice the dose-excess gene theory in the performance of three copies of Chromosome 21..." .

What is dose-excess gene theory? Why was it important to prove her? And when do three copies of chromosome 21 appear in the cells of the human body? In 1866, the English doctor John Langdon Down described a disease that was later named after him Down syndrome. About 50 years ago it became clear that it is related to the appearance of three copies of chromosome 21 (instead of the two copies that exist in the cells of normal humans). Contrary to popular belief, Down syndrome is a fairly common genetic disease. Despite widespread use of prenatal diagnosis methods, one out of every 800 babies born in the Western world suffers from Down syndrome. In addition to intellectual-developmental retardation, patients with this disease suffer from a series of defects, some of which appear in other diseases that are common in the normal population. Among other things, these are deficiencies in muscle function, diabetes, leukemia, and a relatively high frequency of Alzheimer's disease.

The scientists, who sought to understand how an additional, third copy of chromosome 21 causes Down syndrome, examined two possibilities in this matter. One theory, called Developmental Instability, postulated that Down's symptoms are caused by the disruption of the physiological balance as a result of the increase in the number of chromosomes. This concept reconciled with the great similarity between Down syndrome patients and with the fact that most of the defects from which they suffer, also appear in the general population, although with lower frequency and severity. Another theory, called the Gene dosage effect, proposed that each of the symptoms is directly caused by an excess dosage of one gene or several genes from the extra chromosome, which is accompanied by an increase in the amount of protein produced by that gene.

Prof. Gruner, being a molecular biologist, tended to support the "excess gene dose" theory, and set himself the goal of proving it. His findings not only proved the theory, but also laid the foundations for the study of the molecular genetics of Down syndrome. He set himself a challenge: to isolate one particular gene from chromosome 21, and prove that an excess dose of that gene causes the symptoms known in Down syndrome - and in this way prove the correctness of the "excess gene dosage" theory. This approach was at the time (1979) bold and innovative. The information regarding the genes found on chromosome 21 was extremely incomplete, molecular tests that allow monitoring of gene expression were not available, and gene cloning technology was in its infancy.

As in many other cases, in this case too, in order to reach new areas of knowledge, the scientists had to invent new wheels. Test results showed that in the blood of those suffering from Down syndrome there is a relatively large amount of an enzyme called SOD1. But is the gene encoding SOD1 located on chromosome 21? And if so, what is the role of SOD1 in the disease? Is an excess of this enzyme related in one way or another to the symptoms unique to the syndrome? Questions of this type, which are routinely asked today in many studies in the field of genetics, were then, in the early 80s, almost beyond the reach of science.

But Prof. Gruner and the members of his research group did not give up. They set out on a hunting trip for the "suspect" gene, with which they hoped to prove the "excess gene dose" theory. The hunting trip (see diagram) led to the cloning of the first gene from chromosome 21 and the determination of its base sequence, and was an important milestone on the way to achieving the goal. But do the genes that create an excess amount of SOD1 really play a role in causing the defects of Down syndrome? In the first step, the members of Prof. Gruner's group created transgenic cells with several copies of the gene that contained an excess amount of SOD1, similar to cells from patients with Down syndrome.

These transgenic cells showed abnormal physiological properties as a result of creating an excess amount of hydrogen peroxide which is the reaction product of SOD1. One of the defects that resulted from this was damage to the absorption process of nerve mediators (neurotransmitters), which are normally absorbed and stored inside the cell. Later, Prof. Gruner and the members of his group deciphered the molecular mechanism that causes the defect, and showed that the focus of the damage is a special pump whose function is to pump the neurotransmitters into the cell. This finding provided a molecular mechanistic explanation of how excess gene dosage of a gene from chromosome 21 causes a physiologically significant defect in cell function. But what is the connection between this impairment in the process of pumping out neurotransmitters and the symptoms of Down syndrome? To answer this question, Prof. Gruner and the members of his group set out on another pioneering adventure. They inserted the gene encoding SOD1 into mice, creating for the first time a transgenic mouse model with a gene from chromosome 21.

