Nadine Binart

New mutations in non-syndromic primary ovarian insufficiency patients identified via whole-exome sequencing

STUDY QUESTIONnIs it possible to identify new mutations potentially associated with non-syndromic primary ovarian insufficiency (POI) via whole-exome sequencing (WES)?nnnSUMMARY ANSWERnWES is an efficient tool to study genetic causes of POI as we have identified new mutations, some of which lead to protein destablization potentially contributing to the disease etiology.nnnWHAT IS KNOWN ALREADYnPOI is a frequently occurring complex pathology leading to infertility. Mutations in only few candidate genes, mainly identified by Sanger sequencing, have been definitively related to the pathogenesis of the disease.nnnSTUDY DESIGN, SIZE, DURATIONnThis is a retrospective cohort study performed on 69 women affected by POI.nnnPARTICIPANTS/MATERIALS, SETTING, METHODSnWES and an innovative bioinformatics analysis were used on non-synonymous sequence variants in a subset of 420 selected POI candidate genes. Mutations in BMPR1B and GREM1 were modeled by using fragment molecular orbital analysis.nnnMAIN RESULTS AND THE ROLE OF CHANCEnFifty-five coding variants in 49 genes potentially related to POI were identified in 33 out of 69 patients (48%). These genes participate in key biological processes in the ovary, such as meiosis, follicular development, granulosa cell differentiation/proliferation and ovulation. The presence of at least two mutations in distinct genes in 42% of the patients argued in favor of a polygenic nature of POI.nnnLIMITATIONS, REASONS FOR CAUTIONnIt is possible that regulatory regions, not analyzed in the present study, carry further variants related to POI.nnnWIDER IMPLICATIONS OF THE FINDINGSnWES and the in silico analyses presented here represent an efficient approach for mapping variants associated with POI etiology. Sequence variants presented here represents potential future genetic biomarkers.nnnSTUDY FUNDING/COMPETING INTEREST(S)nThis study was supported by the Universidad del Rosario and Colciencias (Grants CS/CIGGUR-ABN062-2016 and 672-2014). Colciencias supported Liliana Catherine Patiño´s work (Fellowship: 617, 2013). The authors declare no conflict of interest.

Familial Multiplicity of Estrogen Insensitivity Associated With a Loss-of-Function ESR1 Mutation

ContextnEstrogens influence many physiological processes in mammals, including reproduction. Estrogen peripheral actions are mainly mediated through estrogen receptors (ERs) α and β, encoded by ESR1 and ESR2 genes, respectively.nnnObjectivenThe studys aim was to describe a family in which 3 members presented with estrogen insensitivity.nnnDesign and SettingnClinical evaluation and genetic and mutational analysis were performed in an academic medical center.nnnPatients and InterventionsnAn ESR1 mutation was identified in 2 sisters and 1 brother, originating from a consanguineous Algerian family, who did not enter puberty and presented with delayed bone maturation consistent with estrogen insensitivity. The 2 sisters had enlarged multicystic ovaries. Hormonal evaluation as well as genetic and mutational analysis were performed.nnnResultsnHormonal evaluation revealed extremely high plasma 17β-estradiol (>50-fold normal range) associated with elevated gonadotropin levels (greater than threefold normal range), highly suggestive of estrogen resistance. The 3 affected patients carried a homozygous mutation of a highly conserved arginine 394 for which histidine was substituted through an autosomal recessive mode of transmission. Structural and functional analysis of the mutant ERα revealed strongly reduced transcriptional activity and the inability to securely anchor the activating hormone, estradiol, compared with wild-type ERα. A group of other potential ER activating ligands were tested, but none overcame the estrogen insensitivity in these patients.nnnConclusionnDescription and analysis of this family of patients with mutant ERα provide additional clinical findings toward identification and characterization of what was previously thought to be a highly rare clinical condition.

