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Effects of diversity

Intense miscegenation makes Brazil a fertile field for hereditary anemias

eduardo cesarOne of the stages of the tests: in search of altered forms of hemoglobineduardo cesar

There is a drop of blood in each poem, once said the poet Mario de Andrade. There is also a drop of blood – in the real sense and the metaphorical one – in each discovery made or supervised by physician Fernando Ferreira Costa, from the State University of Campinas (Unicamp). His team has been reaching conclusions that express and try to treat the consequences of the intense miscegenation of the Brazilian population: the continuous interaction between the descendants of Africans, Europeans, and Asians – groups originally little used to inter-racial relations – has favored the concentration of altered genes, responsible for the appearance of modified forms of hemoglobin, the protein that makes the blood a red liquid and distributes oxygen to all the cells of the body. As a consequence, as the racial mixture intensifies, the possibilities of there occurring some hereditary anemias increase.

From 2000 until now, this group from Unicamp’s School of Medical Sciences – made up of the teams led by Costa and by physician Sara Saad, at the Blood Center, and by biologist Maria de Fátima Sonati, at the Clinical Pathology Department – has identified six new variants, as the abnormal forms of hemoglobin are called, a result of subtle deformations in the structure of this protein. Following a tradition in this area, these variations have been given names of the towns in the interior of São Paulo or south of Minas Gerais when they emerged: Joanópolis, Paulínia and Campinas, only found in the newborn and described in November in the Haematologica magazine, and Rio Claro, Poços de Caldas and Campinas, exclusive to adults. Another two are at the stage of publication, also from adults, found in Itapira, in the interior of São Paulo, and in Boa Esperança, in the south of Minas.

The same group has also detected another 37 variants – very rare, with one or two cases described in the world – of this protein, predominant in the red cells of the blood: there are about 600 millions of molecules of hemoglobin in each one of the 5 million red cells in circulation through the veins and arteries. Hemoglobin is also one of the most studied human proteins, with 882 variations now identified.

Altered forms
In adults, each molecule of hemoglobin is made up of four subunits, usually produced in equal quantities: there are two alpha chains and two beta, while in fetuses there is a slightly different structure, with two alpha chains and two gamma. In each one of the chains there is one atom of iron, which links up temporarily with oxygen every time the red cells pass through the lungs – iron is part of a structure known as the heme group, which gives blood its red color. The meeting of altered genes, facilitated by miscegenation, and genetic mutations originate defects in the chains or make the organism produce more of one of them than the other. An imbalance between the quantities of the alpha and beta chains is fatal: it determines the destruction of the red cells and can cause anemia – there are also other kinds of anemia, such as those resulting from a diet deficient in iron, vitamin B12 or folic acid, a relatively common problem in Brazil, which can often be eliminated by the addition of these nutrients tothe food.

The modified forms of hemoglobin sometimes remain in silence, without causing any apparent symptoms, like the eight variants found recently. Even so, identifying precisely the defect clarifies its seriousness and avoids unnecessary examinations and mistaken treatments, since many silent alterations can be mistaken for those that lead to more serious situations. Some of these mutations, though, are dangerous, and may even make a pregnancy inviable, if not diagnosed in time. In 2003, the team from Unicamp found in a woman eight weeks pregnant a silent and extremely rare form of hemoglobin called Indianápolis, which until then had not manifested itself nor been detected in the usual laboratory examinations.

To treat a urinary infection, the woman had taken a medicine that, very probably, caused an acute hemolytic crisis – a massive destruction of the red cells. “Once the infection was cured and the medicine suspended, the situation went back to normal”, says Maria de Fátima, responsible for the laboratory to diagnose the alterations in hemoglobin, located on the second floor of the Hospital and Clinics. A defect in hemoglobin often only manifests itself when combined with another, the same or otherwise. This is the risk run, unwittingly, by the children of couples who are the bearers of silent and asymptomatic mutations. This is what happened to two brothers, referred to Unicamp’s hospital with an anemia classified as moderately serious. As is her usual procedure in these cases, Maria de Fátima asked for blood tests of the whole family.

