scholarly journals Neuroendocrine Phenotype Analysis in Five Patients with Isolated Hypogonadotropic Hypogonadism due to a L102P Inactivating Mutation of GPR54

2007 ◽  
Vol 92 (3) ◽  
pp. 1137-1144 ◽  
Author(s):  
Yardena Tenenbaum-Rakover ◽  
Monique Commenges-Ducos ◽  
André Iovane ◽  
Chantal Aumas ◽  
Osnat Admoni ◽  
...  
Keyword(s):  
Hypogonadotropic Hypogonadism ◽  
In Vivo Studies ◽  
Homozygous Mutation ◽  
Loss Of Function ◽  
Phenotypic Analysis ◽  
Lh Pulsatility ◽  
Gonadotropic Axis ◽  
Phenotype Analysis ◽  
Inactivating Mutation

Abstract Context: Loss of function of the G protein-coupled receptor of kisspeptins (GPR54) was recently described as a new cause of isolated hypogonadotropic hypogonadism. In vivo studies performed in several species have confirmed the major role of kisspeptins in neuroendocrine regulation of the gonadotropic axis and therefore sexual maturation. Objective: The objective of this study was to specify the exact contribution of kisspeptins and GPR54 to the initiation of puberty in humans. Design: Detailed neuroendocrine descriptions were performed in five patients with isolated hypogonadotropic hypogonadism bearing a new GPR54-inactivating mutation. Results: A homozygous mutation (T305C) leading to a leucine substitution with proline (L102P) was found in the five affected patients. This substitution completely inhibited GPR54 signaling. Phenotypic analysis revealed variable expressivity in the same family, either partial or complete gonadotropic deficiency. LH pulsatility analysis showed peaks with normal frequency but low amplitude. Repeated GnRH tests performed between 12 and 21 yr of age in one affected male revealed progressive changes in pituitary response from an early pubertal to an almost full pubertal pattern. Double GnRH test stimulations performed at a 120-min interval showed reduced dynamic pituitary response in GPR54-mutated patients. Conclusion: GPR54 inactivation does not impede neuroendocrine onset of puberty; rather, it delays and slows down pubertal maturation of the gonadotropic axis. The L102P loss of function mutation in GPR54 results in a more quantitative than qualitative defect of gonadotropic axis activation.

scholarly journals An Isolated Hypogonadotropic Hypogonadism due to a L102P Inactivating Mutation of KISS1R/GPR54 in a Large Family

2019 ◽  
Vol 2019 ◽  
pp. 1-6
Author(s):  
Ahmad J. Alzahrani ◽  
Azzam Ahmad ◽  
Tariq Alhazmi ◽  
Lujin Ahmad
Keyword(s):  
Amino Acid ◽  
Extended Family ◽  
Hypogonadotropic Hypogonadism ◽  
Large Family ◽  
Homozygous Mutation ◽  
Loss Of Function ◽  
Gonadotropin Secretion ◽  
Healthy Baby ◽  
Inactivating Mutation

KISS1R (GPR54) mutations have been reported in several patients with congenital normosmic idiopathic hypogonadotropic hypogonadism (nIHH). We aim to describe in detail nIHH patients with KISS1R (GPR54) mutations belonging to one related extended family and to review the literature. A homozygous mutation (T305C) leading to a leucine substitution with proline (L102P) was found in three affected kindred (2 males and 1 female) from a consanguineous Saudi Arabian family. This residue is localized within the first exoloop of the receptor, affects a highly conserved amino acid, perturbs the conformation of the transmembrane segment, and impairs its function. In the affected female, a combined gonadotropin administration restored regular period and ovulation and she conceived with a healthy baby boy after 4 years of marriage. We showed that a loss-of-function mutation (p.Tyr305C) in the KISS1R gene can cause (L102P) KISS1 receptor dysfunction and familial nIHH, revealing the crucial role of this amino acid in KISS1R function. The observed restoration of periods and later on pregnancy by an exogenous gonadotropin administration further support, in humans, that the KISS1R mutation has no other harmful effects on the patients apart from the gonadotropin secretion impairment.

