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.).

Computational Analysis of Missense Variants of G Protein-Coupled Receptors Involved in the Neuroendocrine Regulation of Reproduction.

INTRODUCTION: Many missense variants in G protein-coupled receptors (GPCRs) involved in the neuroendocrine regulation of reproduction have been identified by phenotype-driven or large-scale exome sequencing. Computational functional prediction analysis is commonly performed to evaluate their impact on receptor function. METHODS: To assess the performance and outcome of functional prediction analyses for these GPCRs, we performed a statistical analysis of the prediction performance of SIFT and PolyPhen-2 for variants with documented biological function as well as variants retrieved from Ensembl. We obtained missense variants with documented biological function testing from patients with reproductive disorders from a comprehensive literature search. Missense variants from individuals with known reproductive disorders were retrieved from the Human Gene Mutation Database. Missense variants from the general population were retrieved from the Ensembl genome database. RESULTS: The accuracies of SIFT and PolyPhen-2 were 83 and 85%, respectively. The performance of both prediction tools was greater in predicting loss-of-function variants (SIFT: 92%; PolyPhen-2: 95%) than in predicting variants that did not affect function (SIFT: 54%; PolyPhen-2: 57%). Concordance between SIFT and PolyPhen-2 did not improve accuracy. Surprisingly, approximately half of the variants retrieved from Ensembl were predicted as loss-of-function variants by SIFT (47%) and PolyPhen-2 (54%). CONCLUSION: Our findings provide new guidance for interpreting the results and limitations of computational functional prediction analyses for GPCRs and will help to determine which variants require biological function testing. In addition, our findings raise important questions regarding the link between genotype and phenotype in the general population.

TACR3 mutations disrupt NK3R function through distinct mechanisms in GnRH-deficient patients.

Neurokinin B (NKB) and its G-protein-coupled receptor, NK3R, have been implicated in the neuroendocrine control of GnRH release; however, little is known about the structure-function relationship of this ligand-receptor pair. Moreover, loss-of-function NK3R mutations cause GnRH deficiency in humans. Using missense mutations in NK3R we previously identified in patients with GnRH deficiency, we demonstrate that Y256H and Y315C NK3R mutations in the fifth and sixth transmembrane domains (TM5 and TM6), resulted in reduced whole-cell (79.3±7.2%) or plasma membrane (67.3±7.3%) levels, respectively, compared with wild-type (WT) NK3R, with near complete loss of inositol phosphate (IP) signaling, implicating these domains in receptor trafficking, processing, and/or stability. We further demonstrate in a FRET-based assay that R295S NK3R, in the third intracellular loop (IL3), bound NKB but impaired dissociation of Gq-protein subunits from the receptor compared with WT NK3R, which showed a 10.0 ± 1.3% reduction in FRET ratios following ligand binding, indicating activation of Gq-protein signaling. Interestingly, R295S NK3R, identified in the heterozygous state in a GnRH-deficient patient, also interfered with dissociation of G proteins and IP signaling from wild-type NK3R, indicative of dominant-negative effects. Collectively, our data illustrate roles for TM5 and TM6 in NK3R trafficking and ligand binding and for IL3 in NK3R signaling.

Involvement of G protein-coupled receptor 30 (GPR30) in rapid action of estrogen in primate LHRH neurons.

