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Molecular Biology Interlude

After sequencing it will be the turn of creating the genomics of pathology and pharmacology

Reinaldo José Lopes

“It began with the double helix and finished up with the human genome”, James Watson used to say when he was heading the enormous effort to sequence the chemical lettering that goes to make up the genetic information of human cells. For the co-discoverer of the DNA structure and at that moment the leader of the Human Genome Project (PGH), one thing was the logical consequence of the other. If Watson and Francis Crick had managed to uncover the secret of life with their elegant model of the double helix, the key to the understanding of how this secret manifests itself in the organism of man, could only be there, in the very sequence of the DNA. Obtaining it would mean the discovery of what it is to be human and would usher in a new era for medicine.

After more than ten years and three billion pairs of nucleotides (the units that make up DNA), the panorama is far from being as simple as Watson had imagined, especially when dealing with using the mass of information obtained with the genome in order to improve human health in the short term. “There were many scientists, whether through innocence or lack of vision, who had believed that the PGH would settle questions such as ?what does it mean to be human?”, Sérgio Danilo Pena, a geneticist at the Federal University of Minas Gerais (UFMG), ponders.

“They are hawks, the Donald Rumsfelds of science”, Pena states, comparing this type of researcher to the hard-line Secretary of Defense in the administration of George W. Bush. “There was complicity of the press and of the public itself in this exaggeration. But the mistake was only temporary: in the medium and long term period, the fruits of the PGH will undoubtedly be extraordinary.” Even then, up until the present and the near future they are boiling with possibilities, scientists suggest.

If the fantastic cures must be discarded as a distant dream or even as being unreal, it is in the understanding and prevention of innumerable diseases that the genomic data can make the difference, denouncing in a much more evident form what is wrong in the organism and suggesting forms of encircling the problem. The Brazilian leap ? Currently it is difficult to imagine Brazil out of the potential scientific revolution that the genomic studies promise to bring about. However, this was exactly the situation of the country up until 1997. At that time, the indicators of Brazilian research production made it clear that something had to be corrected in the area of genomics.

Suffice it to say that in spite of the increase in production, reflected in the number of articles published in scientific periodicals indexed through the data base of the Scientific Information Institute (ISI)and practically doubling between 1981 and 1995, the growth in the area of molecular biology had multiplied by a factor of 1.69 ? less than the world average during the same period that was of 1.89. It was necessary to act to claw back the delay. It was with this strategic vision that FAPESP?s Genome Project came about. But it was not possible to imagine during its launching that success would come so quickly. Willingness was not in short supply.

Proof of this was the initial value destined to the sequencing of the genome of theXylella fastidiosa bacterium, which causes the feared yellowing in the orange groves of the State of Sao Paulo: US$ 12 million, nothing less than the highest value conceded up until that point in time to any scientific project in Brazil. The intention of this pioneering project, officially announced in October of 1997, went beyond sequencing for the first time a microorganism that causes sickness in plants (phytopathogen), an important bacterium for Brazilian agriculture. The idea was to train people and research institutions to deal with this novel situation, at least for we Brazilians, of genomic work on a large scale.

This was only made possible through the integration of almost two hundred researchers from thirty institutions in the ONSA network (Organization of Nucleotide Sequencing Analysis) ? a type of virtual institute whose organization was something as unprecedented in the country as were its objectives. Other projects such as the Sugarcane Genome and the Human Cancer Genome quickly came on line with that of theXylella , and they have progressed at a much faster rhythm than anticipated. Started in 1999 with a partnership between FAPESP and the Ludwig Cancer Research Institute, the Human Cancer Genome highlighted itself by making use of an innovative methodology for identifying genes.

The biochemist Andrew Simpson and the biologist Emmanuel Dias Neto, both at that time researchers with the Ludwig Institute, developed a new system of sequencing, which, instead of analyzing the entire gene, centered its efforts in decoding the truly active part of the gene, its central portion, responsible for the production of proteins ? we are dealing with Orestes, the abbreviation forOpen Reading Frame Expressed Sequence Tags .

The return from this technique in terms of understanding the most common forms of cancer in Brazil ? such as that of breast cancer and those of the head and neck ? will still take a long time to be totally evaluated: more than one million sequences of genes active in human tumors were generated, some of which are already being identified as important indicators of the degree of gravity or the precocious appearance of the cancer. The consecration of these efforts occurred with the conclusion of the sequencing of theXylella genome four months ahead of schedule, in January of 2000.

Honors by the government of the State of Sao Paulo and by the then Brazilian president Fernando Henrique Cardoso, marked the conclusion of the project. But the greater recognition came from the international scientific community itself. For the first time in one hundred and thirty one years, Brazilian research was on the front cover of the prestigious British scientific magazineNature , which published the article about the genome of the bacterium in its issue of the 13th of July 2000.

Another British weekly,The Economist magazine, had no doubts as to the significance of these results: for the magazine, Brazil was now famous for three reasons: samba, football? and now genomics. It was not an exaggeration. With these programs, now engrossed through initiatives such as the Brazilian Genome Project, financed by the National Council for Scientific and Technological Development (CNPq), the country turned itself into the second largest depositor of DNA sequences at the GeneBank, the public data bank used by researchers of sequencing projects throughout the worlds. Even without having been invited, as Andrew Simpson usually jokes, Brazil had entered into the festival of the human genome. The challenge of discovering how to apply this data had begun.

