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ENDOCRINOLOGY

Early awakening

Brazilian researchers have identified the first gene associated with a hereditary form of premature puberty

SANDRA JAVERAIt’s been about 7 years since Dr. Ana Claudia Latronico treated a peculiar case that caught her attention at the pediatric endocrinology outpatient clinic of São Paulo’s Hospital das Clínicas (HC). This case, involving a five-year-old girl who already presented the first signs of puberty, would eventually lead to the identification, in mid-2013, of the first gene associated with precocious puberty of hereditary origin. Her breasts had begun to form and hair was growing more dense in her armpits and pubic region, two signs that sex hormones, produced in greater quantity only in late childhood, had been circulating at high levels in her body. Although uncommon in the general population, such cases of puberty occurring long before the appropriate time are more common at this major hospital in Latin America, to which the most rare and complex of Brazil’s medical problems are referred.

But Dr. Latronico’s interest was aroused for another reason. The girl had been brought to the hospital by her paternal grandmother, then a woman of 69, who had also entered into puberty early and began menstruating at age nine. Weeks later the grandmother returned with a second granddaughter, the daughter of another of her children, and years later with a third, the first child of a second marriage. What they had in common was the fact that all had experienced the bodily changes of puberty well before the time such changes usually appear in most children: at age eight in girls and age nine in boys.

This cluster of cases in the same family—none entered puberty later than age six—led Dr. Latronico to suspect that the problem had a genetic origin, something that few experts thought at the time. She began an active search among the relatives of the children she and her team treated at HC. “We started to talk to the mothers, who are usually the ones who bring the children to their appointments, about the onset of puberty of the fathers, aunts, uncles and grandparents,” Dr. Latronico recalls. Questions such as “how old was your grandmother when she began menstruating?” Or “in your family are there cases of men who began to shave very early?” helped this group from the University of São Paulo School of Medicine (USP) locate 11 more Brazilian families with a case of precocious puberty among first-degree relatives.

Clinical and hormonal tests confirmed that these 12 Brazilian families and another three non-Brazilian families had 32 people who entered puberty very early, on average at age six. In all these cases, presented in an article that appeared in the June 2013 issue of the New England Journal of Medicine, the accelerated body development marking the transition from childhood to adulthood had begun too soon, because of a premature increase in gonadotrophin-releasing hormone production. GnRH controls the body’s sexual maturation, and these cases are called central or true precocious puberty.

Produced in the brain by a small group of neurons in the hypothalamus, GnRH works like a car’s accelerator. This hormone is released in more rapid bursts at puberty, inducing the pituitary gland to produce two other sex hormones: luteinizing hormone (LH) and follicle stimulating hormone (FSH). These hormones are released into the bloodstream and travel to the ovaries and testes, where they trigger the release of other sex hormones that make the body grow and mature from a reproductive point of view (see infographic above).

With the data from these 32 people in hand, the group needed to find out why the bodies of these subjects were secreting more GnRH too early. Dr. Latronico’s group, in partnership with researchers at São Paulo’s Santa Casa Medical School, the Federal University of Minas Gerais (UFMG), Belgium’s University of Leuven and Harvard University in the US, decided to sequence the genetic material of these participants to look for changes that could explain the earlier onset of puberty. One-third of them (eight people) showed defects in the same gene: the MKRN3, now considered the first gene responsible for a hereditary form of precocious puberty.

“This is an important finding, because determining the onset of puberty remains one of biology’s unsolved mysteries,” says Jean-Claude Carel, an endocrinologist at Paris Diderot University and the French-based Reference Center for Rare Endocrine Growth Diseases. “Puberty is associated with a number of physical and psychological long-term outcomes, and a better understanding of what defines its inception creates an opportunity to answer questions surrounding health issues such as cancer, risk behaviors and drug abuse,” observes Carel, an internationally renowned expert who studies central precocious puberty. Erica Eugster of the US-based Indiana University Health says, “this finding represents an important advance in determining the genetic basis of central precocious puberty,” especially because it involves a previously unknown way to control GnRH production.

SANDRA JAVERADr. Latronico says she never expected to find an altered gene in 33% of her subjects with an inherited form of precocious puberty. “Gene alterations generally do not affect more than 10% of people with a specific genetic disease,” she notes. In addition to the very frequent cases in which precocious puberty manifests itself in more than one generation of the same family, MKRN3 mutations are also common in people with central precocious puberty of non-hereditary origin.