Genetic evidence for the transcriptional-activating function of Homothorax during adult fly development

Development ◽  
2001 ◽  
Vol 128 (18) ◽  
pp. 3405-3413 ◽  
Author(s):  
Adi Inbal ◽  
Naomi Halachmi ◽  
Charna Dibner ◽  
Dale Frank ◽  
Adi Salzberg
Keyword(s):  
Transcriptional Activity ◽  
Target Genes ◽  
Genetic Evidence ◽  
In Vivo Studies ◽  
Loss Of Function ◽  
Native Form ◽  
Downstream Target ◽  
Activating Function

Homothorax (HTH) is a homeobox-containing protein, which plays multiple roles in the development of the embryo and the adult fly. HTH binds to the homeotic cofactor Extradenticle (EXD) and translocates it to the nucleus. Its function within the nucleus is less clear. It was shown, mainly by in vitro studies, that HTH can bind DNA as a part of ternary HTH/EXD/HOX complexes, but little is known about the transcription regulating function of HTH-containing complexes in the context of the developing fly. Here we present genetic evidence, from in vivo studies, for the transcriptional-activating function of HTH. The HTH protein was forced to act as a transcriptional repressor by fusing it to the Engrailed (EN) repression domain, or as a transcriptional activator, by fusing it to the VP16 activation domain, without perturbing its ability to translocate EXD to the nucleus. Expression of the repressing form of HTH in otherwise wild-type imaginal discs phenocopied hth loss of function. Thus, the repressing form was working as an antimorph, suggesting that normally HTH is required to activate the transcription of downstream target genes. This conclusion was further supported by the observation that the activating form of HTH caused typical hth gain-of-function phenotypes and could rescue hth loss-of-function phenotypes. Similar results were obtained with XMeis3, the Xenopus homologue of HTH, extending the known functional similarity between the two proteins. Competition experiments demonstrated that the repressing forms of HTH or XMeis3 worked as true antimorphs competing with the transcriptional activity of the native form of HTH. We also describe the phenotypic consequences of HTH antimorph activity in derivatives of the wing, labial and genital discs. Some of the described phenotypes, for example, a proboscis-to-leg transformation, were not previously associated with alterations in HTH activity. Observing the ability of HTH antimorphs to interfere with different developmental pathways may direct us to new targets of HTH. The HTH antimorph described in this work presents a new means by which the transcriptional activity of the endogenous HTH protein can be blocked in an inducible fashion in any desired cells or tissues without interfering with nuclear localization of EXD.

scholarly journals Long Term Pharmacological Perturbation of Autophagy in Mice: Are HCQ Injections a Relevant Choice?

Biomedicines ◽  
2020 ◽  
Vol 8 (3) ◽  
pp. 47 ◽  
Author(s):  
Jean-Daniel Masson ◽  
Benoit Blanchet ◽  
Baptiste Periou ◽  
François-Jérôme Authier ◽  
Baharia Mograbi ◽  
...  
Keyword(s):  
Mouse Models ◽  
In Vivo Studies ◽  
Loss Of Function ◽  
Evolutionarily Conserved ◽  
Atg Genes ◽  
Conditional Inhibition ◽  
The Impact ◽  
Catabolic Process

Macroautophagy (hereafter referred to as autophagy) is an evolutionarily conserved catabolic process whose loss-of-function has been linked to a growing list of pathologies. Knockout mouse models of key autophagy genes have been instrumental in the demonstration of the critical functions of autophagy, but they display early lethality, neurotoxicity and unwanted autophagy-independent phenotypes, limiting their applications for in vivo studies. To avoid problems encountered with autophagy-null transgenic mice, we investigated the possibility of disturbing autophagy pharmacologically in the long term. Hydroxychloroquine (HCQ) ip injections were done in juvenile and adult C57bl/6j mice, at range doses adapted from the human malaria prophylactic treatment. The impact on autophagy was assessed by western-blotting, and juvenile neurodevelopment and adult behaviours were evaluated for four months. Quite surprisingly, our results showed that HCQ treatment in conditions used in this study neither impacted autophagy in the long term in several tissues and organs nor altered neurodevelopment, adult behaviour and motor capabilities. Therefore, we recommend for future long-term in vivo studies of autophagy, to use genetic mouse models allowing conditional inhibition of selected Atg genes in appropriate lineage cells instead of HCQ treatment, until it could be successfully revisited using higher HCQ doses and/or frequencies with acceptable toxicity.

scholarly journals High-resolution mass spectrometry analysis of protein oxidations and resultant loss of function

