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STUDY QUESTION: Is the 24-h urinary gonadotropin assay an effective diagnostic tool in central precocious puberty (CPP) in girls? SUMMARY ANSWER: This study is the first to provide 24-h urinary gonadotropin assay data, using an electrochemiluminescent immunoassay (CMIA), and to report its usefulness as a tool for the diagnosis of CPP. WHAT IS KNOWN ALREADY: Data about the GnRH test in the diagnosis of CPP are variable and there is no consensus regarding its interpretation. The measurement of FSH and LH in urines was previously reported to be an alternative biological tool. STUDY DESIGN, SIZE, DURATION: This is a retrospective two-cohort study, involving a setting and a validation cohort. A total of 516 girls, included between October 2012 and July 2015, and 632 urinary collections were analyzed in the setting cohort. In the validation cohort, 39 girls were included between January 2021 and May 2023, and 49 urinary collections were analyzed. PARTICIPANTS/MATERIALS, SETTING, METHODS: This study included girls who consulted for an investigation of disturbed growth rate or a clinical suspicion of puberty onset in different medical centres across France (setting cohort). Girls with a suspicion of precocious puberty onset were addressed at the expert centre of paediatric endocrinology of the Groupement Hospitalier Lyon Est (validation cohort). Pelvic ultrasonography was performed and enabled their classification according to clinical and morphologic changes criteria (prepubertal or pubertal groups). The parents collected 24-h urine samples (u24) according to standardized instructions. FSH and LH (urinary or plasmatic) were measured using a current and automated CMIA. MAIN RESULTS AND THE ROLE OF CHANCE: The area under the ROC curves for CPP prediction was 0.709 for u24FSH (P < 0.001), 0.767 for u24LH (P < 0.001), and 0.753 for the u24LH/u24FSH ratio (P < 0.001). We retained all possible combinations of the four thresholds in the validation cohort (u24FSH = 1.1 or 2.0 IU/24 h; u24LH = 0.035 or 0.08 IU/24 h). The combination of u24FSH > 1.1 IU/24 h and u24LH > 0.08 IU/24 h had a positive PV of 85.7% and a negative PV of 94.3%, a sensitivity of 85.7% and a specificity of 94.3%, for classifying prepubertal and pubertal girls in this cohort. LIMITATIONS, REASONS FOR CAUTION: This is a retrospective study, in which a margin of error remains due to the inherent uncertainty regarding the clinical assessment of pubertal onset. It must be considered that the thresholds can only apply to the used reagents; measurements without extractions using other reagents are likely to show important heterogeneity. WIDER IMPLICATIONS OF THE FINDINGS: The assay performed herein is a simple, non-invasive, and analytically robust technique meeting the criteria for an alternative to the GnRH test which could be used to supplement its lack of sensitivity. STUDY FUNDING/COMPETING INTEREST(S): No specific funding was used. All authors declared no conflict of interest. TRIAL REGISTRATION NUMBER: In-house #23-5214 registered study.
Assuntos
Hormônio Foliculoestimulante , Hormônio Luteinizante , Puberdade Precoce , Humanos , Feminino , Puberdade Precoce/urina , Puberdade Precoce/diagnóstico , Puberdade Precoce/sangue , Estudos Retrospectivos , Criança , Hormônio Luteinizante/sangue , Hormônio Luteinizante/urina , Hormônio Foliculoestimulante/sangue , Hormônio Foliculoestimulante/urina , Imunoensaio/métodos , Valor Preditivo dos TestesRESUMO
Prior to the meiotic divisions, dynamic chromosome reorganizations including pairing, synapsis, and recombination of maternal and paternal chromosome pairs must occur in a highly regulated fashion during meiotic prophase. How chromosomes identify each other's homology and exclusively pair and synapse with their homologous partners, while rejecting illegitimate synapsis with non-homologous chromosomes, remains obscure. In addition, how the levels of recombination initiation and crossover formation are regulated so that sufficient, but not deleterious, levels of DNA breaks are made and processed into crossovers is not understood well. We show that in Caenorhabditis elegans, the highly conserved Serine/Threonine protein phosphatase PP4 homolog, PPH-4.1, is required independently to carry out four separate functions involving meiotic chromosome dynamics: (1) synapsis-independent chromosome pairing, (2) restriction of synapsis to homologous chromosomes, (3) programmed DNA double-strand break initiation, and (4) crossover formation. Using quantitative imaging of mutant strains, including super-resolution (3D-SIM) microscopy of chromosomes and the synaptonemal complex, we show that independently-arising defects in each of these processes in the absence of PPH-4.1 activity ultimately lead to meiotic nondisjunction and embryonic lethality. Interestingly, we find that defects in double-strand break initiation and crossover formation, but not pairing or synapsis, become even more severe in the germlines of older mutant animals, indicating an increased dependence on PPH-4.1 with increasing maternal age. Our results demonstrate that PPH-4.1 plays multiple, independent roles in meiotic prophase chromosome dynamics and maintaining meiotic competence in aging germlines. PP4's high degree of conservation suggests it may be a universal regulator of meiotic prophase chromosome dynamics.