These mice, who carried SOD1 genes in an excess dose and produced large amounts of SOD1, contained in their blood a very low level of the neurotransmitter serotonin - similar to what is found in babies suffering from Down syndrome. This finding practically proved the "excess gene dosage" theory as the mechanism that causes Down syndrome.

But how exactly does an excess of SOD1 cause a decrease in serotonin in the blood? Later in the study, it was discovered that, similar to the findings in transgenic cells, in mice too, excess SOD1 causes an abnormal production of hydrogen peroxide which damages the special pump that draws serotonin from the blood into the platelet cells, where it is supposed to accumulate.

The failure of this pumping mechanism causes serotonin to remain in the bloodstream and break down. As a result, a low level of serotonin is created in the platelets, and later also in the brain of the transgenic mice, similar to the situation that exists in people with Down syndrome (a similar phenomenon occurs in diabetes, where, as a result of a lack of insulin, or a failure of its effectiveness, the sugar dissolved in the blood does not penetrate into the cells where it is needed, but remains in the bloodstream and causes negative effects).

Thus, a classic cycle of scientific research was completed, starting with a hypothesis that tries to explain a biological phenomenon at the level of the whole organism, continuing with the identification of the molecular cause of the phenomenon, its isolation, the proactive reproduction of the phenomenon using the identified factor, and ending with the deciphering of the mechanism of action in which the factor works - and the proof of the hypothesis.

The process of cloning the gene encoding SOD1

The multi-step process, described in the drawing, which Prof. Gruner and his group members took to clone the gene for SOD1, is based on the ability to translate in vitro, in a non-cellular system, the mRNA that codes for SOD1, and to identify the product through the use of antibodies against SOD1.
In the first step, the scientists captured mRNA molecules that were on their way from the cell nuclei to the ribosomes, where they produce different proteins. Among all the molecules there were of course also those that produce SOD1. With the help of the RT enzyme, they copied the mRNA, that is, "translated" it into DNA. (The RT enzyme, which was discovered by David Baltimore and Howard Thiemin and won them the Nobel Prize in Physiology and Medicine in 1975, is used by viruses that carry their genetic cargo in RNA molecules, to copy the RNA into DNA which they integrate into the chromosomes of the cells
that they attack. This is how, for example, the HIV virus that causes AIDS works.

Prof. Gruner and his group members injected the DNA they created in a test tube into millions of bacteria, assuming that some of them would "get infected" and start producing many copies of the DNA that codes for SOD1. In fact, each bacterium produces DNA of one particular gene inserted into it. To identify those individual bacteria that do produce SOD1 DNA, Gruner and his colleagues extracted the bacterial DNA and fixed it on tiny paper discs. Thus, a kind of "rod" was created with which they "fished" the mRNA that codes for SOD1 and translated it into a protein in a test tube. They identified the protein through a reaction with specific antibodies that bind to SOD1. In this process, they were able to identify the bacteria that contained the DNA that codes for SOD1. Isolating one such bacterium completed the cloning process, that is, Gruner's group had a bacterium producing large amounts of human DNA encoding SOD1. This fact allowed them to decipher the gene's base sequence. Thus, SOD1 became the first gene from chromosome 21 to be cloned and its base sequence determined.

Twenty years later, the circle that opened with the cloning of SOD1 was closed. Prof. Gruner's group participated in the project of mapping and deciphering the human genome in an international corporation that determined the complete base sequence of chromosome 21. This activity was done in the framework of the Crown Genome Center under the leadership of Prof. Doron Lantz from the Department of Molecular Genetics. The Human Genome Project is the largest project in the field of biology ever undertaken. With the completion of the genome project, the need for gene cloning became unnecessary.

Today, every scientist who wants to isolate this or that gene and study it accesses a computer, and in seconds receives all the information he needs.

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