Cintia Tusset

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

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.

New genetic factors implicated in human GnRH-dependent precocious puberty: The role of kisspeptin system

Human puberty is triggered by the reemergence of GnRH pulsatile secretion with progressive activation of the gonadal function. A number of genes have been identified in the complex regulatory neuroendocrine network that controls puberty initiation. KISS1 and KISS1R genes, which encode kisspeptin and its cognate receptor, respectively, are considered crucial factors for acquisition of normal reproductive function. Recently, rare missense mutations and single nucleotide polymorphisms (SNPs) of the kisspeptin system were associated with puberty onset. Two gain-of-function mutations of the KISS1 and KISS1R genes were implicated in the pathogenesis of GnRH-dependent precocious puberty, previously considered idiopathic. These discoveries have yielded significant insights into the physiology and pathophysiology of this important life transition time. Here, we review the current molecular defects that are implicated in human GnRH-dependent precocious puberty.

Absence of functional LIN28B mutations in a large cohort of patients with idiopathic central precocious puberty.

Aim: To investigate LIN28B gene variants in children with idiopathic central precocious puberty (CPP). Patients and Methods: We studied 178 Brazilian children with CPP (171 girls, 16.8% familial cases). A large multiethnic group (1,599 subjects; Multiethnic Cohort, MEC) was used as control. DNA analysis and biochemical in vitro studies were performed. Results: A heterozygous LIN28B variant, p.H199R, was identified in a girl who developed CPP at 5.2 years. This variant was absent in 310 Brazilian control individuals, but it was found in the same allele frequency in women from the MEC cohort, independent of the age of menarche. Functional studies revealed that when ectopically expressed in cells, the mutant protein was capable of binding pre-let-7 microRNA and inhibiting let-7 expression to the same extent as wild-type Lin28B protein. Other rare LIN28B variants (p.P173P, c.198+ 32_33delCT, g.9575731A>C and c.-11C>T) were identified in CPP patients and controls. Therefore, no functional mutation was identified. Conclusion: In vitro studies revealed that the rare LIN28B p.H199R variant identified in a girl with CPP does not affect the Lin28B function in the regulation of let-7 expression. Although LIN28B SNPs were associated with normal pubertal timing, rare variations in this gene do not seem to be commonly involved in the molecular pathogenesis of CPP.

Impact of mutations in kisspeptin and neurokinin B signaling pathways on human reproduction.

The involvement of kisspeptin and neurokinin in B pathways in the reproductive axis was first suspected by linkage analysis in consanguineous families with isolated hypogonadotropic hypogonadism (IHH). Since then, several loss-of-function mutations affecting the kisspeptin receptor and neurokinin B and its receptor were associated with sporadic and familial IHH without olfactory abnormalities or other associated developmental alterations. Clinical manifestations were indistinguishable in individuals with mutations affecting these pathways. Micropenis and cryptorchidism were common findings among male patients. Response to acute GnRH stimulation varied from blunted to normal, and many affected males and females were successfully treated for infertility with either exogenous gonadotropins or long term pulsatile GnRH infusion. More recently, rare activating mutations of the kisspeptin and its receptor were identified in children with idiopathic central precocious puberty, supporting the crucial role of this system in the human pubertal onset. Kisspeptin is a potent excitatory regulator of the GnRH secretion, whereas the role of neurokinin B in the neuroendocrine control of the reproductive axis is still poorly understood. Interestingly, kisspeptin and neurokinin B are coexpressed in the arcuate nucleus in the mammalian hypothalamus, suggesting that these systems are closely related and potential partners of the regulation of the reproductive axis.

Mutational analysis of the necdin gene in patients with congenital isolated hypogonadotropic hypogonadism.

CONTEXT Necdin activates GNRH gene expression and is fundamental for the development, migration, and axonal extension of murine GNRH neurons. In humans, necdin plays a potential role in the hypogonadotropic hypogonadism phenotype in patients with Prader-Willi syndrome. AIM To investigate necdin gene (NDN) variants in patients with isolated hypogonadotropic hypogonadism (IHH). PATIENTS AND METHODS We studied 160 Brazilian patients with IHH, which includes 92 with Kallmann syndrome and 68 with normosmic IHH. Genomic DNA was extracted and the single NDN exon was amplified and sequenced. To measure GNRH transcriptional activity, luciferase reporter plasmids containing GNRH regulatory regions were transiently transfected into GT1-7 cells in the presence and absence of overexpressed wild-type or mutant necdin. RESULTS A heterozygous variant of necdin, p.V318A, was identified in a 23-year-old male with Kallmann syndrome. The p.V318A was also present in affected aunt and his father and was absent in 100 Brazilian control subjects. Previous FGFR1 gene analysis revealed a missense mutation (p.P366L) in this family. Functional studies revealed a minor difference in the activation of GNRH transcription by mutant protein compared with wild type in that a significant impairment of the necdin protein activity threshold was observed. CONCLUSION A rare variant of necdin (p.V318A) was described in a family with Kallmann syndrome associated with a FGFR1 mutation. Familial segregation and in vitro analysis suggested that this non-synonymous variant did not have a direct causative role in the hypogonadism phenotype. NDN mutations are not a frequent cause of congenital IHH.