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Pareamento Cromossômico/genética , Segregação de Cromossomos/genética , Fosfoproteínas Fosfatases/genética , Animais , Caenorhabditis elegans , Troca Genética , Quebras de DNA de Cadeia Dupla , Recombinação Homóloga/genética , Meiose/genética , Complexo Sinaptonêmico/genéticaRESUMO
Telomere repeat binding factor 2 (TRF2) is a component of the shelterin complex that is known to bind and protect telomeric DNA, yet the detection of TRF2 in extra-telomeric regions of chromosomes suggests other roles for TRF2 besides telomere protection. Here, we demonstrate that TRF2 plays a critical role in antagonizing the repressive function of neuron-restrictive silencer factor, also known as repressor element-1 silencing transcription factor (REST), during the neural differentiation of human embryonic stem cells (hESCs) by enhancing the expression of a truncated REST splice isoform we term human REST4 (hREST4) due to its similarity to rodent REST4. We show that TRF2 is specifically upregulated during hESC neural differentiation concordantly with an increase in the expression of hREST4 and that both proteins are highly expressed in NPCs. Overexpression of TRF2 in hESCs increases hREST4 levels and induces their neural differentiation, whereas TRF2 knockdown in hESCs and NPCs reduces hREST4 expression, hindering their ability to differentiate to the neural lineage. Concurrently, we show that TRF2 directly interacts with the C-terminal of hREST4 through its TRF2 core binding motif [F/Y]xL, protecting hREST4 from ubiquitin-mediated proteasomal degradation and consequently furthering neural induction. Thus, the TRF2-mediated counterbalance between hREST4 and REST is vital for both the generation and maintenance of NPCs, suggesting an important role for TRF2 in both neurogenesis and function of the central nervous system.
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Diferenciação Celular/fisiologia , Células-Tronco Neurais/citologia , Neurogênese/fisiologia , Proteínas Repressoras/metabolismo , Proteína 2 de Ligação a Repetições Teloméricas/metabolismo , Linhagem Celular , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Citometria de Fluxo , Humanos , Immunoblotting , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Células-Tronco Neurais/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução Genética , Regulação para CimaRESUMO
The original version of this Article contained an error in the spelling of the author Ambrosius P. Snijders, which was incorrectly given as Ambrosious P. Snijders. This has now been corrected in both the PDF and HTML versions of the Article.
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The formation of haploid gametes from diploid germ cells requires the regulated two-step release of sister chromatid cohesion (SCC) during the meiotic divisions. Here, we show that phosphorylation of cohesin subunit REC-8 by Aurora B promotes SCC release at anaphase I onset in C. elegans oocytes. Aurora B loading to chromatin displaying Haspin-mediated H3 T3 phosphorylation induces spatially restricted REC-8 phosphorylation, preventing full SCC release during anaphase I. H3 T3 phosphorylation is locally antagonized by protein phosphatase 1, which is recruited to chromosomes by HTP-1/2 and LAB-1. Mutating the N terminus of HTP-1 causes ectopic H3 T3 phosphorylation, triggering precocious SCC release without impairing earlier HTP-1 roles in homolog pairing and recombination. CDK-1 exerts temporal regulation of Aurora B recruitment, coupling REC-8 phosphorylation to oocyte maturation. Our findings elucidate a complex regulatory network that uses chromosome axis components, H3 T3 phosphorylation, and cell cycle regulators to ensure accurate chromosome segregation during oogenesis.
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Aurora Quinase B/genética , Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/genética , Organismos Hermafroditas/genética , Oócitos/metabolismo , Anáfase , Animais , Aurora Quinase B/metabolismo , Caenorhabditis elegans/citologia , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromátides/metabolismo , Cromátides/ultraestrutura , Cromatina/metabolismo , Cromatina/ultraestrutura , Proteínas Cromossômicas não Histona/metabolismo , Segregação de Cromossomos , Regulação da Expressão Gênica , Organismos Hermafroditas/citologia , Organismos Hermafroditas/metabolismo , Histonas/genética , Histonas/metabolismo , Oócitos/citologia , Oogênese/genética , Fosforilação , Proteína Fosfatase 1/genética , Proteína Fosfatase 1/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , CoesinasRESUMO
Wapl induces cohesin dissociation from DNA throughout the mitotic cell cycle, modulating sister chromatid cohesion and higher-order chromatin structure. Cohesin complexes containing meiosis-specific kleisin subunits govern most aspects of meiotic chromosome function, but whether Wapl regulates these complexes remains unknown. We show that during C. elegans oogenesis WAPL-1 antagonizes binding of cohesin containing COH-3/4 kleisins, but not REC-8, demonstrating that sensitivity to WAPL-1 is dictated by kleisin identity. By restricting the amount of chromosome-associated COH-3/4 cohesin, WAPL-1 controls chromosome structure throughout meiotic prophase. In the absence of REC-8, WAPL-1 inhibits COH-3/4-mediated cohesion, which requires crossover-fated events formed during meiotic recombination. Thus, WAPL-1 promotes functional specialization of meiotic cohesin: WAPL-1-sensitive COH-3/4 complexes modulate higher-order chromosome structure, while WAPL-1-refractory REC-8 complexes provide stable cohesion. Surprisingly, a WAPL-1-independent mechanism removes cohesin before metaphase I. Our studies provide insight into how meiosis-specific cohesin complexes are regulated to ensure formation of euploid gametes.
