MKRN3 inhibits puberty onset via interaction with IGF2BP1 and regulation of hypothalamic plasticity.

Makorin ring finger protein 3 (MKRN3) was identified as an inhibitor of puberty initiation with the report of loss-of-function mutations in association with central precocious puberty. Consistent with this inhibitory role, a prepubertal decrease in Mkrn3 expression was observed in the mouse hypothalamus. Here, we investigated the mechanisms of action of MKRN3 in the central regulation of puberty onset. We showed that MKRN3 deletion in hypothalamic neurons derived from human induced pluripotent stem cells was associated with significant changes in expression of genes controlling hypothalamic development and plasticity. Mkrn3 deletion in a mouse model led to early puberty onset in female mice. We found that Mkrn3 deletion increased the number of dendritic spines in the arcuate nucleus but did not alter the morphology of GnRH neurons during postnatal development. In addition, we identified neurokinin B (NKB) as an Mkrn3 target. Using proteomics, we identified insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) as another target of MKRN3. Interactome analysis revealed that IGF2BP1 interacted with MKRN3, along with several members of the polyadenylate-binding protein family. Our data show that one of the mechanisms by which MKRN3 inhibits pubertal initiation is through regulation of prepubertal hypothalamic development and plasticity, as well as through effects on NKB and IGF2BP1.

The Congenital and Acquired Mechanisms Implicated in the Etiology of Central Precocious Puberty.

The etiology of central precocious puberty (CPP) is multiple and heterogeneous, including congenital and acquired causes that can be associated with structural or functional brain alterations. All causes of CPP culminate in the premature pulsatile secretion of hypothalamic GnRH and, consequently, in the premature reactivation of hypothalamic-pituitary-gonadal axis. The activation of excitatory factors or suppression of inhibitory factors during childhood represent the 2 major mechanisms of CPP, revealing a delicate balance of these opposing neuronal pathways. Hypothalamic hamartoma (HH) is the most well-known congenital cause of CPP with central nervous system abnormalities. Several mechanisms by which hamartoma causes CPP have been proposed, including an anatomical connection to the anterior hypothalamus, autonomous neuroendocrine activity in GnRH neurons, trophic factors secreted by HH, and mechanical pressure applied to the hypothalamus. The importance of genetic and/or epigenetic factors in the underlying mechanisms of CPP has grown significantly in the last decade, as demonstrated by the evidence of genetic abnormalities in hypothalamic structural lesions (eg, hamartomas, gliomas), syndromic disorders associated with CPP (Temple, Prader-Willi, Silver-Russell, and Rett syndromes), and isolated CPP from monogenic defects (MKRN3 and DLK1 loss-of-function mutations). Genetic and epigenetic discoveries involving the etiology of CPP have had influence on the diagnosis and familial counseling providing bases for potential prevention of premature sexual development and new treatment targets in the future. Global preventive actions inducing healthy lifestyle habits and less exposure to endocrine-disrupting chemicals during the lifespan are desirable because they are potentially associated with CPP.

Development of precocious puberty in children: Surmised medicinal plant treatment.

Precocious puberty in children is one of the common endocrine diseases in paediatrics. Epidemiological surveys have shown that the number of children with precocious puberty has significantly increased globally. Precocious puberty negatively affects the physical and mental health of children and may increase the risk of hypertension, diabetes, obesity and infertility in adulthood. Therefore, the initiating factors of adolescence have become core issues in the study of sexual development in children. Owing to developments in molecular genetics, many studies have been able to show that precocious puberty is mostly resulted in autosomal inheritance. For instance, makorin ring finger protein 3 gene (MKRN3) may be implicated in familial CPP. Gonadotropin-releasing hormone agonist (GnRHa) is the gold standard for treatment, but its safety still requires long-term evaluation and management. Traditional medicinal plants have been used in clinical treatments and in exploring novel treatment methods. From the collected datas, in Asia, traditional Chinese medicine treatment is based on the principles of nourishing Yin, lowering fire and draining fire from the liver to help precocious children and alleviate or delay the onset of precocious puberty by medicinal plants such as Anemarrhena asphodeloides Bge., Phellodendron amurense Rupr., Rehmannia glutinosa and Poria cocos Wolf. They play an important role in exploring the pharmacological mechanisms of precocious puberty treatment effects and drug development. Therefore, by elucidating the occurrence and development of precocious puberty, this review provides novel and valuable insights of paediatric endocrine therapy accessing the published researches on the effectiveness of traditional herbal medicine in the treatment of precocious puberty and its therapeutic mechanisms.

