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The basis of the difference

Study on a mutation that increases the risk of prostate cancer goes on the cover of Cancer Research

EDUARDO CESARHuman DNA has 3 billion nucleotides, the nitrogenized bases that encode all the information stored in our genome. Many people do not understand how swapping one nucleotide for another – there are four basis types of base, adenine (A), cytosine (C), guanine (G) and timine (T) – can raise or lower predisposition to a given disease.

A recent study completed by a group of researchers coordinated by Maria Rita Passos-Bueno, of the Human Genome Study Center, of the Biosciences Institute of the University of São Paulo (USP) – one of the ten Research, Innovation, and Dissemination Centers (Cepids) created in 2000 by FAPESP – revealed that an alteration of this sort, of only one nucleotide, in the gene COL18A1, located on chromosome 21, raises the risk of prostate cancer by two and half times. This type of tumor is the second most common in men in Brazil, recording almost 21,000 new cases a year. The discovery is on the cover of the October 15 issue of the magazine Cancer Research, published by the American Cancer Research Association.

The modification occurs in nucleotide number 4,349 of the gene, which, instead of being of a guanine base, as with most people, has adenine. The swap of the “chemical letters” causes the COL18A1 to produce a different type of amino acid: instead of aspartic acid, synthesized by individuals without this genetic alteration, asparagine is produced. As the proteins are synthesized from a combination of 20 amino acids, the unexpected presence of asparagine alters the manufacturing formula of a compound derived from the COL18A1, endostatin. The mutation can be seen by means of a technique of amplifying a section of DNA called a polymerase chain reaction (PCR).

Using this method, the region of interest is seen in a gel in the form of a fragment. When it contains the genetic alteration, the fragment – of around 170 base pairs – can be cut into two pieces by a restricting enzyme. Without the mutation, the fragment does not divide on contact with the enzyme. For this reason, in prostate cancer carriers, who carry the mutation more frequently than healthy individuals, the presence of two fragments instead of one is more common. “We believe that the defective gene leads to a less functional endostatin”, says Maria Rita. “It is as if the protein were not produced”.

Since 1996, when it was discovered, endostatin has been studied in the oncology field because it has the ability to inhibit the angiogenesis of solid tumors – a process of formation of blood vessels feeding this type of cancer. In mice, management of the protein enables the size of the tumors to be reduced. In the United States, scientists are now testing the treatment in humans. We must not confuse the real meaning of the USP team’s discovery. The mutation in COL18A1 is not the cause of prostate cancer. Its effect is smaller, but still important: the alteration turns the COL18A1 into a high risk gene, a kind of marker for the disease. In other words, whoever carries the modified gene runs a higher risk of developing this form of the tumor, but it does not necessarily mean the person will in fact develop it.

What scientific data enable us to say that the mutation increases the probability of prostate cancer? Answer: Maria Rita and her colleagues observed that the genetic alteration is more common in patients with this disease than in the population as a whole. After mapping the section of the COL18A1 gene responsible for encoding endostatin in 379 men (181 with solid tumors in the prostate and 198 healthy individuals, used as a control), the USP researchers observed that 25% of the cancer carriers also carried the modification in their DNA. Only 12% of the control group had the mutation. “These results support, for the first time, a potential association between a polymorphism in the encoding region of the endostatin and prostate cancer”, says Maria Rita.

The next stage in the work will be to check whether the mutation in COL18A1 can be related to other types of cancer, such as that of the liver and the stomach. The continuation of the study will have the support of colleagues at the Ludwig Cancer Research Institute in São Paulo, USP’s Medicine School, and USP’s Physics Institute at São Carlos, which took part in the discovery of the genetic alteration associated with the prostate tumors. Rats and mice will be used to understand the role of the genetic alteration in the development of solid tumors.

Polymorphism
The type of mutation found in the COL18A1 is technically called SNP, the acronym for single nucleotide polymorphism. The term refers to the various forms that a nucleotide can take. These possibilities, in fact are restricted to the four nitrogenized bases: A, C, G or T. When they announce that they have discovered an SNP related to a given disease, the scientists are saying that they have found a variation in just one base in a section of the genome. To be classified as an SNP, the variation has to be present in at least 1% of the population.

The researchers estimate that there are more than 2 million SNPs in the human genome. This type of alteration is the commonest form of genetic variation in our species. The practical implication of the existence of an SNP is not always bad, as we can understand from the news of the discovery of the mutation in gene COL18A1. Apparently, most SNPs produce innocuous results, since they occur in parts of the genome that do not code proteins. Others merely determine certain physical characteristics, not necessarily good or bad. Still others – and this is where the danger lies – cause or accentuate a predisposition to given diseases.

One of medicine’s dreams is to locate the highest possible number of risk genes for the main diseases. This valuable information could lead to predictive tests being created. These would show the real chances, for example, of a tumor occurring in an individual. The pharmaceutical industry is also interested in establishing the incidence of this type of genetic variation for other reasons. “People’s response to a medication may be associated with their SNPs”, comments Mayana Zatz, director of the Human Genome Study Center.