Previously, we have reported that 17beta-estradiol (E(2)) induces an increase in firing activity of primate LH-releasing hormone (LHRH) neurons. The present study investigates whether E(2) alters LHRH release as well as the pattern of intracellular calcium ([Ca(2+)](i)) oscillations and whether G protein-coupled receptor 30 (GPR30) plays a role in mediating the rapid E(2) action in primate LHRH neurons. Results are summarized: 1) E(2), the nuclear membrane-impermeable estrogen, estrogen-dendrimer conjugate, and the plasma membrane-impermeable estrogen, E(2)-BSA conjugate, all stimulated LHRH release within 10 min of exposure; 2) whereas the estrogen receptor antagonist, ICI 182,780, did not block the E(2)-induced LHRH release, E(2) application to cells treated with pertussis toxin failed to induce LHRH release; 3) GPR30 mRNA was expressed in olfactory placode cultures, and GPR30 protein was expressed in a subset of LHRH neurons; 4) pertussis toxin treatment blocked the E(2)-induced increase in [Ca(2+)](i) oscillations; 5) knockdown of GPR30 in primate LHRH neurons by transfection with small interfering RNA (siRNA) for GPR30 completely abrogated the E(2)-induced changes in [Ca(2+)](i) oscillations, whereas transfection with control siRNA did not; 6) the estrogen-dendrimer conjugate-induced increase in [Ca(2+)](i) oscillations also did not occur in LHRH neurons transfected with GPR30 siRNA; and 7) G1, a GPR30 agonist, resulted in changes in [Ca(2+)](i) oscillations, similar to those observed with E(2). Collectively, E(2) induces a rapid excitatory effect on primate LHRH neurons, and this rapid action of E(2) appears to be mediated, in part, through GPR30.

Prolactin regulation of neonatal ovine uterine gland morphogenesis.

Uterine gland development or adenogenesis in the neonatal ovine uterus involves budding, proliferation, and branching morphogenesis of the glandular epithelium (GE) from the luminal epithelium (LE) between birth (postnatal day or PND 0) and PND 56. This critical developmental event is coincident with increases in serum PRL and expression of long and short PRL receptors specifically in the nascent and proliferating GE. In study one, ewes were treated with a placebo pellet as a control (CX) or a bromocryptine mesylate pellet from PNDs 0-56. On PND 56, the endometrium of bromocryptine mesylate ewes contained fewer glands, particularly in the stratum spongiosum that contained numerous coiled and branched glands in CX uteri. In study two, ewes were treated with saline as a CX or recombinant ovine PRL from PNDs 0-56. Treatment with PRL increased gland number and density on PND 14 and PND 56. In study three, expression of signal transducers and activators of transcription (STAT) 1, 3, and 5 proteins was detected in the developing glands from PNDs 7-56. In study four, Western blot analyses indicated that PRL increased levels of phosphorylated STATs 1 and 5, but not STAT 3, and phosphorylated ERK 1 and 2 MAPKs and c-Jun N-terminal kinase/stress-activated protein kinase proteins in explanted PND 28 ovine uteri. Collectively, results indicate that PRL regulates endometrial adenogenesis in the neonatal ovine uterus.

Evidence of the importance of the first intracellular loop of prokineticin receptor 2 in receptor function.

Prokineticin receptors (PROKR) are G protein-coupled receptors (GPCR) that regulate diverse biological processes, including olfactory bulb neurogenesis and GnRH neuronal migration. Mutations in PROKR2 have been described in patients with varying degrees of GnRH deficiency and are located in diverse functional domains of the receptor. Our goal was to determine whether variants in the first intracellular loop (ICL1) of PROKR2 (R80C, R85C, and R85H) identified in patients with hypogonadotropic hypogonadism interfere with receptor function and to elucidate the mechanisms of these effects. Because of structural homology among GPCR, clarification of the role of ICL1 in PROKR2 activity may contribute to a better understanding of this domain across other GPCR. The effects of the ICL1 PROKR2 mutations on activation of signal transduction pathways, ligand binding, and receptor expression were evaluated. Our results indicated that the R85C and R85H PROKR2 mutations interfere only modestly with receptor function, whereas the R80C PROKR2 mutation leads to a marked reduction in receptor activity. Cotransfection of wild-type (WT) and R80C PROKR2 showed that the R80C mutant could exert a dominant negative effect on WT PROKR2 in vitro by interfering with WT receptor expression. In summary, we have shown the importance of Arg80 in ICL1 for PROKR2 expression and demonstrate that R80C PROKR2 exerts a dominant negative effect on WT PROKR2.

