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Early diagnosis

New tests help with the prevention of hereditary diseases and with the control of opportunist infections

MIGUEL BOYAYANDozens of genetic tests are done weekly at USPMIGUEL BOYAYAN

Discovering in advance the possibility of a genetic problem or the incidence of an opportunistic virus may be the key for suitable treatments, prevention of future diseases, or of rejection of organs in transplanted patients. The methodology for this, such as the measurement of viral loads and genetic tests, until a short time ago restricted to laboratory research, is beginning to reach the general population, through the transfer to laboratories specialized in clinical examinations of the technology developed at two of the Research, Innovation and Diffusion Centers (Cepids), funded by FAPESP.

The first of the Cepids to transfer this kind of technology is the Antonio Prudente Cancer Research and Treatment Center, linked to the AC Camargo Cancer Hospital and the Ludwig Cancer Research Institute, which entered into a partnership with Diagnósticos da América (Dasa), a company responsible for the laboratories Delboni Auriemo, Lavoisier and Elkis e Furlanetto, in São Paulo, and Lâmina and Bronstein, in Rio de Janeiro, besides the Curitiba Charity Hospital Laboratory.

Viral load
Based on the agreement, Dasa is now making available to the public two kinds of examination, one for measuring the viral load of two types of virus – the cytomegalovirus (CMV) and the Epstein-Barr virus (EBV), which can cause infections in several organs of immunodepressed persons – and another for genetic tests. The tests for measuring viral load aim at reducing rejection and complications in transplanted patients. And the genetic tests are intended to identify individuals with a probability of developing tumors of a hereditary origin.

The viral load test, developed by the group from the Cancer Hospital/Ludwig Institute, is different from the usual examinations, which merely confirm the presence of the viruses. With this new methodology, it is possible to find out whether there are viral particles or not, but also to know the quantity of these particles that are present. This differential can be better understood by taking as an example the EBV, present in 80% of the population, but usually without any activity in the organism.

When, however, the individual who is carrying the EBV becomes immunodepressed, as a result of a disease such as cancer or Aids, for example, or because he has undergone a transplant that obliged him to a treatment with drugs, to lower the levels of rejection of the transplanted organ, this virus can multiply in the organism, causing infections that can be fatal if not detected at the outset.According to André Luiz Vettore, the coordinator of the cancer genetics laboratory at the Ludwig Institute, the efficiency of this examination lies in measuring the viral load and revealing whether it is increasing or not. “If a patient with a given viral load undergoes a transplant and this load remains the same, measured at short intervals of time, there is no danger. But if there is a hike, the patient will have to be treated with antiviral drugs as soon as possible, to increase his chances of survival”, he says. The test that is being offered by Dasa can measure and monitor the viral load for days, weeks, or months, as need be.

Vettore says that the idea of developing viral load exams of EBV and CMV arose from the collaboration with researchers from the Federal University of São Paulo (Unifesp) and from the Tropical Medicine Institute of the University of São Paulo. Both tests use the technique of assessing in real time the reaction of the polymerase chain, known as Real Time-PCR, short for Real Time Polymerase Chain Reaction. The samples of DNA are obtained from the lymphocytes of the blood, collected in the process commonly used for simple viral load detection. The novelty lies in adapting the technique to measure and to accompany the presence of EBV and CMV. “This was an exam that was not available in Brazil, the importance of which is being able to monitor the patient, preventing the development of infections that can kill him”, Vettore says.

For him, the importance of the tests is highlighted by the fact that, due to their cost, the antiviral medicines are often administered only after they have been found to be absolutely necessary. “The advantage of the test and of the monitoring lies in the doctor knowing earlier on what the patient’s needs are, increasing the speed of the diagnosis and the precision of the accompaniment, even with a lower viral load”. He points out that, in the case of the CMV, studies were carried out on patients with bone marrow and kidney transplants, while the EBV had its behavior monitored in patients with a lymphoproliferative post-transplant disease.

The development of the tests and their assessment in over 400 patients lasted about one year. The research into the EBV was part of an article based on the doctoral thesis of researcher Otávio Baiocchi, from Unifesp, published in the American magazine Haematologica. Another two works, this time on CMV, are also to be published.

Genetic maps
Another technology developed at the Cancer Hospital and which is also undergoing a transfer process is for genetic tests, used for diagnosing people who were born with some kind of hereditary syndrome, that is, with alterations in some genes, which increases the possibility of developing one or more tumors in the course of their lives. Even though only 5% to 10% of cancer cases are hereditary, the importance of the genetic tests lies in being able to detect the change in the patient and accompany other people from the same family before the disease arises.

In some cases, it is now possible to know which genes are related to certain syndromes, as in hereditary breast tumors, where the genes associated are the BRCA1 and the BRCA2. The genetic test consists of fragmenting the patient’s gene into small pieces and assessing them in high performance liquid chromatography equipment, which reveals which fragments may contain an alteration. The “suspect” pieces are forwarded to another apparatus, the DNA sequencer, where the sequences are deciphered and compared with a normal one, to find out whether an alteration really exists. By selecting beforehand the pieces that can show an alteration, the cost of the examination is brought down, since, on average, only 10% of the gene needs to be sequenced.

