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Needle in a haystack

Unicamp team identifies the genetic mutation that produces a rare blood syndrome

The Rh null syndrome has two causes. The most common results from a gene mutation that does not have a direct relationship with the Rh antigens, but is responsible for the synthesis of a protein that interacts with the Rh protein in the membrane of the red blood cells. With this alteration, the antigens are not expressed. The second cause, rarer, occurs through a defect in the gene responsible for the Rh system. There had been only  three cases of this type detected in the world. Leticia’s mother was the fourth. The genetic sequencing of blood samples of Leticia’s mother, her grandparents and aunts, led to another discovery. The mutation that originated the problem in the Curitiba family had never been described. It is installed in a chromosome region different from the other three cases observed in the world, one in France, one in the United States and one in Japan. Such alterations were described starting from 1998, thanks to improvements in molecular diagnostics.

The researcher Lilian Castilho specialized in immunohematology at the National Reference Center for Blood Groups, in France, and carried out her post-doctoral work at the New York Blood Center, in the United States. At Unicamp’s Blood Bank she coordinates the only public laboratory that carries out the molecular biology of blood groups, acquired thanks to the assistance of FAPESP. “Blood Banks of various cities search us out because they know that we’re a molecular biology center for blood groups”, explains Lilian. The group’s major ambition is to set up a national bank of blood donors capable of improving the safety of transfusions in the country. The idea is to submit blood samples of donors to the technique of type casting of DNA and to register the donors of rare blood types.

The genetic composition of the blood of 500 donors in the Campinas region can already be found registered at the Unicamp Blood Bank. This process will gain agility with the recent acquisition of a piece of equipment, based on the microarray technique, or chip technology. By way of this technique genetic blood samples or secretions are investigated by microscopic DNA probes contained in sensors set in special glass slides with different genes of blood groups. Specific equipment and software monitor the process and, when the genes being looked for in the samples are located, the chips emit a fluorescent signal.

“We need to study the Brazilian population much more”, says Lilian Castilho. “There are variations of groups that predominate amongst us and are not frequent in other countries”, she points out. The mapping of rare donors, Lilian says, as well as pushing forward research in the field of hematology, will give more security to transfusions and will help in the prevention of dramas such as that of Ana Maria da Silva, who gave blood for young Leticia. After her gesture of solidarity, Ana Maria herself needed a blood transfusion. She was only saved because the doctors who had treated her managed to obtain from England a sample of the extremely rare blood, Rh null.

On October 18th, a few hours after having been born in the city of Curitiba, Leticia Laudino was taken to the neo-natal ICU with hemolytic anemia – her red blood cells were being destroyed by antibodies inherited from her mother. The immediate diagnosis was of erythroblastosis fetalis, the illness that attacks children with blood type Rh positive whose mothers are Rh negative. In favor of this thesis had been the fact that Leticia’s mother, Debora, had had her blood classified as A Rh negative. The doctors applied the standard treatment – an Rh negative blood transfusion capable of blocking the hereditary antibodies and interrupting the attack. But the anemia, instead of chilling down, got worse. The action of the doctors over the next 72 hours not only saved the young girl’s life but gained registration in international medical literature. After the second crisis, blood samples from Leticia and Debora were submitted to advanced tests at the Blood Bank of Curitiba, which revealed an extremely rare characteristic. They were not Rh positive or Rh negative, but or Rh null, a condition that affects one in every 6 million people and which becomes a nightmare when a transfusion is necessary. An Rh null can only receive blood from another Rh null.

The Rh blood group system has 49 antigens, proteins that can lead to the formation of antibodies capable of interacting with red blood cells. The most important of them is known as RhD. When one says that an individual is Rh positive, it means that the RhD protein is present in the membrane of its erythrocytes – this is the case of 85% of human beings. Rh negative individuals do not possess this specific protein – but the erythrocytes, in spite of this, are normal. However, Rh null individuals, as well as having the absence of the RhD antigen, also do not express other antigens of the Rh system – and, for this reason, suffer from a fragility of red blood cells that causes a kind of anemia that is more serious.

Research made upon the family showed that as well as Leticia’s mother an aunt of the young girl also had Rh null blood – but had an incompatibility in the ABO antigen system. Leticia had blood type O, while her mother and her aunt were blood type A. In search of a solution, the Curitiba doctors called the biologist Lilian Maria de Castilho, a professor at the State University of Campinas (Unicamp), responsible for a pioneering study in the field of molecular biology into the Rh system. Lilian had just completed her participation in a congress where she had learned that a Rh null  carrier in Rio de Janeiro with the same ABO type as Leticia had been registered. The drama continued. The telephone of Ana Maria da Silva, who lives in the town of Nova Iguaçu, in the greater Rio de Janeiro rang and she did not back away from the required humanitarian gesture. Her blood was quickly collected and taken to Curitiba. Leticia survived.

Two years later, in November of 2005, research based on Leticia’s case was published in the scientific magazine Transfusion, signed by professor Lilian Castilho, her master’s degree student Karina Rosa, as well as doctors from Curitiba and Rio de Janeiro along with the New York researchers who participated in the investigation. By way of DNA analysis, the original genetics of the illness was discovered – Leticia’s mother and aunt acquired the Rh null syndrome because they are the fruit of a consanguinity marriage. The grandparents of the child were cousins. The characteristic, amplified thanks to the union between relations, gained expression in the blood of two of the women and was only discovered with the birth of Leticia. The child’s mother was advised not to become pregnant again. In a second pregnancy the chances are very high that the attack of the antibodies would be even more intense.