2008 ◽  
Vol 36 (5) ◽  
pp. 1037-1044 ◽  
Author(s):  
Stephen Barnes ◽  
Erin M. Shonsey ◽  
Shannon M. Eliuk ◽  
David Stella ◽  
Kerri Barrett ◽  
...  
Keyword(s):  
Singlet Oxygen ◽  
High Resolution Mass Spectrometry ◽  
Catalytic Triad ◽  
Mass Spectrometry Analysis ◽  
In Vivo Studies ◽  
Ion Cyclotron Resonance ◽  
Loss Of Function ◽  
Human Bile ◽  
Tryptophan Residues

MS, with or without pre-analysis peptide fractionation, can be used to decipher the residues on proteins where oxidative modifications caused by peroxynitrite, singlet oxygen or electrophilic lipids have occurred. Peroxynitrite nitrates tyrosine and tryptophan residues on the surface of actin. Singlet oxygen, formed by the interaction of UVA light with tryptophan, can oxidize neighbouring cysteine, histidine, methionine, tyrosine and tryptophan residues. Dose–response inactivation by 4HNE (4-hydroxynonenal) of hBAT (human bile acid CoA:amino acid N-acyltransferase) and CKBB (cytosolic brain isoform of creatine kinase) is associated with site-specific modifications. FT-ICR (Fourier-transform ion cyclotron resonance)–MS using nanoLC (nano-liquid chromatography)–ESI (electrospray ionization)–MS or direct-infusion ESI–MS with gas-phase fractionation identified 14 4HNE adducts on hBAT and 17 on CKBB respectively. At 4HNE concentrations in the physiological range, one member of the catalytic triad of hBAT (His362) was modified; for CKBB, although all four residues in the active site that were modifiable by 4HNE were ultimately modified, only one, Cys283, occurred at physiological concentrations of 4HNE. These results suggest that future in vivo studies should carefully assess the critical sites that are modified rather than using antibodies that do not distinguish between different modified sites.

scholarly journals A Crucial Role of ACBD3 Required for Coxsackievirus Infection in Animal Model Developed by AAV-Mediated CRISPR Genome Editing Technique

Viruses ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 237
Author(s):  
Hye Jin Shin ◽  
Keun Bon Ku ◽  
Soojin Kim ◽  
Heon Seok Kim ◽  
Yeon-Soo Kim ◽  
...  
Keyword(s):  
Animal Model ◽  
Genome Editing ◽  
Cultured Cells ◽  
Host Factor ◽  
In Vivo Studies ◽  
Loss Of Function ◽  
Host Interaction ◽  
Cvb3 Infection

Genetic screens using CRISPR/Cas9 have been exploited to discover host–virus interactions. These screens have identified viral dependencies on host proteins during their life cycle and potential antiviral strategies. The acyl-CoA binding domain containing 3 (ACBD3) was identified as an essential host factor for the Coxsackievirus B3 (CVB3) infection. Other groups have also investigated the role of ACBD3 as a host factor for diverse enteroviruses in cultured cells. However, it has not been tested if ACBD3 is required in the animal model of CVB3 infection. Owing to embryonic lethality, conventional knockout mice were not available for in vivo study. As an alternative approach, we used adeno-associated virus (AAV)-mediated CRISPR genome editing to generate mice that lacked ACBD3 within the pancreas, the major target organ for CVB3. Delivery of sgRNAs using self-complementary (sc) AAV8 efficiently induced a loss-of-function mutation in the pancreas of the Cas9 knock-in mice. Loss of ACBD3 in the pancreas resulted in a 100-fold reduction in the CVB3 titer within the pancreas and a noticeable reduction in viral protein expression. These results indicate a crucial function of ACBD3 in CVB3 infection in vivo. AAV-mediated CRISPR genome editing may be applicable to many in vivo studies on the virus–host interaction and identify a novel target for antiviral therapeutics.

scholarly journals hsa_circ_0013401 Accelerates the Growth and Metastasis and Prevents Apoptosis and Autophagy of Neuroblastoma Cells by Sponging miR-195 to Release PAK2

2021 ◽  
Vol 2021 ◽  
pp. 1-21
Author(s):  
Shibo Zhu ◽  
Xiangliang Tang ◽  
Xiaofeng Gao ◽  
Jingqi Zhang ◽  
Yanhong Cui ◽  
...  
Keyword(s):  
Regulatory Gene ◽  
Neuroblastoma Cells ◽  
Tumor Formation ◽  
In Vivo Studies ◽  
Luciferase Assay ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Loss Of Function ◽  
Downstream Target