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Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/antagonistas & inibidores , Estruturas Cromossômicas , Meiose , Animais , Caenorhabditis elegans , Proteínas de Ciclo Celular/antagonistas & inibidores , CoesinasRESUMO
Progeny of mice treated with the mutagen N-ethyl-N-nitrosourea (ENU) revealed a mouse, designated Longpockets (Lpk), with short humeri, abnormal vertebrae, and disorganized growth plates, features consistent with spondyloepiphyseal dysplasia congenita (SEDC). The Lpk phenotype was inherited as an autosomal dominant trait. Lpk/+ mice were viable and fertile and Lpk/Lpk mice died perinatally. Lpk was mapped to chromosome 15 and mutational analysis of likely candidates from the interval revealed a Col2a1 missense Ser1386Pro mutation. Transient transfection of wild-type and Ser1386Pro mutant Col2a1 c-Myc constructs in COS-7 cells and CH8 chondrocytes demonstrated abnormal processing and endoplasmic reticulum retention of the mutant protein. Histology revealed growth plate disorganization in 14-day-old Lpk/+ mice and embryonic cartilage from Lpk/+ and Lpk/Lpk mice had reduced safranin-O and type-II collagen staining in the extracellular matrix. The wild-type and Lpk/+ embryos had vertical columns of proliferating chondrocytes, whereas those in Lpk/Lpk mice were perpendicular to the direction of bone growth. Electron microscopy of cartilage from 18.5 dpc wild-type, Lpk/+, and Lpk/Lpk embryos revealed fewer and less elaborate collagen fibrils in the mutants, with enlarged vacuoles in the endoplasmic reticulum that contained amorphous inclusions. Micro-computed tomography (CT) scans of 12-week-old Lpk/+ mice revealed them to have decreased bone mineral density, and total bone volume, with erosions and osteophytes at the joints. Thus, an ENU mouse model with a Ser1386Pro mutation of the Col2a1 C-propeptide domain that results in abnormal collagen processing and phenotypic features consistent with SEDC and secondary osteoarthritis has been established.
Assuntos
Colágeno Tipo II/genética , Mutação de Sentido Incorreto/genética , Osteoartrite/complicações , Osteoartrite/genética , Osteocondrodisplasias/congênito , Sequência de Aminoácidos , Animais , Sequência de Bases , Condrócitos/metabolismo , Condrócitos/patologia , Condrócitos/ultraestrutura , Cromossomos de Mamíferos/genética , Colágeno Tipo II/química , Modelos Animais de Doenças , Embrião de Mamíferos/anormalidades , Embrião de Mamíferos/patologia , Loci Gênicos/genética , Lâmina de Crescimento/anormalidades , Lâmina de Crescimento/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Proteínas Mutantes/metabolismo , Tamanho do Órgão , Osteocondrodisplasias/complicações , Osteocondrodisplasias/genética , Osteogênese , Fenótipo , Mapeamento Físico do Cromossomo , Processamento de Proteína Pós-TraducionalRESUMO
BACKGROUND: Chromosome segregation and the repair of DNA double-strand breaks (DSBs) by homologous recombination require cohesin, the protein complex that mediates sister chromatid cohesion (SCC). In addition, cohesin is also required for the integrity of DNA damage checkpoints in somatic cells, where cohesin loading depends on a conserved complex containing the Scc2/Nipbl protein. Although cohesin is required for the completion of meiotic recombination, little is known about how cohesin promotes the repair of meiotic DSBs and about the factors that promote loading of cohesin during meiosis. RESULTS: Here we show that during Caenorhabditis elegans meiosis, loading of cohesin requires SCC-2, whereas the cohesin-related complexes condensin and SMC-5/6 can be loaded by mechanisms independent of both SCC-2 and cohesin. Although the lack of cohesin in scc-2 mutants impairs the repair of meiotic DSBs, surprisingly, the persistent DNA damage fails to trigger an apoptotic response of the conserved pachytene DNA damage checkpoint. Mutants carrying an scc-3 allele that abrogates loading of meiotic cohesin are also deficient in the apoptotic response of the pachytene checkpoint, and both scc-2 and scc-3 mutants fail to recruit the DNA damage sensor 9-1-1 complex onto persistent damage sites during meiosis. Furthermore, we show that meiotic cohesin is also required for the timely loading of the RAD-51 recombinase to irradiation-induced DSBs. CONCLUSIONS: We propose that meiotic cohesin promotes DSB processing and recruitment of DNA damage checkpoint proteins, thus implicating cohesin in the earliest steps of the DNA damage response during meiosis.