A novel non genomic glucocorticoid signaling mediated by a membrane palmitoylated glucocorticoid receptor cross talks with GnRH in gonadotrope cells

Glucocorticoid hormones (GC) are the main stress mediators associated with reproductive disorders. GC exert their effects through activation of the glucocorticoid receptor (GR) principally acting as a transcription factor. Beside well-established GR-mediated genomic actions, several lines of evidence suggest a role for rapid membrane-initiated GC signaling in gonadotrope cells triggered by a membrane-associated GR. Herein, we demonstrate the existence of a specific membrane-initiated GC signaling in LβT2 gonadotrope cells involving two related phosphoproteins: Ca2+/Calmodulin-dependent protein kinase II (CaMKII) and synapsin-I. Within 5u2009min, LβT2 cells treated with stress range of 10−7u2009M Corticosterone or a membrane impermeable-GC, BSA-conjugated corticosterone, exhibited a 2-fold increase in levels of phospho-CaMKII and phospho-synapsin-I. Biochemical approaches revealed that this rapid signaling is promoted by a palmitoylated GR. Importantly, GC significantly alter GnRH-induced CaMKII phosphorylation, consistent with a novel cross-talk between the GnRH receptor and GC. This negative effect of GC on GnRH signaling was further observed on LH release by mouse pituitary explants. Altogether, our work provides new findings in GC field by bringing novel understanding on how GR integrates plasma membrane, allowing GC membrane-initiated signaling that differs in presence of GnRH to disrupt GnRH-dependent signaling and LH secretion.

The tyrosine kinase inhibitor sunitinib affects ovulation but not ovarian reserve in mouse: A preclinical study

The aim of the study was to evaluate ovarian toxicity of tyrosine kinase inhibitor (TKI) sunitinib, since only scarce data are available on gonadal function after this treatment. Six-week-old female mice received orally, once daily, vehicle or sunitinib (50 mg/kg/d) during 5 weeks. Fertility parameters were analyzed from ovulation to litter assessment. Sunitinib exposure significantly reduced (i) corpora lutea number per ovary (1.1 ± 0.38 in sunitinib group versus 4 ± 0.79 in control group, p<0.01) and (ii) serum Anti Müllerian hormone (AMH) levels in sunitinib treated mice (12.01 ± 1.16) compared to control mice (14.33 ± 0.87 ng/ml, p< 0.05). However, primordial and growing follicles numbers per ovary were not different in both groups. After treatment withdrawal, female mice in both groups were able to obtain litters. These data could be helpful to counsel clinicians and patients, when fertility preservation methods are discussed, before TKI treatment in girls and young women.

Autocrine actions of prolactin contribute to the regulation of lactotroph function in vivo

Prolactin (PRL), whose principal role is regulation of lactation, is mainly synthesized and secreted by lactotroph anterior pituitary cells. Its signaling is exerted via a transmembrane PRL receptor (PRLR) expressed in a wide variety of tissues, including the anterior pituitary. Dopamine, which is secreted by tuberoinfundibular hypothalamic neurons, is the major inhibitory regulator of prolactin secretion. Although PRL is well established to stimulate hypothalamic dopamine secretion, thereby exerting a negative feedback regulation on its own release, autocrine or paracrine actions of PRL on lactotroph cells have also been suggested. Within the pituitary, PRL may inhibit both lactotroph proliferation and secretion, but in vivo evaluation of these putative functions is limited. To determine whether the autocrine actions of prolactin have a significant role in the physiologic function of lactotrophs in vivo, we examined the consequences of conditional deletion of Prlr in lactotroph cells using a novel mouse line with loxP sites flanking the Prlr gene (Prlrloxl/lox) and Cre‐recombinase (Cre) expressed under the control of the pituitary‐specific Prl promoter. Prlrlox/lox/Prl‐Cre mice have normal PRL levels and did not develop any pituitary lactotroph adenoma, even at 20 mo of age. Nevertheless, Prlrloxl/loxlPrl‐Cre mice displayed an increased dopaminergic inhibitory tone compared with control Prlrlox/lox mice. These results elegantly confirm an autocrine/paracrine feedback of PRL on lactotroph cells in vivo, which can be fully compensated by an intact hypothalamic feedback system.—Bernard, V., Lamothe, S., Beau, I., Guillou, A., Martin, A., Le Tissier, P., Grattan, D., Young, J., Binart, N. Autocrine actions of prolactin contribute to the regulation of lactotroph function in vivo. FASEBJ. 32, 4791–4797 (2018). www.fasebj.org

