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Unambiguous identity

Laboratory at USP discovers the genetic causes and the forms of treatment of rare hormonal alterations

An award granted in the United States for the first time to a Brazilian researcher, 39-year old endocrinologist Ana Claudia Latronico, called the attention to the scientific productivity of a team from the University of São Paulo (USP) that is dealing with an extremely rich material. Every month, the group from the Endocrinology of Development Unit at USP’s Hospital and Clinics attends to 400 people who come from all over the country with hormonal disturbances that prevent children from growing normally, interfere with puberty in adolescents or generate anomalies in the development of the sexual organs, such as hermaphroditism, in which the individual is born with ovaries and testicles, as well as poorly defined external genitals.

Such a diversity of patients and the opportunity for treating and accompanying them has transformed the unit into an international point of reference as a research center. It is there that, under the supervision of Berenice Bilharinho de Mendonça, Ana Claudia has been taking part for 12 years in a series of pioneering studies that are describing new genetic mutations that cause hormonal diseases. It is the set of these works that has earned Ana Claudia the Richard E. Weitzman Award, granted in June by The Endocrine Society, of the United States, to researchers who are under 40 years old.

The team of 25 researchers led by Berenice most of them women, as it is common in the area of endocrinology faces every day the challenge of showing those who turn up there in search of assistance that, in general, it is possible to treat these genetic problems that are marked by strong prejudice. This is the case of identifying the sex of individuals who are born with ambiguous genitals with male and female structures, whether complete or not. This problem comprises three groups of different diseases.

One is the true hermaphroditism, in which the individual has ovaries and testicles and external genital organs with male and female structures, known as genital ambiguity. In genetical terms, the majority of true hermaphrodites have, in each cell, two X chromosomes normal men have one X chromosome and one Y, and women have two Xs. They ought, therefore, to be women. But how ist he development of testicles to be explained? This is probably due to alterations in as yet unknown genes that act like the SRY gene of the Y chromosome, which is responsible for the formation of the testicles.

The second group is the male pseudohermaphroditism: from the genetical point of view, the individual is a man (XY), but the penis does not develop completely and the opening of the urethra is in an abnormal position. “Pseudohemaphrodites ought to have been normal men, but they were not virilized completely, because of defects that occurred in the course of embryonic sexual development”, Berenice says. Up to the second month of gestation, she reminds us, men and women have identical genitalia, what differentiates them, from this moment onwards, is the action of the male sexual hormone, testosterone.

Pseudohermaphrodite women constitute the third category: they have two x chromosomes and the complete female reproductive apparatus, like any woman, but during intrauterine life they underwent a process of virilization of the genitals: the clitoris grows excessively and appears like a structure similar to the penis.Since 1976, the cases of genital ambiguity diagnosed at this unit of USP’s Hospital and Clinics have reached 500. The most frequent cause responsible for one in every three cases is a disease with a complicated name, congenital virilizing adrenal hyperplasia. Mutations in the CYP21A2 gene result in the deficiency of an enzyme, 21-hydroxylase.

The shortage of this enzyme prevents the synthesis of cortisol, a hormone produced in the suprarenal glands. In a chain reaction, the lack of this hormone activates a gland at the base of the brain, the pituitary gland, which intensifies the production of another hormone, the corticotropin, which stimulates the activity of the suprarenals. In response to this stimulus, the suprarenals increase in size and produce more testosterone, the male hormone. In fetuses of the female sex, the excess of this hormone causes virilization: women are born with a hypertrophied clitoris, which is reminiscent of a penis, and a scrotum without testicles, which completely covers the vagina.

But the internal reproductive apparatus and the chromosomes are those of a woman, characterizing a classic case of female pseudohermaphroditism. “Sometimes, even in the nurseries of large maternity hospitals, this kind of baby is registered as a boy”, Berenice says. Without the help of hormonal examinations and determining the sexual chromosomes, doctors are mistaken and imagine that they are dealing with a baby of the male sex whose testicle are only going to come down later into the scrotum. In the fetuses of the male sex, mutations in this gene only mean that there is an abnormal growth of the suprarenal glands, without any alterations to the external genitals.

