Vinicius Nahime Brito

A GPR54-activating mutation in a patient with central precocious puberty.

Gonadotropin-dependent, or central, precocious puberty is caused by early maturation of the hypothalamic-pituitary-gonadal axis. In girls, this condition is most often idiopathic. Recently, a G protein-coupled receptor, GPR54, and its ligand, kisspeptin, were described as an excitatory neuroregulator system for the secretion of gonadotropin-releasing hormone (GnRH). In this study, we have identified an autosomal dominant GPR54 mutation--the substitution of proline for arginine at codon 386 (Arg386Pro)--in an adopted girl with idiopathic central precocious puberty (whose biologic family was not available for genetic studies). In vitro studies have shown that this mutation leads to prolonged activation of intracellular signaling pathways in response to kisspeptin. The Arg386Pro mutant appears to be associated with central precocious puberty.

Central Precocious Puberty Caused by Mutations in the Imprinted Gene MKRN3

BACKGROUND The onset of puberty is first detected as an increase in pulsatile secretion of gonadotropin-releasing hormone (GnRH). Early activation of the hypothalamic-pituitary-gonadal axis results in central precocious puberty. The timing of pubertal development is driven in part by genetic factors, but only a few, rare molecular defects associated with central precocious puberty have been identified. METHODS We performed whole-exome sequencing in 40 members of 15 families with central precocious puberty. Candidate variants were confirmed with Sanger sequencing. We also performed quantitative real-time polymerase-chain-reaction assays to determine levels of messenger RNA (mRNA) in the hypothalami of mice at different ages. RESULTS We identified four novel heterozygous mutations in MKRN3, the gene encoding makorin RING-finger protein 3, in 5 of the 15 families; both sexes were affected. The mutations included three frameshift mutations, predicted to encode truncated proteins, and one missense mutation, predicted to disrupt protein function. MKRN3 is a paternally expressed, imprinted gene located in the Prader-Willi syndrome critical region (chromosome 15q11-q13). All affected persons inherited the mutations from their fathers, a finding that indicates perfect segregation with the mode of inheritance expected for an imprinted gene. Levels of Mkrn3 mRNA were high in the arcuate nucleus of prepubertal mice, decreased immediately before puberty, and remained low after puberty. CONCLUSIONS Deficiency of MKRN3 causes central precocious puberty in humans. (Funded by the National Institutes of Health and others.).

Mutations of the KISS1 Gene in Disorders of Puberty

CONTEXT Kisspeptin, encoded by the KISS1 gene, is a key stimulatory factor of GnRH secretion and puberty onset. Inactivating mutations of its receptor (KISS1R) cause isolated hypogonadotropic hypogonadism (IHH). A unique KISS1R-activating mutation was described in central precocious puberty (CPP). OBJECTIVE Our objective was to investigate KISS1 mutations in patients with idiopathic CPP and normosmic IHH. PATIENTS Eighty-three children with CPP (77 girls) and 61 patients with IHH (40 men) were studied. The control group consisted of 200 individuals with normal pubertal development. METHODS The promoter region and the three exons of KISS1 were amplified and sequenced. Cells expressing KISS1R were stimulated with synthetic human wild-type or mutant kisspeptin-54 (kp54), and inositol phosphate accumulation was measured. In a second set of experiments, kp54 was preincubated in human serum before stimulation of the cells. RESULTS Two novel KISS1 missense mutations, p.P74S and p.H90D, were identified in three unrelated children with idiopathic CPP. Both mutations were absent in 400 control alleles. The p.P74S mutation was identified in the heterozygous state in a boy who developed CPP at 1 yr of age. The p.H90D mutation was identified in the homozygous state in two unrelated girls with CPP. In vitro studies revealed that the capacity of the P74S and H90D mutants to stimulate IP production was similar to the wild type. After preincubation of wild-type and mutant kp54 in human serum, the capacity to stimulate signal transduction was significantly greater for P74S compared with the wild type, suggesting that the p.P74S variant is more stable. Only polymorphisms were found in the IHH group. CONCLUSION Two KISS1 mutations were identified in unrelated patients with idiopathic CPP. The p.P74S variant was associated with higher kisspeptin resistance to degradation in comparison with the wild type, suggesting a role for this mutation in the precocious puberty phenotype.