Increased neurokinin B (Tac2) expression in the mouse arcuate nucleus is an early marker of pubertal onset with differential sensitivity to sex steroid-negative feedback than Kiss1.

At puberty, neurokinin B (NKB) and kisspeptin (Kiss1) may help to amplify GnRH secretion, but their precise roles remain ambiguous. We tested the hypothesis that NKB and Kiss1 are induced as a function of pubertal development, independently of the prevailing sex steroid milieu. We found that levels of Kiss1 mRNA in the arcuate nucleus (ARC) are increased prior to the age of puberty in GnRH/sex steroid-deficient hpg mice, yet levels of Kiss1 mRNA in wild-type mice remained constant, suggesting that sex steroids exert a negative feedback effect on Kiss1 expression early in development and across puberty. In contrast, levels of Tac2 mRNA, encoding NKB, and its receptor (NK3R; encoded by Tacr3) increased as a function of puberty in both wild-type and hpg mice, suggesting that during development Tac2 is less sensitive to sex steroid-dependent negative feedback than Kiss1. To compare the relative responsiveness of Tac2 and Kiss1 to the negative feedback effects of gonadal steroids, we examined the effect of estradiol (E(2)) on Tac2 and Kiss1 mRNA and found that Kiss1 gene expression was more sensitive than Tac2 to E(2)-induced inhibition at both juvenile and adult ages. This differential estrogen sensitivity was tested in vivo by the administration of E(2). Low levels of E(2) significantly suppressed Kiss1 expression in the ARC, whereas Tac2 suppression required higher E(2) levels, supporting differential sensitivity to E(2). Finally, to determine whether inhibition of NKB/NK3R signaling would block the onset of puberty, we administered an NK3R antagonist to prepubertal (before postnatal d 30) females and found no effect on markers of pubertal onset in either WT or hpg mice. These results indicate that the expression of Tac2 and Tacr3 in the ARC are markers of pubertal activation but that increased NKB/NK3R signaling alone is insufficient to trigger the onset of puberty in the mouse.

Mutational analysis of TAC3 and TACR3 genes in patients with idiopathic central pubertal disorders.

OBJECTIVE: To investigate the presence of variants in the TAC3 and TACR3 genes, which encode NKB and its receptor (NK3R), respectively, in a large cohort of patients with idiopathic central pubertal disorders. SUBJECTS AND METHODS: Two hundred and thirty seven patients were studied: 114 with central precocious puberty (CPP), 73 with normosmic isolated hypogonadotropic hypogonadism (IHH), and 50 with constitutional delay of growth and puberty (CDGP). The control group consisted of 150 Brazilian individuals with normal pubertal development. Genomic DNA was extracted from peripheral blood and the entire coding region of both TAC3 and TACR3 genes were amplified and automatically sequenced. RESULTS: We identified one variant (p.A63P) in NKB and four variants, p.G18D, p.L58L (c.172C>T), p.W275* and p.A449S in NK3R, which were absent in the control group. The p.A63P variant was identified in a girl with CPP, and p.A449S in a girl with CDGP. The known p.G18D, p.L58L, and p.W275* variants were identified in three unrelated males with normosmic IHH. CONCLUSION: Rare variants in the TAC3 and TACR3 genes were identified in patients with central pubertal disorders. Loss-of-function variants of TACR3 were associated with the normosmic IHH phenotype.

G protein-coupled receptors involved in GnRH regulation: molecular insights from human disease.