The m6A mRNA demethylase FTO regulates GnRH secretion in Mn-induced precocious puberty.

The GABAA receptor (GABAAR) plays important roles in the regulation of Mn-induced GnRH secretion in immature female rats. However, the underlying molecular mechanisms remain unknown. Here, we assessed whether FTO and its substrate m6A are correlated with GABAAR expression in GnRH neurons after treatment with Mn in vitro and in vivo. Our study indicated that Mn treatment increased the expression of GnRH mRNA and decreased the levels of GABAAR protein but had no effect on GABAAR mRNA. Moreover, Mn upregulated the levels of FTO and inhibited global cellular m6A levels and GABAAα2 mRNA m6A levels. Knockdown of FTO increased the expression of GABAAR protein and GABAAα2 mRNA m6A levels. Data from rat models further demonstrate that inhibition of FTO suppressed GABAAR protein expression in the hypothalamus, causing delayed puberty onset. Collectively, our findings suggest that FTO-dependent m6A demethylation plays a critical role in regulating GABAAR mRNA processing in GnRH neurons.

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.

Mutational analysis of the genes encoding RFamide-related peptide-3, the human orthologue of gonadotrophin-inhibitory hormone, and its receptor (GPR147) in patients with gonadotrophin-releasing hormone-dependent pubertal disorders.

RFamide-related peptide-3 (RFRP-3), the orthologue of avian gonadotrophin-inhibitory hormone, and its receptor GPR147 have been recently identified in the human hypothalamus, and their roles in the regulation of reproductive axis has been studied. The present study aimed to investigate whether the presence of variants in the genes encoding human RFRP-3 (NPVF gene) and its receptor, GPR147 (NPFFR1 gene), is associated with the occurrence of gonadotrophin-releasing hormone-dependent pubertal disorders. Seventy-eight patients with idiopathic central precocious puberty (CPP) and 51 with normosmic isolated hypogonadotrophic hypogonadism (nIHH) were investigated. Fifty healthy subjects comprised the control group. The coding sequences of the NPVF and NPFFR1 genes were amplified and sequenced. Odds ratios (OR) were used to estimate the likelihood of CPP or nIHH in the presence of the described polymorphisms. All such polymorphisms have already been registered in the National Center for Biotechnology Information database. A three-nucleotide in frame deletion was identified in the NPVF gene (p.I71_K72), with a smaller proportion in the CPP (5%) compared to the nIHH (15%) group (P = 0.06). This results in the deletion of the isoleucine at position 71, adjacent to lysine at an endoproteolytic cleavage site of the precursor peptide. This polymorphism was associated with a lower risk of CPP (OR = 0.33; 95% confidence interval = 0.08-0.88); interestingly, only two men with nIHH were homozygotes for this variant. A total of five missense polymorphisms were found in the NPFFR1 gene, which encodes GPR147, with similar frequencies among groups and no association with pubertal timing. Our data suggest that RFRP-3/GPR147 may play secondary, modulatory roles on the regulation of pubertal development; a restraining modulatory effect of the NPVF p.I71_K72 variant on the activation of the gonadotrophic axis cannot be ruled out and deserves further investigation.

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.).

Virilizing sclerosing-stromal tumor of the ovary in a young woman with McCune Albright syndrome: clinical, pathological, and immunohistochemical studies.