The São Paulo group and its collaborators follow 215 children—treated in São Paulo, Ribeirão Preto, Campinas, and in Macedonia, Eastern Europe—where premature puberty manifested itself in isolation, without affecting other family members. Even so, according to a study submitted for publication to the Journal of Clinical Endocrinology and Metabolism, the proportion of people with the defective MKRN3 gene is high: about 3%.

“While changes in this gene explain only 3% of cases, it is still a major breakthrough,” says Dr. Gil Guerra Junior, pediatrician at the University of Campinas (Unicamp) and colleague of Dr. Latronico. “All pediatric endocrinology practices see cases of precocious puberty, and the biggest frustration for clinicians is not knowing the source of the problem in most cases,” he says.

Brazil has no population data on cases of precocious puberty, which is ten times more common in girls than in boys. But international statistics indicate that on average one child per group of 5,000 or 10 000 enter puberty much earlier than expected. Ninety per cent of the time the cause of early puberty is unknown, and doctors have to be content to say that its origin is idiopathic (unknown). Often these cases are isolated to only one family member, which eliminates the suspicion of heredity. Genetic analyses tend to reveal changes in one gene or another. But as far as we know, they are defects that arise randomly, with no evidence that they were transmitted from the parents. At least, that was the thinking.

Based on the work presented in the New England Journal of Medicine, researchers began to suspect that many familial cases were going undetected because doctors were not asking questions about the rest of the family. “Our data indicate that cases of family origin are not that rare,” says Dr. Bilharinho Berenice Mendonça of USP. It was she who, in the 1980s, created HC’s Endocrinology of Development Unit, where, in addition to cases of precocious puberty, the clinic diagnoses and treats disorders of sexual differentiation and growth.

Having worked with Dr. Mendonça since 1987, Dr. Latronico and her group have by now identified mutations in two other genes linked to the biochemical pathway of GnRH production. These mutations also caused the early onset of puberty. But, recalls Dr. Latronico, in “isolated cases.”

One of these altered genes was found in a 1-year-old boy, who had already grown body hair, and had enlarged testicles and penis size. Radiography of his hands revealed that his bones were as developed as those of a child of 3. During the time she spent in Dr. Ursula Kaiser’s laboratory at Harvard, Dr. Leticia Gontijo Silveira found that the boy’s cells carried damaged copies of the gene that encodes kisspeptin-1, the brain protein that activates the release of GnRH. Two years earlier, Milena Teles, also a member of Dr. Latronico’s team, had encountered, in a girl whose breasts had begun to develop during her first year of life and were fully formed by age seven, a mutation in another gene of the same family, which encodes the kisspeptin-1 receptor. In both cases the defective version of the gene precipitated the early release of GnRH and brought on premature puberty. These mutations functioned like a heavy foot on the accelerator of a car.

If kisspeptin-1 and its receptor form the acceleration system, the protein produced by the MKRN3 gene seems to act as a brake. This role only started to become clear with the work of Dr. Ana Paula de Abreu, of the USP team. As part of her postdoctoral studies at Harvard University, she recorded MKRN3 expressions in the brains of mice from the 10th to 60th day of life, which corresponds to the period from childhood to early adulthood in humans. At around the 20th day, at the onset of puberty, gene expression fell to 5% of the initial level. For Dr. Abreu, these data support the idea that the protein produced by healthy MKRN3 functions as a temporary block to puberty. Nine changes already found in familial and isolated cases of precocious puberty produce the opposite effect: it would be like having no brake.

Analysis of the familial cases revealed an unusual pattern of inheritance and expression of these defects. Just one altered copy of the gene (there are two in each cell) is enough to speed up the onset of puberty. But that copy must come from the father. “Copies of maternal origin are silenced by epigenetic mechanisms,” says Dr. Latronico.

Puberdade_215-2Even in cases that are considered isolated, with no family history, the researchers found that the defective gene was inherited from the father. “Fathers are asymptomatic carriers,” says Dr. Abreu. “These data show that cases considered sporadic from a clinical point of view are actually hereditary.”

The problem with having no brake is that in early childhood the car is not ready to run. “The premature increase in GnRH production accelerates very early growth, but the child grows for less time,” says Dr. Guerra. Among primates, humans take the most time to reach maturity. “Human beings grow from birth to 20 years, he says. “Imagine the consequences of growth stopping at age eight or nine.”