Central Precocious Puberty that appears to be sporadic caused by Paternally inherited mutations in the imprinted GENE makorin ring finger 3

CONTEXT Loss-of-function mutations in makorin ring finger 3 (MKRN3), an imprinted gene located on the long arm of chromosome 15, have been recognized recently as a cause of familial central precocious puberty (CPP) in humans. MKRN3 has a potential inhibitory effect on GnRH secretion. OBJECTIVES The objective of the study was to investigate potential MKRN3 sequence variations as well as copy number and methylation abnormalities of the 15q11 locus in patients with apparently sporadic CPP. SETTING AND PARTICIPANTS We studied 215 unrelated children (207 girls and eight boys) from three university medical centers with a diagnosis of CPP. All but two of these patients (213 cases) reported no family history of premature sexual development. First-degree relatives of patients with identified MKRN3 variants were included for genetic analysis. MAIN OUTCOME MEASURES All 215 CPP patients were screened for MKRN3 mutations by automatic sequencing. Multiplex ligation-dependent probe amplification was performed in a partially overlapping cohort of 52 patients. RESULTS We identified five novel heterozygous mutations in MKRN3 in eight unrelated girls with CPP. Four were frame shift mutations predicted to encode truncated proteins and one was a missense mutation, which was suggested to be deleterious by in silico analysis. All patients with MKRN3 mutations had classical features of CPP with a median age of onset at 6 years. Copy number and methylation abnormalities at the 15q11 locus were not detected in the patients tested for these abnormalities. Segregation analysis was possible in five of the eight girls with MKRN3 mutations; in all cases, the mutation was inherited on the paternal allele. CONCLUSIONS We have identified novel inherited MKRN3 defects in children with apparently sporadic CPP, supporting a fundamental role of this peptide in the suppression of the reproductive axis.

Update on the etiology, diagnosis and therapeutic management of sexual precocity

Precocious puberty is defined as the development of secondary sexual characteristics before the age of 8 years in girls and 9 years in boys. Gonadotropin-dependent precocious puberty (GDPP) results from the premature activation of the hypothalamic-pituitary-gonadal axis and mimics the physiological pubertal development, although at an inadequate chronological age. Hormonal evaluation, mainly through basal and GnRH-stimulated LH levels shows activation of the gonadotropic axis. Gonadotropin-independent precocious puberty (GIPP) is the result of the secretion of sex steroids, independently from the activation of the gonadotropic axis. Several genetic causes, including constitutive activating mutations in the human LH-receptor gene and activating mutations in the Gs protein a-subunit gene are described as the etiology of testotoxicosis and McCune-Albright syndrome, respectively. The differential diagnosis between GDPP and GIPP has direct implications on the therapeutic option. Long-acting gonadotropin-releasing hormone (GnRH) analogs are the treatment of choice in GDPP. The treatment monitoring is carried out by clinical examination, hormonal evaluation measurements and image studies. For treatment of GIPP, drugs that act by blocking the action of sex steroids on their specific receptors (cyproterone, tamoxifen) or through their synthesis (ketoconazole, medroxyprogesterone, aromatase inhibitors) are used. In addition, variants of the normal pubertal development include isolated forms of precocious thelarche, precocious pubarche and precocious menarche. Here, we provide an update on the etiology, diagnosis and management of sexual precocity.

Causes, diagnosis, and treatment of central precocious puberty

Central precocious puberty results from the premature activation of the hypothalamic-pituitary-gonadal axis. It mimics physiological pubertal development, although at an inappropriate chronological age (before 8 years in girls and 9 years in boys). It can be attributable to cerebral congenital malformations or acquired insults, but the cause in most cases in girls remains unknown. MKRN3 gene defects have been identified in familial disease, with important basic and clinical results. Indeed, genetic analysis of this gene should be included in the routine clinical investigation of familial and idiopathic cases of central precocious puberty. Gonadotropin-releasing hormone agonists are the gold-standard treatment. The assessment and management of this disease remain challenging for paediatric endocrinologists. In this Series paper, we describe current challenges involving the precise diagnosis and adequate treatment of this disorder.

Early onset of primary hypogonadism revealed by serum anti-Müllerian hormone determination during infancy and childhood in trisomy 21