Functional evidence implicating NOTCH2 missense mutations in primary ovarian insufficiency etiology

Primary ovarian insufficiency (POI) is a frequently occurring disease affecting women under 40 years old. Recently, we have analyzed unrelated POI women via whole exome sequencing (WES) and identified NOTCH2 mutations underlying possible functional effects. The present study involved reanalyzing of WES assays. We used in the KGN granulosa‐like cell model, a synthetic gene reporter construct driving luciferase gene expression to assess the functional effects of five NOTCH2 mutations identified in POI patients. We found that NOTCH2‐p.Ser1804Leu, p.Ala2316Val, and p.Pro2359Ala mutations had a functional impact on the proteins transcriptional activity. The results have demonstrated for the first time that NOTCH2 mutations contribute to POI etiology. We therefore recommend sequencing NOTCH2s open reading frame in large panels of POI patients to establish an accurate genotype–phenotype correlation. We cannot rule out the fact that patients affected by Alagille syndrome carrying NOTCH2 mutations may suffer ovarian dysfunction.

A novel mutation in KHDRBS1 in a patient affected by primary ovarian insufficiency

Recently, it has been reported the first mutation in the KHDRBS1 gene related to primary ovarian insufficiency (POI) aetiology. Next generation sequencing (whole-exome sequencing-WES) experiments were performed in that study involving a familial case of POI (mother and daughter); this led to proposing that KHDRBS1-c.263C>T (p.Pro88Leu) and c.460A>G (pMet154Val) heterozygous sequence variants could be related to the phenotype. KHDRBS1 (also called Sam68) is an RNA-binding protein participating in several key cell processes, such as pre-mRNA splicing, mRNA translation, cell cycle regulation, signal transduction and apoptosis. This article is protected by copyright. All rights reserved.

R-spondin2, a novel target of NOBOX: identification of variants in a cohort of women with primary ovarian insufficiency

BackgroundR-spondin2 (Rspo2) is a secreted agonist of the canonical Wnt/β-catenin signaling pathway. Rspo2 plays a key role in development of limbs, lungs and hair follicles, and more recently during ovarian follicle development. Rspo2 heterozygous deficient female mice become infertile around 4xa0months of age mimicking primary ovarian insufficiency (POI). The study aimed to investigate the regulation of RSPO2 and its potential involvement in pathophysiology of POI.MethodsWe cloned the RSPO2 promoter and performed transcriptional assays to determine if RSPO2 can be regulated by NOBOX, an ovarian transcription factor. Then, we evaluated 100 infertile women after obtaining a detailed history of the disease and follicle-stimulating hormone measurements, besides karyotype determination and fragile-X premutation syndrome investigation. All exons, intron-exon boundaries and untranslated regions of the RSPO2 gene were identified by sequencing, and the results were statistically analyzed.ResultsWe found that RSPO2 can be regulated by NOBOX via the presence of NOBOX Binding Element in its promoter. Among 9 identified variants in POI women, 4 of them were equally homozygous, 4 have never been described (c.-359Cxa0>xa0G, c.-190Gxa0>xa0A, c.-170xa0+xa013Cxa0>xa0T and c.-169-8xa0Txa0>xa0A), only one c.557xa0Txa0>xa0C was predicted to alter a single amino acid in the RSPO2 protein (p.Leu186Pro).ConclusionsRSPO2 is a novel target gene of the NOBOX key transcription factor, confirming its important role during the follicular growth in ovary. However, RSPO2 mutations are rare or uncommon in women with POI.