Aspectos clínicos e moleculares do hipogonadismo hipogonadotrófico isolado congênito

Congenital isolated hypogonadotropic hypogonadism (IHH) is characterized by partial or complete lack of pubertal development due to defects in migration, synthesis, secretion or action of gonadotropin-releasing hormone (GnRH). Laboratory diagnosis is based on the presence of low levels of sex steroids, associated with low or inappropriately normal levels of pituitary gonadotropins (LH and FSH). Secretion of other pituitary hormones is normal, as well magnetic resonance imaging of the hypothalamohypophyseal tract, which shows absence of an anatomical defects. When IHH is associated with olfactory abnormalities (anosmia or hyposmia), it characterizes Kallmann syndrome. A growing list of genes is involved in the etiology of IHH, suggesting the heterogeneity and complexity of the genetic bases of this condition. Defects in olfactory and GnRH neuron migration are the etiopathogenic basis of Kallmann syndrome. Mutations in KAL1, FGFR1/FGF8, PROK2/PROKR2, NELF, CHD7, HS6ST1 and WDR11 are associated with defects in neuronal migration, leading to Kallmann syndrome. Notably, defects in FGFR1, FGF8, PROKR2, CHD7 and WDR11 are also associated with IHH, without olfactory abnormalities (normosmic IHH), although in a lower frequency. Mutations in KISS1R, TAC3/TACR3 and GNRH1/GNRHR are described exclusively in patients with normosmic IHH. In this paper, we reviewed the clinical, hormonal and genetic aspects of IHH.

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.—Noel, S. D., Abreu, A. P., Xu, S., Muyide, T., Gianetti, E., Tusset, C., Carroll, J., Latronico, A. C., Seminara, S. B., Carroll, R. S., Kaiser, U. B. TACR3 mutations disrupt NK3R function through distinct mechanisms in GnRH‐deficient patients. FASEB J. 28, 1924–1937 (2014). www.fasebj.org

Pesquisa de mutações na neurocinina B e no seu receptor em pacientes com distúrbios puberais centrais idiopáticos

Mutacoes inativadoras nos genes TAC3 e TACR3, os quais codificam a neurocinina B (NKB) e o seu receptor NK3R, respectivamente, foram descritas em pacientes com hipogonadismo hipogonadotrofico isolado (HHI) normosmico. A partir desse achado, hipotetizamos que mutacoes ativadoras na NKB e/ou NK3R resultariam na secrecao prematura de GnRH e, consequentemente, no desenvolvimento de puberdade precoce dependente de gonadotrofinas (PPDG). Nesse estudo, investigamos a presenca de mutacoes ativadoras e/ou polimorfismos nos genes TAC3 e TACR3 em pacientes com PPDG, bem como mutacoes inativadoras e/ou polimorfismos nesses genes em pacientes com retardo constitucional do crescimento e desenvolvimento (RCCD), e HHI normosmico. Duzentos e trinta e sete pacientes com disturbios puberais centrais idiopaticos foram selecionados, sendo 114 com PPDG, 50 com RCCD, e 73 com HHI normosmico. Um grupo de 150 individuos que apresentaram desenvolvimento puberal normal foi utilizado como controle. As regioes codificadoras dos genes TAC3 e TACR3 foram amplificadas pela reacao em cadeia da polimerase, seguido de purificacao enzimatica e sequenciamento automatico direto. Analises in silico e in vitro foram realizadas. Um nova variante foi identificada no gene TAC3, p.A63P, em uma paciente do sexo feminino com PPDG, a qual desenvolveu puberdade aos sete anos de idade. Essa variante (p.A63P) esta localizada na proneurocinina B, e analises in silico sugeriram que ela nao altera sitios constitutivos de splicing e e benigna para a estrutura da proteina. A analise de segregacao familiar mostrou que a mae da paciente, a qual apresentou um desenvolvimento puberal normal, tambem apresentava a alteracao p.A63P em heterozigose, sugerindo que essa variante nao desempenha um papel direto no fenotipo de PPDG. Uma nova variante em heterozigose no gene TACR3, p.A449S, foi identificada em uma paciente do sexo feminino com RCCD, que teve inicio puberal aos treze anos de idade. A analise do grau de... Inactivating mutations of the TAC3 and TACR3 genes, which encode the neurokinin B (NKB) and its receptor, NK3R, respectively, were described in patients with normosmic isolated hypogonadotropic hypogonadism (IHH). Based on these observations, we hypothesized that gain-of-function mutations in the NKB and/or NK3R might be associated with premature activation of GnRH release, leading to gonadotropin-dependent precocious puberty (GDPP). In this study, we investigated the presence of activating mutations and/or polymorphisms in the TAC3 and TACR3 genes in patients with GDPP, and inactivating mutations and/or polymorphisms in these genes in patients with constitutional delay of growth and puberty (CDGP) and normosmic IHH. It was selected 237 patients with idiopathic central pubertal disorders: 114 with GDPP, 50 with CDGP, and 73 with normosmic IHH. Indeed, a group 150 individuals who had puberty at adequate age was used as controls. The coding regions of TAC3 and TACR3 genes were amplified by polymerase chain reaction followed by enzymatic purification and direct automatic sequencing. In silico and in vitro analyses were performed. A new heterozygous variant in the TAC3 gene, p.A63P, was identified in a Brazilian girl with GDPP who had puberty onset at seven years of age. The p.A63P variant was located in the proneurokinin B and in silico analysis suggested that this variant does not alter constitutive splice sites, and it was benign to the protein. The segregation analysis revealed that her mother was heterozygous for the p.A63P variant (who had a normal pubertal development), suggesting that this variant does not play a role in the GDPP phenotype. It was identified a new heterozygous variant, p.A449S, in the TACR3 gene in a Brazilian girl with CDGP, who had puberty onset at thirteen years of age. Conservation degree analysis of alanine at position 449 showed that this amino acid is not a conserved residue among different species. In silico analyses suggested...