Down’s Syndrome
Besides mapping the mutation of the COL18A1 gene, Maria Rita and her group from the Human Genome Study Center carried out another unprecedented study related to endostatin, to be published in European Journal of Human Genetics. They showed for the first time that patients with Down’s syndrome (the technical name for mongolism) had more of this protein that the population as a whole. Measurements carried out in 35 carriers of the syndrome (16 men and 19 women) showed levels of endostatin ranging from 20 to 38 nanograms per milliliter of blood. In the experiment’s control group (54 healthy individuals, 22 men and 32 women), the rates of this protein ranged between 11 and 20 nanograms per milliliter.

Why did the researcher decide to take these measurements specifically in Down’s syndrome patients? According to scientific literature, carriers of this syndrome – clinically marked by a peculiar facial look and mental retardation, frequently accompanied by cardiac problems and immunological deficiency – had a low incidence of solid tumors. It is not known the reason for this apparent natural protection enjoyed by these individuals from this type of cancer. Some researchers however, have always thought that the answer to the mystery lies in the 21st chromosome. While healthy people have a pair of these chromosomes, Down’s syndrome patients have an extra copy. Therefore, there must be something in these DNA fragments that makes it difficult for the disease to spread.

With this information available, Maria Rita formulated the following hypothesis: since there was an extra copy of the COL18A1 gene, supplied by the third chromosome 21, and which practically never developed solid tumors, Down’s syndrome patients must carry a high amount of endostatin. The results of the study show that the reasoning was correct: high amounts of this protein really do seem to prevent tumors from developing.

“Based on the data produced in the work, we believe that a rise of about a third over the normal levels of endostatin would be enough to inhibit the growth of many solid tumors”, suggests Maria Rita. For Mayana Zatz, who took part in the research with Down’s syndrome patients, monitoring the levels of endostatin may, perhaps, give rise to a predictive test on the occurrence of tumors. “According to the results of the examination, we would increase or not the level of people’s endostatin”, says Mayana.

Genome sequencing
The identification of the mutation of the COL18A1 which alters the production of endostatin illustrates the importance of the mass of information provided by the huge international effort unleashed by the Human Genome Project, which, in February this year, produced a high quality draft of the contents of all 23 pairs of our species’ chromosomes. The COL18A1 was described for the first time in 1994, but it is only recently that its role in the human organism has begun to be better understood.

Maria Rita discovered the alteration in the region that codes the endostatin almost by chance. The researcher has been studying the genetic roots of Knobloch’s syndrome for almost ten years. This is a rare hereditary disease causing severe myopia and blindness and it may lead to the formation of lumps on the head. Based on an analysis of the DNA of 12 members of a family in the town in Bahia, Euclides da Cunha, who had the disease, Maria Rita began her work. Around 1996 she saw that the syndrome was caused by some gene, or genes, located on chromosome 21.

The study progressed until, in 1999, there was only one genetic region that could house the cause of the syndrome. The problem is that there were three genes in this region, with approximately 250,000 base pairs, and it was necessary to determine which one (or which ones) was the cause of the pathology. “We encountered difficulty in expanding these genes”, recalls Maria Rita. But then the sequencing of chromosome 21 was done, published in the May18, 2000 issue of the magazine Nature, and that helped solve the problem.

With the help of the new information, the cause of the syndrome was finally established. It was the COL18A1 gene, which, as well as the endostatin, produces collagen 18, a fibrous protein present in the connecting tissue of the skin, tendons and bone. Going further into the genetic origin of the syndrome, Maria Rita began probing the COL18A1 in search of SNPs. She found 20 mutations in the part of the gene that encodes the collagen 18 and one in the region giving rise to the endostatin. Although it had nothing to do with her original line of research, the SNP of the endostatin caught her attention. The geneticist saw that she could be facing an important discovery. She went ahead with her research and the rest, as they say, is history.

Testing for cancer
Four clinical tests with cancer patients that no longer respond to conventional therapy against the disease assess the feasibility of using endostatin in fighting more than 20 types of solid tumor. In the United States, where three studies began in 1999, the research involved the National Cancer Institute, universities and EntreMed, a US company that has the technology and licenses to manufacture endostatin on a large scale. The fourth clinical test is being conducted by the Academic Hospital of the Free University of Amsterdam, in the Netherlands, where research has been undertaken since the second half of last year.

All the studies are in the early stages and so far, there are no conclusive results on the efficiency of the procedure of inhibiting the cancer’s angiogenesis, a process that consists of forming the blood vessels essential to the growth of the tumors. Meanwhile, tests in humans have essentially shown that use of the protein, which, in mice, has managed to avoid the development of certain types of cancer, is not toxic. At this stage, this was precisely the kind of response that the experiments were designed to produce.

According to EntreMed, the tests have also already came up with clues about the possible mechanism of the action of the endostatin as an inhibitor of angiogenesis. In a work published in the July/August issue of the Journal of Biological Chemistry, the company’s researchers say that the compound is associated with another protein, tropomyosin, connected with the movement, growth and reproduction of endothelioid cells, which cover the blood vessels. Interference in the action of the tropomyosin, such as that produced by the presence of endostatin, according to EntreMed, affects the programming of the endothelioid cells, leading to death. Hence, without its source of nutrients, the tumor may shrink.

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