Over the last four years, two researchers from this team, Tânia Bachega and Ana Elisa Billerbeck, have discovered five new mutations of CYP21A2, as part of the results of the two thematic projects supported by FAPESP. Accordingly, the analysis of genetic mutations connected with sexual diseases has become integrated with prenatal diagnosis, thus making it possible for women who have already had a pseudohermaphrodite daughter to prevent the problem from repeating itself. In the third month of pregnancy, the mother-to-be is submitted to an examination of a fragment of the placenta, which will reveal whether the embryo is of the female sex and if it shows any alteration in the CYP21A2 gene.

If the test indicates mutations, Berenice’s team knows how to avoid the consequences: the mother has to take a medicine, the dexamethasone, which prevents the girl’s sexual organs from looking like a boy’s. Reaching the fetus through the bloodstream, the dexamethasone swallowed by the mother inhibits excessive production of the adrenocorticotrophic hormone (ACTH), which impedes the exaggerated production of the male sexual hormone. In this case, the Brazilian team carried out molecular studies and has attested to the effectiveness of this treatment, created in the mid-60’s in France, in Brazil.

Early puberty
Sexual identity is crystallized around the age of 2, and it is very complicated to change it. For this reason, Berenice warns, it is essential to identify any dysfunction early, to avoid traumas to the parents and child. But, regrettably, late diagnosis still occurs frequently in Brazil. Half the cases of genital ambiguity that arrive at the HC’s clinic are adults. In these cases, defining sex becomes an issue that is not limited to genetics. It may be written in the nucleus of each cell whether an individual is a woman, if two X chromosomes are shown, but what can be done if the person was brought up as a man and developed a male identity? Berenice’s team came up against a case of this kind in June, when a 20-year old true hermaphrodite appeared, with male and female genitals.

She had been registered as a woman, but brought up as a man. He (the name cannot be disclosed, out of respect for privacy) arrived at the HC after a calvary of public embarrassments. Born in the interior of Pernambuco, he came to São Paulo in search of treatment, and ended up in one of those evening television programs, which dealt with such anomalies like an attraction in a circus. The program offered him medical treatment, but he would have to tell his girlfriend, on the air, that he was a hermaphrodite, she did not know.

Taking advantage of the exposure from the program, a doctor said that he was willing to treat him, for free. This moral violence only ended when the young man did the examinations to start treatment, and another doctor, an anonymous hero, passed him on to the Hospital and Clinics. The analysis of chromosomes identified two X chromosomes, despite the ambiguous genitals shown. Genetically, then, he was of the female sex. As he had adopted a male identity, he himself opted to submit himself to the removal of uterus, ovaries and vagina, and to what is known as masculinization (transposing the urethra to the glans) of the external genitals.

With some frequency, researches by this group from USP reveal the origin of diseases that are rare or are mistaken for others, and this encourages the search for new treatments. For example, a study carried out by physician Sorahia Domenice, published in September 2001 in the Journal of Clinical Endocrinology and Metabolism, showed that an already known mutation of the DAX1 gene produces broader manifestations than used to be believed. The main symptoms of the disease were the precarious functioning of the suprarenal glands and delayed puberty. Two years ago, the team from the HC described another mutation in the DAX1 gene, found in a boy, which brought about, not a delay, but early puberty, with sexual features arising before the age of 3.

In a study that had great repercussion, published in 1996 in the New England Journal of Medicine, the researchers from the HC described a genetic defect in the receptor of the luteinizing hormone (or LH) produced by the pituitary gland, which stimulates the testicles to make testosterone. With the mutation in the receptor of LH, the organism does not produce testosterone in adequate quantities, and the fetuses, despite having male chromosomes, would be born with female or ambiguous genitalia. One year after researchers from University Hospital Nijmegen, in Holland, had published the first description of a mutation in the receptor of LH causing male pseudohermaphroditism, the Brazilian study showed that the spectrum of this anomaly was wider, when it described a boy with a micropenis and a hitherto unknown effect: the sisters of the bearers of male pseudohermaphroditism, due to the mutation of the receptor of LH, are infertile, with irregular or absent menstruation.

This year, physician Regina Martin proved the genetic origin of another rare ailment, the excess of aromatase, an enzyme that transforms testosterone into estrogen, the female hormone. This genetic dysfunction results from the excess production of estrogen, and it makes boys show a micropenis and to develop breasts, while girls suffer from an increased uterus and exaggerated growth of the breasts. In all cases, the syndrome causes short stature. The study that describes the fifth family found in the world with this disease, and the first in Brazil came out in June in the Journal of Clinical Endocrinology and Metabolism.