The father was Chinese and the woman, from the state of Bahia, probably of African descent. Neither of them had reported any problems related to hemoglobin, but three of the couple’s four children showed an unusual form of thalassemia, the hemoglobim H disease. They had inherited from their father a gene with a more serious mutation, common in the Chinese population, and another gene from the mother, with a silent mutation, found in one in four descendants of blacks in Brazil. At the beginning of this year, a case came up – probably the first documented in this country – of the most serious form of alpha thalassemia: all the hemoglobin molecules of the fetus are defective and fail to irrigate with oxygen the organism in formation, with remote chances of survival.

Having been studied at Unicamp for over 20 years, since Costa created the first laboratory of the group that he leads today, the hemoglobins that depart from the standard are an expression of the history of the racial miscegenation that started five centuries ago with the arrival of the Europeans and the practice that anthropologist Darcy Ribeiro, in O povo brasileiro [published in English with title The Brazilian People – the formation and the meaning of Brazil], calls in-lawism – a habit of the natives of incorporating strangers into their family group. As early as the 16th century, the slaves began to arrive, and with them the gene of hemoglobin S, which, in a double dose, causes falciform anemia, a disease that is more common amongst the descendants of Africans and is marked by the intense destruction of red cells, fevers and muscular pains.

Almost 300 years afterwards, now at the end of the 19th century, the ships docked bringing immigrants from Europe, from Asia, and from the Middle East, which broadened the country’s ethnic diversity and helped to form the Brazilian people. It was also at that time that the genes responsible for thalassemia disembarked in Brazil; this is another form of hereditary anemia, which causes the destruction, also very intense, of the red cells, which in the more serious cases are replaced by means of regular blood transfusions. Little by little, the ethnic diversity and the intense miscegenation of the population produced diseases with manifestations similar to those of falciform anemia, even in pale skinned people, as a result of the association between the gene of hemoglobin S and the thalassemia gene.

At the Clinical Pathology unit, biologist Elza Kimura and technician Sirley Gervásio take care of from 150 to 200 diagnoses a month, for people suspected of hemoglobin alterations. In parallel, this same laboratory has been running for five years a program for the triage of rare variants, in which over a hundred blood samples are examined every day, chosen at random amongst the patients at Unicamp’s hospital, without any symptoms related to hemoglobin alterations. This was how the largest database in the country was formed, with some 100,000 persons analyzed to date.

The sharp eyes of the team from Campinas detected, last year, a new mutation that causes thalassemia of the B type, and one case of an unusual form of alpha thalassemia, the so-called hemoglobin H disease, previously described a single time, on an island of the Azores, in 1991. Also in 2003, the researchers identified an asymptomatic and very rare mutation of hemoglobin, the Osu-Christiansborg variant, described for the first time 13 years ago in Ghana, in Africa. The Brazilian case, reported in February this year in the Hemoglobin magazine, was caused by a type of mutation known as de novo: it was not brought from other peoples or lands, but originated in the 10 year old boy himself, without either of the parents being carriers of the genes that could cause it.

De novo mutations can also cause anemia, as happened with a girl who was treated at Unicamp a few years ago. The father was a bearer of an asymptomatic variant called Porto Alegre, while the mother did not have any alteration in the hemoglobin genes. The daughter inherited from her father the gene of the Porto Alegre hemoglobin and suffered from a de novo mutation, which generated the Santa Ana hemoglobin, which, on its own, is asymptomatic. But the sum of the two forms caused a serious anemia, to the point of the doctors opting for a radical treatment to avoid the continued destruction of the red cells: the removal of the spleen, an organ the size of a clenched fist in which the old or defective red cells are eliminated.

