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Syndrome

The first map of Down

Team from São Carlos begins to relate genetic origins to the different manifestations of the syndrome

If it were a football team, the Molecular Biology Laboratory of the Federal University of São Carlos (UFSCar) would be like the São Caetano  (A team formed 11 eleven years ago and reached the final stages of the Brazilian Championship): young and modest, but well equipped and efficient, ready to surprise the stars of genetics. Flávio Henrique da Silva, who coordinated the 18 laboratory researchers, started off the formation of an ambitious genetic database: in an unprecedented work for Brazil, he concluded the map of the gene expression of a patient with Down’s syndrome, one of the most common genetic accidents, which happens once for every 700 births.

The recently concluded map is the first of a series of others, now under way, the target for which is to understand the different manifestations of this genetic problem, through a search for the correlation between the genotype (the set of genes) and the phenotype (how they are expressed physically – in the shape of short or tall people, for example). Mental retardation and muscular weakness are signs common to all the patients, but the syndrome frequently causes gastrointestinal problems and in the endocrine glands, as well as deficiencies in the immune system. There is a high level of uncertainty over its effects: almost half the individuals affected can show cardiac problems, leukemia, with more probability than the population at large, and, prematurely, between the ages of 30 and 40, Alzheimer’s disease, a neurodegenerative disease marked by the growing loss of memory.

The gene expression map – or transcriptome -, the way by which an understanding is being sought of the origins of the symptoms -, is a functional interpretation of the genes, an assessment of the quantity of RNA (ribonucleic acid) that the cell uses in the synthesis of proteins from the sequences of DNA (deoxyribonucleic acid, the carrier of the genetic code) of each gene. “If an individual has less RNA of a specific gene, he will produce less protein”, as Silva sums it up. As the proteins codified by the genes run the workings of all the cells in the organism, this association indicates those that are involved with the syndrome, and makes it possible to understand more clearly the evolution of the problem.

This first map consisted in the mapping of, so far, 12,000 expressed (active) genes in one kind of blood cells, the leukocytes, of a 29-year-old patient who lives in Passos, in Minas Gerais, the same city where the researcher was born. He was chosen precisely for the possibility of elucidating the relationship between the syndrome and aging. One of the most notable results of this study, carried out using the Sage (serial analysis of gene expression) technique, is the indication that there is a reduction in the expression of at least 15 genes that codify proteins known as cytokines, involved in differentiating cells and the immune response, and others, called transcription factors, which regulate the expression of other genes.

To confirm that these genes show a differentiated expression from those who do not have the syndrome and that they therefore effectively signpost the different manifestations of the disease, Silva is working on the analysis of the gene expression of another 20 patients of Down’s syndrome who reside in São Carlos. This time, they are children and teenagers, like Pedro Pinheiro Fagian, a 13-year-old boy who studies, like any of his colleagues, in the seventh grade of the Oca dos Curumins School. The sample may be expanded even more, through the collaboration of researchers from the College of Medicine of the University of São Paulo (USP) in Ribeirão Preto, so that in future, separating the patients by groups with genes more or less expressed, a range of patterns for gene expression can be established. Using these standards, the intention is to understand more accurately the mechanisms responsible for the syndrome, and to arrive at new therapeutic strategies.

To reach their objectives, the team from São Carlos has to calculate the number of genes expressed in a cell. It is no easy task: besides the different cells expressing different genes, expression also varies in accordance with the situation in which the cell finds itself. Nevertheless, the genes detected can be used to compare the overall standard of gene expression amongst sufferers with the syndrome and other individuals.

Triple chromosome
Described in 1866 by the English doctor John Langdon Down (1828-1896), the syndrome was originally called mongolism, and those who suffered from it mongoloids, due to the characteristic facial traits – terms with a racist connotation that were excluded in 1965 from the publications of the World Health Organization. Its causes were only unveiled in 1959 by the Frenchman Jérôme Lejeune (1926-1994), who identified an anomaly called the trisomy 21: instead of 46 chromosomes (23 inherited from the mother and 23 from the father), the bearer has 47, because there is an extra chromosome in pair 21. That is, instead of two, the bearer has three number 21 chromosomes.