Potential

One of the immediate advantages of having a complete map of human genetic material is to multiply the chances of finding a gene involved in a malady. “Let?s say that I had attempted to identify a gene on a determined region of a chromosome. Without the sequencing it would be as if I had arrived at a town district about which I had no information and I had to find a specific house”, Mayana Zatz, from the Study Center for the Human Genome of the University of Sao Paulo (USP), compares. “With the sequencing, I can investigate the region that interests me and find the most probable candidates ? and not only find the house, but the bricks that are missing from it”, Mayana, who studies neuromuscular diseases of genetic origin, explains. “The identification of genes linked to various genetic illnesses will be shortened since the genes are already physically mapped”, Fabrício Santos, from UFMG, says.

“What is missing is to discover their functions.” “Genomic analysis has been compared to attempting to open a door by testing thousands of keys, one by one”, Sérgio Verjovski-Almeida, from the Chemical Institute of USP, stated. “Before the sequencing of the human genome, we had no idea at all where even the keyhole was.” On the other hand, the study of the genome is revealing unexpected complexities in the functioning of genetic material, some of it with direct impact on the health of human beings.

“We are working out mysterious mechanisms”, Mayana says. One of these processes can be observed with the so-called dynamic genes, which increase in size from one generation to the next. This mechanism is associated to more than twelve illnesses such as myotonic dystrophy ? a neuromuscular disorder that, in general, leads to the loss of strength in the hands. “This disease seemed to worsen for each generation in the same family, with forms that run from the precocious appearance of cataracts and baldness to a generalized muscular weakness that can reach the stage of incapacitating the victim”, Mayana explains.

“What has been discovered is that an increase in the number of a trio of nucleotides (the units that go to make up DNA) was involved”, the geneticist says. While healthy people have from 5 to 37 of these trios in the gene, the carriers of the infirmity show from 50 to thousands of repetitions of this small sequence. The problem is that when there are more than 50 trios the gene becomes unstable and the repetition tends to increase from generation to generation, aggravating the illness. On the other hand, alterations in different genes can cause the same clinical problem. “For example, this occurs in muscular dystrophy of the waist, to which fifteen genes that codify different proteins, have been associated”, Mayana explains.

“Some of these proteins act in conjunction, forming a complex. If there is a defect in one of them the working of the complex as a whole is jeopardized”, the researcher explains. In spite of the fact that the working of the genes is showing greater and greater complexity, the abundance of data about the genome can precociously and precisely denounce the most complicated illnesses from the genetic point of view, brought about by various factors, such as the innumerable forms of cancer. “With this information it is possible to study thousands of genes that act together”, explains Verjovski-Almeida, who investigates factors that determine the gravity of prostrate cancer.

“Today we know of one hundred and seventy genes related to the malignity of prostrate cancer”, the researcher from USP relates. “Sixty percent of them are new genes, identified through sequencing on a large scale of the genetic material extracted from the tissue affected by the cancer.” The simultaneous analysis of hundreds of these sequences, carried out by way of microchips of DNA (small glass laminates that show up the activity of the genes), has already allowed the creation of a molecular profile of individuals and indicates the probability of their showing more severe or bland forms of the illness.

“A microchip for breast cancer, created by Laura van?t Veer, from the Dutch Cancer Institute, has already been transformed by the company Rosetta Inpharmatics into a method of diagnosis in the United States”, the researcher goes on. The very genetic standard of a tumor itself allows for anticipating its gravity and the chance that it will spread itself into other organs. The molecular diagnosis of the more simple genetic illnesses, caused by a single gene, has obvious advantages. “In practical terms”, Mayana Zatz states, “the identification of these infirmities through a DNA test avoids painful and complicated procedures, such as biopsy or electromyography.”

In spite of there being confusion in the relationships behind multifactor diseases, an understanding of the genetic propensity for the development of an illness can also be helpful. “I would not like to know if I have an increased risk of developing Alzheimer?s disease, for which there is currently no known treatment”, Mayana suggests. “But I would certainly like to know if I have a tendency towards diabetes (a multi-factorial illness) because I could look after myself and reduce the influence of the environmental factors”, the geneticist ponders. “With a more reliable and precise diagnosis, the chances of success of the treatment are enhanced”, Fabrício Santos sums up.

Another important point, according to the researchers, is that molecular diagnosis can liberate doctors from relatively gross methods of detecting illnesses. “In the case of prostrate cancer, even today you depend on the alteration of the morphology of the tissue. There is not a single molecular marker really specific to it”, Verjovski-Almeida explains. “We speak of breast cancer as a single infirmity. However, more recently we have learned through the course of genomic understanding that breast cancer is not one but various illnesses”, Sérgio Pena says.