CONTEXT: McCune-Albright syndrome (MAS) is characterized by polyostotic fibrous dysplasia, café-au-lait skin pigmentations, and gonadotropin-independent sexual precocious puberty, resulting from a somatic postzygotic activating mutation of the GNAS1 gene. SETTING: We report a virilizing sclerosing-stromal tumor of the ovary in a young female with MAS. PATIENT: She presented polyostotic fibrous dysplasia of the left upper and lower limbs and a café-au-lait skin spot in the posterior area of the neck. She had a history of precocious puberty, diagnosed at the age of 6 years and treated with cyproterone acetate until the age of 10 years; then she developed central puberty with severe oligomenorrhea. At the age of 23 years, she was hospitalized for a virilization syndrome including hirsutism, acne, deepening of the voice, amenorrhea, and clitoromegaly. Serum levels of T were dramatically increased (1293 ng/dl; normal range, 10-80). The abdominal computed tomography scan revealed a solid mass located on the left ovary. INTERVENTION: An ovariectomy was performed, and histological examination revealed a sclerosing-stromal tumor with pseudolobular pattern. RESULTS: Immunohistochemical studies revealed that the tumor cells expressed all steroidogenic enzymes involved in androgen synthesis. Molecular analysis revealed that ovarian tumor cells harbored the Arg 201 activating mutation in the GNAS1 gene. After surgery, T levels returned to normal, the patient retrieved a normal gonadal function, and she was able to become pregnant. CONCLUSION: This observation extends the clinical spectrum of ovarian pathology of women with MAS. However, the mechanisms causing this ovarian tumor remain unclear, even if the gsp oncogene has been implicated in the pathogenesis of some gonadal tumors.

Analyzing the functional and structural consequences of two point mutations (P94L and A368D) in the CYP11B1 gene causing congenital adrenal hyperplasia resulting from 11-hydroxylase deficiency.

CONTEXT: Congenital adrenal hyperplasia is a group of autosomal recessive inherited disorders of steroidogenesis. The deficiency of steroid 11-hydroxylase (CYP11B1) resulting from mutations in the CYP11B1 gene is the second most frequent cause. OBJECTIVE: We studied the functional and structural consequences of two CYP11B1 missense mutations, which were detected in a 1.8-yr-old boy with acne and precocious pseudopuberty, to prove their clinical relevance and study their impact on CYP11B1 function. RESULTS: The in vitro expression studies in COS-7 cells revealed an almost complete absence of CYP11B1 activity for the P94L mutant to 0.05% for the conversion of 11-deoxycortisol to cortisol. The A368D mutant severely reduced the CYP11B1 enzymatic activity to 1.17%. Intracellular localization studies by immunofluorescence revealed that the mutants were correctly localized. Introducing these mutations in a three-dimensional model structure of the CYP11B1 protein provides a possible explanation for the effects measured in vitro. We hypothesize that the A368D mutation interferes with structures important for substrate specificity and heme iron binding, thus explaining its major functional impact. However, according to structural analysis, we would expect only a minor effect of the P94L mutant on 11-hydroxylase activity, which contrasts with the observed major effect of this mutation both in vitro and in vivo. CONCLUSION: Analyzing the in vitro enzyme function is a complementary procedure to genotyping and a valuable tool for understanding the clinical phenotype of 11-hydroxylase deficiency. This is the basis for accurate genetic counseling, prenatal diagnosis, and treatment. Moreover, the combination of in vitro enzyme function and molecular modeling provides valuable insights in cytochrome P450 structural-functional relationships, although one must be aware of the limitations of in silico-based methods.

In boys with abnormal developmental tempo, maturation of the skeleton and the hypothalamic-pituitary-gonadal axis remains synchronous.

The primary mechanism that initiates puberty is unknown. One possible clue is that pubertal maturation often parallels skeletal maturation. Conditions that delay skeletal maturation also tend to delay the onset of puberty, whereas conditions that accelerate skeletal maturation tend to hasten the onset of puberty. To examine this relationship, we studied boys with congenital adrenal hyperplasia (n = 13) and familial male-limited precocious puberty (n = 22), two conditions that accelerate maturational tempo, and boys with idiopathic short stature (n = 18) in which maturational tempo is sometimes delayed. In all three conditions, the onset of central puberty generally occurred at an abnormal chronological age but a normal bone age. Boys with the greatest skeletal advancement began central puberty at the earliest age, whereas boys with the greatest skeletal delay began puberty at the latest age. Furthermore, the magnitude of the skeletal advancement or delay matched the magnitude of the pubertal advancement or delay. This synchrony between skeletal maturation and hypothalamic-pituitary-gonadal axis maturation was observed among patients within each condition and also between conditions. In contrast, the maturation of the hypothalamic-pituitary-gonadal axis did not remain synchronous with other maturational processes including weight, height, or body mass index. We conclude that in boys with abnormal developmental tempo, maturation of the skeleton and the hypothalamic-pituitary-gonadal axis remains synchronous. This synchrony is consistent with the hypothesis that in boys, skeletal maturation influences hypothalamic-pituitary-gonadal axis maturation.