The first signs of puberty observed by parents and pediatricians, apart from hair growth, are the development of breasts girls and genitalia in boys. Almost always, however, the entire body is already growing at a faster rate—it is the growth spurt, which in normal puberty occurs at the end of the first decade of life. Increased levels of estradiol, one of the sex hormones, cause bones to lengthen much faster. But their extremities consolidate earlier, halting growth. “If puberty is not blocked early on, the child can not reach his or her full growth potential, and as an adult will be 10-12 centimeters shorter than people of the same age,” says Dr. Latronico.

Body changes are accompanied by behavioral changes. “Many children assume pre-adolescent attitudes,” says psychologist Marlene Inácio, who for more than 20 years has been following the cases treated at HC. Well before normal, they begin to question their parents and want to boss around children of the same age. Dr. Inácio says that it is common for girls to arrive at appointments with painted ​​nails and wearing makeup. Boys become withdrawn and more restless and aggressive. “The child realizes that the body has changed, but does not understand it from a subjective point of view,” she explains.

“During childhood boys and girls act like enemies,” says Dr. Durval Damiani, a pediatrician at USP’s Children’s Institute. But, as puberty begins, so does interest in the opposite sex. “Girls, for example, begin to like their male classmates,” says Dr. Damiani. And parents, especially of girls, start to fear the risk of sexual violence and possibly a pregnancy.

Although there has been a trend toward earlier puberty in Western countries in recent years—European data indicate that the age of first menstruation declined from age 17 in the early 19th century to age 13 in the mid-20th century— such early body development does not always present a health problem. “Many cases of precocious puberty are a variant of normal and need not be treated,” says Dr. Damiani. “Often a girl begins to exhibit the first signs of puberty at age seven, such as breast development, but her bone age is normal, and she will only begin to menstruate at age 12.” In these circumstances it is best to closely monitor the case.

At an age where interaction with other children is common, those who enter puberty too early may feel rejected. Girls who menstruate very early, for example, start to avoid going to the bathroom with classmates out of fear of discovery. “Being different at that age brings emotional distress,” says Dr. Latronico.

As soon as they identify signs of early puberty and confirm the need for treatment, physicians prescribe quarterly or monthly injections of a compound with a chemical structure similar to GnRH. This medication, which is provided by the public health system, but considered expensive at R$500.00 to R$800.00 per month, temporarily stops the hormone’s action. The goal of the treatment, which lasts until around age 12, is to preserve the child’s ability to grow, and make the signs of puberty retreat. “Some months after the start of treatment, the child begins to behave like others of the same age,” says Dr. Inácio.

Recently Dr. Vinicius Nahime de Brito, an endocrinologist, and Tais Menk, a psychologist, began a study at HC with 60 girls between the ages of six and 11 to assess how premature body changes affect emotional development. By using psychological tests they evaluated the character and degree of stress before, during, and after treatment. Preliminary results suggest that girls with early onset puberty have poor body images, are socially isolated and have heightened sexuality, in addition to fear and feelings of inferiority more intense than children of the same age with normal development, signs that are alleviated when GnRH is blocked. “The stress level was higher in the pre-treatment group than in the post-treatment group,” says Menk. “Although the number of children evaluated is  small,” adds Dr. Brito, “the data reinforce our hypothesis that early puberty causes a higher level of stress.”

Having treated cases of early puberty for almost three decades, Dr. Mendonça values the identification of gene defects in MKRN3 as an asset. “Previously, we only knew about genetic alterations with stimulatory action on GnRH,” says Dr. Mendonça. “This discovery provides a way for us to one day be able to act in an inhibitory fashion.” Although the current treatment is effective, 5% of children are allergic to the medication. If this line of research is successful, it may become possible to delay puberty by not only by taking the foot off the accelerator, but also by stepping on the brake.

Project
Molecular characterization of congenital endocrine diseases that affect growth and development (nº 05/04726-0); Grant Mechanism Thematic Project; Coord. Ana Claudia Latronico – FM / USP; Investment R$ 1,372,370.77 (FAPESP).

Scientific articles
ABREU, A.P. et al. Central precocious puberty Caused by mutations in the imprinted gene MKRN3. New England Journal of Medicine. June 27, 2013.
TELES, M.G. et al. A GPR54-activating mutation in a patient with central precocious puberty. New England Journal of Medicine. February 14, 2008.