In the past two decades, an increasing body of evidence has demonstrated that several G protein-coupled receptor (GPCR)-ligand pairs are critical for normal human reproductive development and function. Patients harboring genetic insults in either the receptors or their cognate ligands have presented with reproductive disorders characterized by varying degrees of GnRH deficiency. These disorders include idiopathic hypogonadotropic hypogonadism (IHH) and Kallmann Syndrome (KS). Conversely, mutations in some of these ligand-receptor pairs have been associated with accelerated reproductive maturation, manifested as central precocious puberty (CPP). To date, a series of elegant studies have characterized four GPCRs that play important roles in the neuroendocrine control of human reproductive development and function: GnRHR, KISS1R, PROKR2 and NK3R. Furthermore, these studies provide insights into the mechanisms by which mutations in these receptors give rise to reproductive disease phenotypes. This report will review mutations identified in GPCRs involved in the neuroendocrine control of the human reproductive axis with the aims of elucidating structure-function relationships of these GPCRs and identifying correlations between these structure-function relationships and the genotypic-phenotypic characterization of the patients.

TAC3/TACR3 mutations reveal preferential activation of gonadotropin-releasing hormone release by neurokinin B in neonatal life followed by reversal in adulthood.

CONTEXT: Mutations in TAC3 and TACR3 (encoding neurokinin B and its receptor) have been identified in Turkish patients with idiopathic hypogonadotropic hypogonadism (IHH), but broader populations have not yet been tested and genotype-phenotype correlations have not been established. OBJECTIVE: A broad cohort of normosmic IHH probands was screened for mutations in TAC3/TACR3 to evaluate the prevalence of such mutations and define the genotype/phenotype relationships. DESIGN AND SETTING: The study consisted of sequencing of TAC3/TACR3, in vitro functional assays, and neuroendocrine phenotyping conducted in tertiary care centers worldwide. PATIENTS OR OTHER PARTICIPANTS: 345 probands, 18 family members, and 292 controls were studied. INTERVENTION: Reproductive phenotypes throughout reproductive life and before and after therapy were examined. MAIN OUTCOME MEASURE: Rare sequence variants in TAC3/TACR3 were detected. RESULTS: In TACR3, 19 probands harbored 13 distinct coding sequence rare nucleotide variants [three nonsense mutations, six nonsynonymous, four synonymous (one predicted to affect splicing)]. In TAC3, one homozygous single base pair deletion was identified, resulting in complete loss of the neurokinin B decapeptide. Phenotypic information was available on 16 males and seven females with coding sequence variants in TACR3/TAC3. Of the 16 males, 15 had microphallus; none of the females had spontaneous thelarche. Seven of the 16 males and five of the seven females were assessed after discontinuation of therapy; six of the seven males and four of the five females demonstrated evidence for reversibility of their hypogonadotropism. CONCLUSIONS: Mutations in the neurokinin B pathway are relatively common as causes of hypogonadism. Although the neurokinin B pathway appears essential during early sexual development, its importance in sustaining the integrity of the hypothalamic-pituitary-gonadal axis appears attenuated over time.

Mutational analysis of TAC3 and TACR3 genes in patients with idiopathic central pubertal disorders / Análise de mutações nos genes TAC3 e TACR3 em pacientes com distúrbios puberais centrais idiopáticos

OBJECTIVE: To investigate the presence of variants in the TAC3 and TACR3 genes, which encode NKB and its receptor (NK3R), respectively, in a large cohort of patients with idiopathic central pubertal disorders. SUBJECTS AND METHODS: Two hundred and thirty seven patients were studied: 114 with central precocious puberty (CPP), 73 with normosmic isolated hypogonadotropic hypogonadism (IHH), and 50 with constitutional delay of growth and puberty (CDGP). The control group consisted of 150 Brazilian individuals with normal pubertal development. Genomic DNA was extracted from peripheral blood and the entire coding region of both TAC3 and TACR3 genes were amplified and automatically sequenced. RESULTS: We identified one variant (p.A63P) in NKB and four variants, p.G18D, p.L58L (c.172C>T), p.W275* and p.A449S in NK3R, which were absent in the control group. The p.A63P variant was identified in a girl with CPP, and p.A449S in a girl with CDGP. The known p.G18D, p.L58L, and p.W275* variants were identified in three unrelated males with normosmic IHH. CONCLUSION: Rare variants in the TAC3 and TACR3 genes were identified in patients with central pubertal disorders. Loss-of-function variants of TACR3 were associated with the normosmic IHH phenotype. Arq Bras Endocrinol Metab. 2012;56(9):646-52.