Tailor-made medicine

Always provocative, the scientist -entrepreneur, Craig Venter, responsible for a parallel sequencing of the human genome carried out by the company Celera Genomics, says that in the future a person could obtain the sequencing of his or her very own genome at a cost of US$ 2,000. Thus he could understand with anticipation the problems that he could face during his life and bring about strategies to combat them. “Perhaps this goal will be unreachable, but it would be interesting to find an intermediary path”, Verjovski-Almeida evaluates.And, it is in the genetic variation between people that the key for more efficient medicines could lie.

“If the sequencing of the genome helps to demonstrate that we are all equal, or all equally different, it also reveals the differences that may be important for treating illnesses”, Mayana Zatz advises. Since everyone has descended from a small African population that lived close to one hundred thousand years ago (a mere blink of the evolutionary eye), the human beings from any region of the planet are all very similar. But the short-term adaptation to the very different environments has created varied profiles of resistance or susceptibility to diseases. And it is this variation that genomic medicine, the science that relates genetic profile to the response to medicine, promises to explore in favor of human health.

“We know that the doses of medicines recommended on the directions are only gross suggestions, made on a basis of population averages”, Sérgio Pena says. Genomic philosophy – “Ethical differences can influence the manner in which people respond to a medicine”, Mayana says. The researcher tells that, in a study carried out by her and her colleagues at USP, it was verified that a gene linked to the transporting of a neurotransmitter, named serotonin, has two distinct forms within the Brazilian population.

One of them, the so-called long allele that rapidly breaks up serotonin, appears in 80% of the people, while the other, short allele, only appears in 20% of them. “But in the people of Japanese origin, the proportion is inverted”, Mayana reveals. This could be extremely important when projecting a medicine capable of interfering in this process. There are some genes in which variations of this type are already known and could be tested to avoid adverse reactions. At the same time, the map that shows the genetic predisposition of a person to acquire illnesses cannot become public property, the researcher alerts. “Insurance companies, employers, everyone will want to know my chances of getting a disease”, she says.

Mechanisms for genetic privacy will have to be created so as to avoid discrimination carried out on the basis of the genome coming into the labor market. The optimism of researchers runs into a brick wall when one deals with the use of DNA sequencing data for therapies that have as their target the genome itself. “I am skeptical as to the viability of gene therapy”, Verjovski-Almeida recognizes. Carlos Menck, from the Biomedical Sciences Institute of USP, closely understands the difficulty of applying the technique even in more preliminary tests. “We managed to treatXeroderma pigmentosum , an illness of the skin that causes terrible lesions and even cancer because the patient doesn?t manage to repair his DNA”, Menck tells.

“However, we ended up confronting limitations of the adenovirus itself that serves as a vector for the corrected gene. After some time, the patient?s immune system creates resistance and the treatment no longer functions.” It could be that this changes and it will be possible to carry out prevention, creating some sort of palliative for the patient. “But it will not be a panacea”, Menck underlines. Even when things seem to work well, as in the case of the “bubble babies” in the United States and France, who suffer from a severe form of immunology deficiency, the genome is a system so complex that even supposed beneficial alterations have an unforeseeable effect: some of these children, cured of the disease, contracted leukemia because of the gene therapy. “Our understanding with respect to cellular regulation and its interaction with the genome is still at the minimum level”, Fabrício Santos says.

“Any genetic therapy, such as that of the bubble babies, will be through trial and error, since we do not have sufficient control of the variables with which we are dealing.” Sérgio Pena also recognizes that there is as yet a huge uncertainty in genomic manipulation and reinforces the idea that we might “perhaps we will abstain forever from attempting to make modifications in germinating human lineage”. Modifications in germinating lineage, or that is to say, our eggs and spermatozoids, which transmit the genetic material to the next generation, will make genetic alterations transmissible, with potential effects that are even more dangerous.

For the time being genomics should restrict itself to providing assistance for the invention of more specific drugs that act directly on the coded protein of one gene involved in a disease. Another hope, to be corroborated, is the technique known as RNAi, interference of the RNA, another type of genetic material. Potent and specific, it acts upon a type of RNA, called messenger RNA, which conducts the information contained on the DNA and starts the production of proteins.

Tests on plants, on the wormC. elegans and on cellular strains suggest that RNAi will be capable of deactivating almost totally the desired gene without directly affecting and without influencing other genes. “It is perhaps one of the most important discoveries of modern biology”, Pena suggestes. One thing seems certain: even without new sequencings the data already obtained through the human genome and other important organisms has only just begun to be interpreted in an adequate manner. “Large scale sequencing is likely to continue for some time but we shall have to use more refined hypotheses in order to justify them”, Menck evaluates. “In particular, I am a fan of comparative genomics that places different organisms side by side in order to see what regions of the genome are conserved and therefore are important to them”, he says.

In this manner, winning the challenge of identifying all of the human genes whose very number appears uncertain should become much easier. For Menck, an effort to strengthen and diversify bioinformatic studies in Brazil is also required, so that these computer analyses of the genome accelerate the task of identifying and understanding the differences in genes. “I look upon the genomic efforts as an interlude in the history of molecular biology”, Pena philosophizes. “The part of the sequencing of the PGH is partially finished and is giving us the anatomy of the human genome. Now we are going to spend the next century developing genomic physiology, genomic pathology and genomic pharmacology.”

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