Short stature
One month before, in this same magazine, Maria Cândida Fragoso published research into the identification of two mutations that can cause the ACTH-independent Cushing’s syndrome, a disease characterized by the appearance in the suprarenals of nodules that produce in excess the cortisol hormone, which is a regulator of the metabolism of sugar and of the blood pressure. The excess of this hormone in the circulation causes a series of side effects, such as obesity localized in the middle of the body, a face the shape of a full moon, atrophy of the skin, and high blood pressure. According to Berenice, the discovery of the genetic mutations does not alter the course of the treatment which consists of the removal of the suprarenal glands, followed by hormone replacement, but it does help to understand this disease, which can be caused by several different mechanisms.

Until then, only one cause of the ailment was known: some people would produce cortisol in excess, due, for example, to the peptide called GIP, secreted by the pancreas in response to the arrival of food in the digestive apparatus. Another specialty of the group from the HC is the study of endocrinological diseases that lead to short stature, which affects 3% of the population. In this area, a work which had a great repercussion because of its applicability was carried out last year by Alexander Jorge, who is studying for his doctorate. He showed the fragility of the test most used to diagnose cases in which the applications of growth hormone do not seem to work in correcting short stature. Jorge found out that it lacked reproducibility: two tests with identical samples did not show the same results.

This year, in another research project, Eveline Gadelha, a former pupil of Berenice and today a professor at the Federal University of Ceará, assessed the precision of the exams that formed the basis for diagnosing a growth hormone deficiency and came to a conclusion: the diagnosis in the laboratory may often be wrong. The researchers, who published the results of this study in May, in Hormone Research, assessed 30 normal children and 26 with a growth hormone deficiency, to define the cut point for the most modern methods: hormonal deficiency can only really be attested when the hormone concentration is below 3.3 nanograms per milliliter of blood serum. According to Berenice, doctors still use a cut point of 7 nanograms, valid for older dosage methods, with less sensitivity.

“There are children with a diagnosis of short stature who are taking hormones without having any endocrinological problem”, the researcher warns. “They are shorter than average due to the genetic heritage from their parents.” For boys or girls who are 4 or 5 years old, for example, growth hormone deficiency means being 7 or 8 centimeters below the average expected height. A student of Ivo Arnhold studying for a doctorate at the same laboratory, Luciani Carvalho, recently discovered a new mutation in HESX1, a gene that interferes with the development of the pituitary gland.

The defect was already associated with a very serious manifestation called septo-optic dysplasia: its victims are born blind and with alterations in the central nervous system. The contribution from this study, to be published in the Journal of Clinical Investigation, was the discovery of a blander manifestation of the same disease, when the error occurs in a different region of the gene. The case that provided the basis for this pioneering research is a typical example of the routine at the HC. In 1985, Berenice’s team attended to a little 5 year old girl who came from Recife, with a growth deficiency she was 87 centimeters tall, 20 less than she ought to be. It was a serious deficiency in the production of hormones, which it was possible to treat with replacement. To grow, the girl took growth hormone and today, at the age of 23, she has reached a height of 1.65 meters. She developed a uterus and breasts and menstruates regularly.

The researchers have now discovered in this girl’s DNA sample the exact origin of the disease: a mutation in the HESX1 gene. Its altered version only expresses itself when it is inherited both from the father and from the mother, which rarely occurs. It happened in this case because the girl was the fruit of a consanguineous marriage, the parents are cousins. Before this discovery, her case would be lost in the midst of hundreds of others. In the last decade, Berenice’s team has accumulated some 300 DNA samples of Brazilians of short stature of unknown origin and knows that it has in its hands a genetic treasure that hides stories like the one of the little girl from Recife.

The projects
1. Molecular Diagnosis of Sexual Differentiation Disorders (nº 97/01196-1); Modality Thematic project Coordinator Berenice Bilharinho de Mendonça; Investment R$ 648,094.20
2. Molecular Diagnosis of the Alterations of the GHRH, GH and IGF1 axis (nº 00/14092-4); Modality Thematic project; Coordinator Ivo Jorge Prado Arnhold – USP; Investment  R$ 1,565,450.00