Even so, the presence of genetic alterations in the family does not mean that all the individuals may come to develop the same kind of ailment, since cancer is very much connected with the natural aging of the organism. “That is why attention has to be paid to the age at which the tumors appears”, Vettore observes. He reckons that early diagnosis facilitates the treatment of the disease. At the moment, these tests are only done outside the country, by laboratories that receive the material collected here.

The researcher’s estimates indicate a cost of up to R$ 10,000 with this procedure, which could fall to one third of this amount, when commercially available. “These examination s are expensive today and are not covered by the health insurance plans, but with time this is going to be worthwhile, since the costs with an early diagnosis are lower than the expenses with treating the diseases”.

This line of thought is shared with Nelson Gaburo Júnior, the coordinator of Dasa’s molecular diagnosis laboratory. For him, the methodology achieved with the researches centralized on the Cancer Hospital should attain great applicability, since besides benefiting patients, it may increase the level of knowledge about diseases and their possible treatments. “To provide the service, Dasa intends to involve as many technicians as necessary. The tests are already being offered, and our expectation is that the number of requests will grow rapidly”.

Lower costs
Reducing the costs of the genetic tests and making them accessible to the population is also the expectation of the researchers from another Cepid, USP’s Human Genome Studies Center (CEGH-USP). To do so, the center is seeking to sign an agreement with the Ministry of Health, for patients of the Public Health System (SUS in the Portuguese acronym) to be able to carry out genetic tests for neuromuscular diseases and other genetic diseases without additional costs.While a nationwide agreement is not established, the CEGH is already carrying out free tests, thanks to an agreement signed with the São Paulo Secretariat for Health, in partnership with the Brazilian Muscular Dystrophy Association (Abdim). Furthermore, a partnership is now being formed with the Fleury Laboratory, also in São Paulo, which will start to offer the tests to its associates. The attempt is thereby being made to obtain resources to help maintain the center, where molecular studies have been carried out for 15 years, with support from FAPESP.

The CEGH attends to between 50 and 100 patients a week, besides carrying out dozens of genetic tests, a quantity that from now onwards tends to increase, according to Mayana Zatz, the coordinator of the center. “The agreement with the Fleury provides for the laboratory to collect the samples and forward them to the center, where the tests will be done. The transfer of technology will take place by means of courses, but the results of the tests, at a first instance, will continue to be produced here at the genome center”, the researcher says. For her, the greatest benefit of this methodology will be the reduction in the costs of the examinations, which may fall by more than 50%.

International landmark
There are currently ten research groups in activity at the CEGH, from which specific exams for over 30 diseases have resulted. Among them is one for neuromuscular diseases that has become a benchmark in Latin America. The group developed genetic tests applied to dozens of neuromuscular diseases. One of them is Duchenne’s muscular dystrophy, a disease caused by a gene that causes progressive weakness of the muscles and manifests itself in the first years of life, leading to the impossibility of movement.

Located in the X chromosome, the gene that causes the disease is only found in women, and it may or not be passed on to the children. By detecting a genetic alteration in a boy, the exam reveals whether the child’s mother is a carrier of the gene (which occurs in two thirds of the cases) and if there is a risk of hereditary transmission to other children.The test uses PCR equipment to analyze the DNA and to locate the exact spot where the mutation happens that causes the disease. In the majority of cases, what occurs is a deletion, that is, the lack of a piece of the gene, the most common molecular defect in this case of muscular dystrophy.

Besides Duchenne’s dystrophy, the group is also researching into dozens of other forms of muscular dystrophies (there are over 30 of them) and other neuromuscular diseases, like the Charcot-Marie neuropathy, which causes atrophy in the lower members. Unlike Duchenne’s dystrophy, the most frequent cause of the Charcot-Marie disease, which usually attacks adults, is the duplication of a gene, that is, there is an excess of genetic material. This happens because, instead of having two copies of the gene (one inherited from the mother and the other from the father), the person affected has three copies. This extra copy alters the myelin sheath (which covers the nerves), causing a secondary atrophy of the muscle. Another neuromuscular disease that is being researched is progressive spinal atrophy, which in its more serious forms causes difficulties for holding the head up, for sucking and swallowing, and even for breathing, due to the weakness of the respiratory muscles.

All the examinations available at the Human Genome Study Center were developed from research projects, which works like a sort of quality control. Some 80% to 90% of the cases of neuromuscular diseases can be diagnosed with molecular examinations, which is extremely important for the timely treatment and prevention of new cases following genetic counseling. Furthermore, molecular diagnosis avoids invasive and not very informative procedures being carried out.

The coordinator’s prospect now is for the tests to be offered to the population without additional costs. “It was FAPESP’s support for the projects that has made it viable for the university to contract technicians. We were able to discover which the most frequent mutations in the population are and to develop tests to detect them, but for these benefits to reach everybody, it is still necessary for SUS to decide to offer them in its program for attending to the public.”

The projects
1. Human Genome Study Center; Modality Research, Innovation and Diffusion Centers (Cepids); Coordinator Mayana Zatz – USP; Investment R$ 1,000,000.00 a year
2. Antonio Prudente Cancer Research and Treatment Center; Modality Research, Innovation and Diffusion Center (Cepids); Coordinator Ricardo Brentani – Cancer Hospital/Ludwig Institute; Investment R$ 1,100,000.00 a year