In recent times, a climate of intense competition has been marking research into Down syndrome. At the front, two wizards. One is the Greek, Stylianos Antonarakis, from the University of Geneva, Switzerland, who coordinated the complete sequencing of chromosome 21. The other is Xavier Estivill, of the Institut de Recerca Oncologica, in Barcelona, Spain, who in 1995 isolated the first gene suspected of having a part in the clinical condition of the Down syndrome, resulting from he action of an as yet uncertain set of genes.

Yet the team from São Carlos, running in the outside lane, has found its own room for recognition in the search for the mechanisms that can explain the manifestations of the syndrome. The pattern of gene expression of an individual with Down syndrome is very different from that of a non-bearer, but Silva hopes to find relevant differences among patients with the syndrome as well. In a parallel approach to the gene expression map, Silva is coordinating the in-depth study of the proteins suspected of involvement in the clinical condition of Down syndrome. In work carried out with the Structural Molecular Biotechnology Center of USP’s Institute of Physics in São Carlos, Silva’s team is currently analyzing 11 proteins from the Down Syndrome Critical Region – abbreviated to DSCR -, the part of the chromosome that is considered a suspect for being responsible for the production of the proteins involved in the disease.

The recognition of the DSCR arose from the finding that there does not have to be an entire extra chromosome 21 for the syndrome to manifest itself: some bearers have only a fragment in triplicate, usually adhering to chromosome 21 or to number 14. The genes of this fragment correspond to the critical region, and it is believed that some of them command the activity of other genes, causing the vast range of the manifestations of the disease. To confirm this hypothesis, they are trying to discover the function of the proteins of the DSCR. Studies are more advanced with the DSCR-1, the first protein of the critical region, expressed preferentially in the brain, the heart and the muscles, precisely the areas most affected in the syndrome.

Destinations
One clue to confirm these mechanisms is the localization of the protein, which defines its role in the cell – the proteins involved in gene regulation, for example, are to be found in the nucleus. In view of this situation, the team coupled the DSCR-1 with a green fluorescent protein (GFP), which works as a marker, inserted it into various kinds of cells and observed: it is to the nucleus that the DSCR-1 goes, an indication of its regulatory role.When studying the interaction between the DSCR-1 and other proteins, the team from São Carlos, in parallel with groups abroad, made a surprising discovery: the DSCR-1 diminished the action of the calcineurin enzyme.

This enzyme is a phosphatase it takes phosphate out of the proteins. One of these proteins, NFAT (nuclear factor of activated T-cells) has to lose a phosphate to reach the nucleus of the cells and activate the genes that are behind the production of the cytokines. Little by little, the relationships are clarified. “Calcineurin is responsible, for example, for the dephosphorization of the Tau protein, which builds up, hyperphosphorylated, in patients with Alzheimer’s disease”.

Aging
The premature evolution of Alzheimer’s disease in patients with Down Syndrome is also a theme for the group led by Marília Cardoso Smith, from the Federal University of São Paulo (Unifesp). She is looking into the relationship between Alzheimer’s disease and the partial loss of chromosome 21, a curious phenomenon that occurs in the process of the premature aging of these patients. With time, Marília explains, between 2% and 4% of the cells of patients with Down syndrome lose the extra chromosome 21. But they do not become normal: they have two chromosomes 21 from the same parent (father or mother), when it is normal to have one chromosome from each parent. The supposition is that this phenomenon interferes in the expression of the genes and contributes towards the degeneration of the brain.

Understanding the mechanisms for the development of the syndrome, the researchers hope to combat its manifestations and to improve the quality of life of its bearers. They also hope to arrive at valuable information about the structure of activated or blocked proteins, so as to permit the development of new medicines. At this point of the game, the competitors are on the same side.

The project
Expression and Intracellular Location of the DSCR-1 – A Protein Related to Down Syndrome (nº 96/11018-0); Modality Young Researcher Program; Coordinator Flávio Henrique da Silva – UFSCar; Investment R$ 261,050.01

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