Deletion hybrid genes, due to unequal crossing over between CYP11B1 (11beta-hydroxylase) and CYP11B2(aldosterone synthase) cause steroid 11beta-hydroxylase deficiency and congenital adrenal hyperplasia.

Chromosomal rearrangements are natural experiments that can provide unique insights into in vivo regulation of genes and physiological systems. We have studied a patient with congenital adrenal hyperplasia and steroid 11beta-hydroxylase deficiency who was homozygous for a deletion of the CYP11B1 and CYP11B2 genes normally required for cortisol and aldosterone synthesis, respectively. The genes were deleted by unequal recombination between the tandemly arranged CYP11B genes during a previous meiosis, leaving a single hybrid gene consisting of the promoter and exons 1-6 of CYP11B2 and exons 7-9 of CYP11B1. The hybrid gene also carried an I339T mutation formed by intracodon recombination at the chromosomal breakpoint. The mutant complementary DNA corresponding to this gene was expressed in COS-1 cells and was found to have relatively unimpaired 11beta-hydroxylase and aldosterone synthase activities. Apparently the 11beta-hydroxylase deficiency and the adrenal hyperplasia are due to the lack of expression of this gene in the adrenal zona fasciculata/reticularis resulting from replacement of the CYP11B1 promoter and regulatory sequences by those of CYP11B2.

A new constitutively activating point mutation in the luteinizing hormone/choriogonadotropin receptor gene in cases of male-limited precocious puberty.

A single point mutation that encodes an aspartic acid (Asp578) to glycine substitution in the LH/CG receptor (LH/CGR) gene, D578G, was recently found in American patients with familial male-limited precocious puberty and in a Japanese patient with a sporadic form of the disorder. Transfection of the mutant, compared to the wild-type, LH/CGR complementary DNA into COS-7 cells results in higher basal cAMP production, but a normal agonist-induced response; the mutation is, therefore, proposed to constitutively activate Leydig cells and elevate serum testosterone, despite low levels of gonadotropin. In the current study we examined two additional Japanese patients with male-limited precocious puberty without a family history of the disease. We describe a heterozygous cytosine (C) to thymine (T) transition at nucleotide 1715 in both; the mutation encodes an alanine to valine substitution in codon 572 of transmembrane helix 6, A572V. Transfected into COS-7 cells, the A572V mutant exhibited the same constitutively high basal cAMP levels and normal agonist-induced cAMP response as the D578G mutant. We conclude that the constitutively higher cAMP levels caused by the A572V mutation led to Leydig cell activation and male-limited precocious puberty, as in the previously described D578G mutation. As the mother of one of the two patients had the same heterozygous mutation, this patient represents the first recognized case of inherited male-limited precocious puberty in the Japanese population. The previously described D578G mutant did not increase basal or agonist-induced inositol phosphate production in transfected COS-7 cells, or the number of LH/CGRs or their affinity for LH/CG. In contrast, transfection of the A572V mutation in COS-7 cells exhibited significantly higher inositol phosphate levels basally and at 10(-11) mol/L hCG, but significantly lower inositol phosphate levels at 10(-7) mol/L hCG. These data suggest that the A572V mutation of the LH/CGR may have effects on the guanine nucleotide binding protein which activates phospholipase C (Gq) coupling and phospholipase-C activation in addition to its effects on Gs coupling and activation of adenylyl cyclase. A572V-transfected cells also exhibited a higher affinity, despite an apparent decrease in the number of binding sites, for [125I]hCG, compared to transfectants with the wild-type LH/CGR. We hypothesize that these differences between the A572V and D578G mutations reflect a greater impact of the A572V mutation on receptor conformation.

Genetic heterogeneity of constitutively activating mutations of the human luteinizing hormone receptor in familial male-limited precocious puberty.