Male patients with an extra sex chromosome or autosome are expected to present primary hypogonadism at puberty owing to meiotic germ-cell failure. Scarce information is available on trisomy 21, a frequent autosomal aneuploidy. Our objective was to assess whether trisomy 21 presents with pubertal-onset, germ-cell specific, primary hypogonadism in males, or whether the hypogonadism is established earlier and affects other testicular cell populations. We assessed the functional status of the pituitary-testicular axis, especially Sertoli cell function, in 117 boys with trisomy 21 (ages: 2months-20year). To compare with an adequate control population, we established reference levels for serum anti-Müllerian hormone (AMH) in 421 normal males, from birth to adulthood, using a recently developed ultrasensitive assay. In trisomy 21, AMH was lower than normal, indicating Sertoli cell dysfunction, from early infancy, independently of the existence of cryptorchidism. The overall prevalence rate of AMH below the 3rd percentile was 64.3% in infants with trisomy 21. Follicle-stimulating hormone was elevated in patients <6months and after pubertal onset. Testosterone was within the normal range, but luteinizing hormone was elevated in most patients <6months and after pubertal onset, indicating a mild Leydig cell dysfunction. We conclude that in trisomy 21, primary hypogonadism involves a combined dysfunction of Sertoli and Leydig cells, which can be observed independently of cryptorchidism soon after birth, thus prompting the search for new hypotheses to explain the pathophysiology of gonadal dysfunction in autosomal trisomy.

A new pathway in the control of the initiation of puberty: the MKRN3 gene

Pubertal timing is influenced by complex interactions among genetic, nutritional, environmental, and socioeconomic factors. The role of MKRN3, an imprinted gene located in the Prader-Willi syndrome critical region (chromosome 15q11-13), in pubertal initiation was first described in 2013 after the identification of deleterious MKRN3 mutations in five families with central precocious puberty (CPP) using whole-exome sequencing analysis. Since then, additional loss-of-function mutations of MKRN3 have been associated with the inherited premature sexual development phenotype in girls and boys from different ethnic groups. In all of these families, segregation analysis clearly demonstrated autosomal dominant inheritance with complete penetrance, but with exclusive paternal transmission, consistent with the monoallelic expression of MKRN3 (a maternally imprinted gene). Interestingly, the hypothalamic Mkrn3 mRNA expression pattern in mice correlated with a putative inhibitory input on puberty initiation. Indeed, the initiation of puberty depends on a decrease in factors that inhibit the release of GnRH combined with an increase in stimulatory factors. These recent human and animal findings suggest that MKRN3 plays an inhibitory role in the reproductive axis to represent a new pathway in pubertal regulation.

Long-Term Surgical Outcome of Masculinizing Genitoplasty in Large Cohort of Patients With Disorders of Sex Development

PURPOSE We evaluated the results of masculinizing genitoplasty in a large cohort of patients with disorders of sex development treated at a single public tertiary center. MATERIALS AND METHODS We evaluated 52 patients with 46,XY and 7 with 46,XX disorders of sex development with proximal hypospadias and genital ambiguity reared as males who had undergone surgery between 1965 and 2008. Mean +/- SD followup was 14.1 +/- 9.2 years and median age at last examination was 22 years, with 38 patients having reached adulthood. Morphological result and urinary stream were evaluated by a physician. Urinary and sexual symptoms, and satisfaction with surgical results were assessed by questionnaire. RESULTS Mean penile length at diagnosis was compared between 46,XY patients and showed that those with 5alpha-reductase 2 deficiency had the shortest penile length (-5.4 +/- 1.2 SD). At the last clinical evaluation following surgical and hormonal treatment mean +/- SD penile length in 38 adults was 7.5 +/- 2.1 cm (range 4 to 12), corresponding to -4.3 +/- 1.3 SD (-6.5 to -1.5). All but 2 patients had penile length less than -2 SD. At that time mean penile length remained shorter in patients with 5alpha-reductase 2 deficiency (-5.4 +/- 1 SD) compared to those with testosterone production deficiency or indeterminate disorders of sex development (p <0.05). There was no statistical difference between mean penile length before and after treatment in all etiological groups (p >0.05). Morphological results were good in 43% of patients, fair in 54% and poor in 3%. The most common complications were urethral fistula (51%) and urethral stenosis (22%). Dribbling after voiding was the most frequent urinary symptom. Satisfaction with surgical results was reported by 89% of patients. Among adults 87% were sexually active, with 64% reporting normal sexual activity. CONCLUSIONS Most patients with 46,XY disorders of sex development were satisfied with long-term results of masculinizing genitoplasty, although specific complaints about small penile length, sexual activity and urinary symptoms were frequent. New surgical approaches should be developed to ensure full satisfaction in adulthood among patients with disorders of sex development.

The effect of distinct activating mutations of the luteinizing hormone receptor gene on the pituitary-gonadal axis in both sexes.

Familial or sporadic male‐limited precocious puberty is a distinct and unusual gonadotrophin‐independent form of sexual precocity caused by constitutively activating mutations of the luteinizing hormone receptor (LHR). In the present study, we evaluated the effect of known activating mutations at different sites of the LHR gene on the pituitary–gonadal axis in both sexes.