Alert to the problems that result from this history, the team from Unicamp may have laid the foundations for new treatments for falciform anemia, which in Brazil is taking on proportions similar to those of some countries from Africa: from 6% to 8% of the descendants of Africans born in Brazil are bearers of the gene that leads to the production of hemoglobin S – a person has to have the two genes for the disease to appear. In the population at large – mixed -, according to a study from the Federal University of Minas Gerais, falciform anemia occurs in one child in each group that varies, according to the region, of from 1,500 to 5,000 births.

In search of alternative
At the moment, one of the only alternatives for combating this ailment is hydroxurea, a medicine also used against cancer, with serious side effects. If the dosage is not strictly controlled, the drug can damage the bone marrow, affecting the production of the red and white blood cells, which make up the immune system. Last year, Nicola Conran, from the team at the Hemocenter, discovered one of the possible mechanisms for the action of this compound. After comparing the chemical reaction of healthy persons with those from two groups of bearers of falciform anemia – one was taking medication and the other not -, she concluded that hydroxurea acts through the intermediary of nitric oxide, raising by as much as five times the quantity of cyclic guanosine monophosphate (cGMP) inside the cells. The cGMP intensifies the production of fetal hemoglobin, predominant in the intra-uterine period, which keeps separate the abnormal hemoglobins of the S type.

Unless they are contained, the S hemoglobins agglomerate in fibers that make the red cell lose its usual shape, like a dried apricot, and turns them into something like a sickle. That is where the name falciform anemia comes from, and is the cause of one of its most serious consequences: the red cells cannot cross through the smaller caliber vessels and oxygenate the tissues. According to Costa, who supervised this study, published in February in the British Journal of Haematology, there are at least five chemical compounds that increase the production of cGMP and may perhaps be less toxic and more efficient that hydroxurea.

But they all have to undergo more tests before being released for treating anemia. Another study, published in Experimental Hematology, also in February, is helping the origin and the development of thalassemia to be understood; it is more frequent in populations of a Mediterranean origin, like Italians, Spaniards, Greeks, and Arabs. In the state of São Paulo, 6% of those descended for Italians are bearers of the gene of thalassemia of the beta type, which, in a double dose, causes serious anemia and an increase in the spleen and the liver, amongst other symptoms.

Camila Santos, who is studying for a doctorate under Costa, has demonstrated for the first time in human beings the role of a protein known by the abbreviation ahsp , which keeps separate the alpha chains that are formed in excess in beta thalassemia and can generate lighter or more serious clinical states. “The ahsp, the production of which proportional to the production of the alpha chains in human cells, is perhaps important for understanding why thalassemia generates more or less serious forms of anemia”, comments Costa, currently Unicamp’s pro-rector for research.

An analysis of the variations in the genes of hemoglobin also reveals the distant history of some ethnic groups. Years ago, genetic analyses confirmed the origin of Brazilian Blacks – Angola or Mozambique, in the case of those who live in São Paulo, or Benin, in the case of those who live in Bahia, as the historians had already documented. In a study published in 2003 in the American Journal of Physical Anthropology, Daniela Ribeiro, a student of Maria de Fátima, confirmed the Asian origin of present-day indigenous Brazilian. She analyzed the sequence of DNA that controls the expression of the genes of the alpha chain of hemoglobin – the largest regulatory element – from two native groups from Amazonia, the Parakanas and the Xikrins.

Following the study of this sequence of DNA, Daniela found that the Parakanas have a genetic affinity both with peoples from Asia and with peoples from Oceania, while the Xikrins show greater similarity with the populations from Oceania. They are more alike the more distant peoples than the neighboring groups, in an indication that amongst these two Brazilian groups miscegenation is almost zero. The problem with them is another one: consanguineous marriages, between uncles and nieces, which increase the risks of hereditary problems arising.

The Project
Hereditary Alterations of the Hemoglobins: Molecular Genetics, Aspects of Clinical Evolution and Production of Transgenic Animals (nº 02/13801-7); Modality Thematic Project; Coordinator
Fernando Ferreira Costa – Unicamp; Investment R$ 1,333,900.00 (FAPESP)