Genomic DNA from 32 unrelated families with male-limited precocious puberty was examined for the previously described Asp-578-->Gly, Met-571-->Ile, and Thr-577-->Ile mutations in transmembrane helix 6 of the human luteinizing hormone receptor (hLHR). Twenty-eight families had the inherited form of the disorder, and of these, 24 were found to have the Asp-578-->Gly mutation. Four additional mutations were found among the remaining four families with the inherited form and in four sporadic cases of the disorder: an A-->C transversion resulting in substitution of leucine for Ile-542 in the fifth transmembrane helix, an A-->G transition resulting in substitution of glycine for Asp-564 in the third cytoplasmic loop, a G-->T transversion resulting in substitution of tyrosine for Asp-578 in the sixth transmembrane helix, and a T-->C transition resulting in substitution of arginine for Cys-581 in the sixth transmembrane helix. Human embryonic kidney cells transfected with cDNAs for each of the mutant hLHRs, created by PCR-based mutagenesis of the wild-type hLHR cDNA, exhibited increased levels of basal cAMP production in the absence of agonist, indicating constitutive activation of the mutation hLHRs. Three of the additional mutations had specific features: Ile-542-->Leu and Cys-581-->Arg appeared ligand-unresponsive, whereas Asp-578-->Tyr appeared to correlate genotype with phenotype. We conclude that the region spanning nt 1624-1741 of exon 11 is a hotspot for heterogeneous point mutations that constitutively activate the hLHR and cause male-limited precocious puberty.

A sporadic case of male-limited precocious puberty has the same constitutively activating point mutation in luteinizing hormone/choriogonadotropin receptor gene as familial cases.

Familial male-limited precocious puberty (FMPP) is an autosomal dominant disorder characterized by marked elevation of serum testosterone despite low levels of gonadotropin. Recently, a single point mutation in the LH/hCG receptor (LH/CGR) gene was found in FMPP families that constitutively activates the LH/CGR, causing Leydig cell activation and precocious puberty. Among the Japanese population, only four sporadic cases of male-limited precocious puberty have been reported. In the current study, we examined one of the four reported Japanese patients with sporadic male-limited precocious puberty and found the same mutation as that in the FMPP families. Genomic DNA was isolated, and the polymerase chain reaction (PCR) was performed to amplify a fragment of LH/CGR DNA encoding amino acid residues that include transmembrane helixes 5 and 6. Sequencing of the PCR products revealed a heterozygous adenosine-guanine transition at nucleotide 1733 in codon 578. The mutation encodes an aspartic acid578-glycine substitution in transmembrane helix 6. The mutant LH/CGR, created by site-directed mutagenesis in vitro, exhibited constitutively higher cAMP levels in transfected COS-7 cells than the wild-type LH/CGR, as described previously; however, basal inositol phosphate levels were not increased by transfection with complementary DNA for the mutant receptor. The concentration and affinity of [125I]hCG-binding sites were similar in cells transfected with the mutant and wild-type LH/CGR complementary DNAs, indicating that the mutant did not alter the production of receptor or its ability to bind human LH/CG. The sporadic occurrence of this case was confirmed by further studies. The mutation creates a recognition site for the restriction endonuclease MspI. Restriction digestion was positive for the mutant not digested by MspI, indicating that the patient's mutant allele was not inherited from his parents. DNA analysis of the patient and the parents, using microsatellite repeat markers, was compatible with biological paternity and maternity. We conclude that the aspartic acid578-->glycine mutation in the LH/CGR has arisen in the Japanese population and is the cause of a sporadic case of male-limited precocious puberty.

Testicular changes in gonadotropin-independent familial male sexual precocity. Familial testotoxicosis.

A recently described form of male sexual precocity characterized by active Leydig cell differentiation and premature onset of spermatogenesis in the absence of pituitary gonadotropin stimulation has been termed familial testotoxicosis. The clinical and endocrine findings in the condition are consistent with an inherited intratesticular defect rather than central or true precocious puberty. In this report, testicular changes in biopsy specimens from a series of affected patients are presented. In all of the cases, Leydig cells demonstrated nuclear and cytoplasmic features characteristic of fully differentiated steroidogenic cells. Reinke crystals were absent. Germ cells at all stages of spermatogenesis were present, but there was evident disorganization of maturation. Spermatids exhibited a variety of structural abnormalities. Sertoli cells were characterized by complex cytoplasmic differentiation, Charcot-Böttcher crystals, and tight junction formation. The morphologic changes indicate premature differentiation of all of the major testicular cell types and are consistent with a distinctive type of intratesticular abnormality.