OBJETIVO: Investigar a presença de variantes nos genes TAC3 e TACR3, os quais codificam a NKB e seu receptor (NK3R), respectivamente, em uma coorte de pacientes com distúrbios puberais centrais idiopáticos. SUJEITOS E MÉTODOS: Duzentos e trinta e sete pacientes foram estudados: 114 com puberdade precoce central (PPC), 73 com hipogonadismo hipogonadotrófico isolado normósmico (HHI) e 50 com retardo constitucional do crescimento e desenvolvimento (RCCD). O grupo controle consistiu de 150 indivíduos brasileiros que apresentaram desenvolvimento puberal normal. O DNA genômico foi extraído de sangue periférico, e as regiões codificadoras dos genes TAC3 e TACR3 foram amplificadas e sequenciadas automaticamente. RESULTADOS: Uma variante (p.A63P) foi identificada na NKB, e quatro variantes, p.G18D, p.L58L (c.172C>T), p.W275X e p.A449S, foram identificadas no NK3R, as quais foram ausentes no grupo controle. A variante p.A63P foi identificada em uma menina com PPC, e a variante p.A449S, em uma menina com RCCD. As variantes previamente descritas, p.G18D, p.L58L e p.W275X, foram identificadas em três indivíduos com HHI normósmico do sexo masculino não relacionados. CONCLUSÃO: Variantes raras nos genes TAC3 e TACR3 foram identificadas em pacientes com distúrbios puberais centrais idiopáticos. Mutações de perda de função no gene TACR3 foram associadas com o fenótipo de HHI normósmico. Arq Bras Endocrinol Metab. 2012;56(9):646-52.

Ovine placental lactogen specifically binds to endometrial glands of the ovine uterus.

A hormonal servomechanism has been proposed to regulate differentiation and function of the endometrial glandular epithelium (GE) in the ovine uterus during pregnancy. This mechanism involves sequential actions of estrogen, progesterone, ovine interferon tau (IFNtau), placental lactogen (oPL), and placental growth hormone (oGH). The biological actions of oPL in vitro are mediated by homodimerization of the prolactin receptor (oPRLR) and heterodimerization of the oPRLR and oGH receptor. The objectives of the study were to determine the effects of intrauterine oPL, oGH, and their combination on endometrial histoarchitecture and gene expression and to localize and characterize binding sites for oPL in the ovine uterus in vivo using an in situ ligand binding assay. Intrauterine infusion of oPL and/or oGH following IFNtau into ovariectomized ewes treated with progesterone daily differentially affected endometrial gland number and expression of uterine milk proteins and osteopontin. However, neither hormone affected PRLR, insulin-like growth factor (IGF)-I, or IGF-II mRNA levels in the endometrium. A chimeric protein of placental secretory alkaline phosphatase (SEAP) and oPL was used to identify and characterize binding sites for oPL in frozen sections of interplacentomal endometrium from pregnant ewes. Specific binding of SEAP-oPL was detected in the endometrial GE on Days 30, 60, 90, and 120 of pregnancy. In Day 90 endometrium, SEAP-oPL binding to the endometrial GE was displaced completely by oPL and prolactin (oPRL) but only partially by oGH. Binding experiments using the extracellular domain of the oPRLR also showed that iodinated oPL binding sites could be competed for by oPRL and oPL but not by oGH. Collectively, results indicate that oPL binds to receptors in the endometrial glands and that oPRL is more effective than oGH in competing for these binding sites. Thus, effects of oPL on the endometrial glands may be mediated by receptors for oPRL and oGH.