Background. Increased levels of circRNAs have been identified in a variety of cancers. However, the specific functions and mechanisms of circRNAs in neuroblastoma (NB) have not been fully explored. Methods. The levels of hsa_circ_0045997, hsa_circ_0080307, hsa_circ_0013401, hsa_circ_0077578, and microRNA-195 were confirmed by RT-qPCR in NB. Gain- and loss-of-function assays and rescue experiments were conducted to determine the influence of hsa_circ_0013401, miR-195, and P21-activated kinase 2 (PAK2) on the proliferation, apoptosis, autophagy, migration, and invasion of NB cells. Regulatory gene targets were validated by the luciferase assay. A xenograft mouse model was used to determine the in vivo effects of hsa_circ_0013401. Results. hsa_circ_0013401 was highly expressed, miR-195 was lowly expressed, and there was a negative correlation between hsa_circ_0013401 and miR-195 in NB. The inhibitory effects of hsa_circ_0013401 knockdown suppressed the proliferation, migration, and invasion and induced the apoptosis and autophagy of NB cells by targeting miR-195 to downregulate PAK2 expression. Luciferase reporter assays showed that miR-195 was a direct target of hsa_circ_0013401, and PAK2 was the downstream target gene of miR-195. In vivo studies showed that hsa_circ_0013401 promotes tumor formation. Conclusions. hsa_circ_0013401 induced NB progression through miR-195 to enhance PAK2. Therefore, we might highlight a novel regulatory axis (hsa_circ_0013401/miR-195/PAK2) in NB.

scholarly journals Molecular and Phenotypic Analysis of 25 Recessive, Homozygous-Viable Alleles at the Mouse agouti Locus

Genetics ◽  
2002 ◽  
Vol 160 (2) ◽  
pp. 659-674
Author(s):  
Rosalynn J Miltenberger ◽  
Kazumasa Wakamatsu ◽  
Shosuke Ito ◽  
Richard P Woychik ◽  
Liane B Russell ◽  
...  
Keyword(s):  
Loss Of Function ◽  
Wild Type ◽  
Allelic Series ◽  
Phenotypic Analysis ◽  
Protein Coding ◽  
Agouti Locus ◽  
Expression Control ◽  
Gene Expression Control ◽  
Specific Locus

Abstract Agouti is a paracrine-acting, transient antagonist of melanocortin 1 receptors that specifies the subapical band of yellow on otherwise black hairs of the wild-type coat. To better understand both agouti structure/function and the germline damage caused by chemicals and radiation, an allelic series of 25 recessive, homozygous-viable agouti mutations generated in specific-locus tests were characterized. Visual inspection of fur, augmented by quantifiable chemical analysis of hair melanins, suggested four phenotypic categories (mild, moderate, umbrous-like, severe) for the 18 hypomorphs and a single category for the 7 amorphs (null). Molecular analysis indicated protein-coding alterations in 8 hypomorphs and 6 amorphs, with mild-moderate phenotypes correlating with signal peptide or basic domain mutations, and more devastating phenotypes resulting from C-terminal lesions. Ten hypomorphs and one null demonstrated wild-type coding potential, suggesting that they contain mutations elsewhere in the ≥125-kb agouti locus that either reduce the level or alter the temporal/spatial distribution of agouti transcripts. Beyond the notable contributions to the field of mouse germ cell mutagenesis, analysis of this allelic series illustrates that complete abrogation of agouti function in vivo occurs most often through protein-coding lesions, whereas partial loss of function occurs slightly more frequently at the level of gene expression control.

scholarly journals Influence of hormonal control on LH pulsatility and secretion in women with classical congenital adrenal hyperplasia

2012 ◽  
Vol 167 (4) ◽  
pp. 499-505 ◽  
Author(s):  
Anne Bachelot ◽  
Zeina Chakhtoura ◽  
Geneviève Plu-Bureau ◽  
Mathieu Coudert ◽  
Christiane Coussieu ◽  
...  
Keyword(s):  
Menstrual Cycle ◽  
Congenital Adrenal Hyperplasia ◽  
Polycystic Ovary ◽  
Hypogonadotropic Hypogonadism ◽  
Pulse Amplitude ◽  
Hormonal Control ◽  
Adrenal Hyperplasia ◽  
Salt Wasting ◽  
Lh Pulsatility ◽  
Gonadotropic Axis

ObjectiveWomen with classical congenital adrenal hyperplasia (CAH) exhibit reduced fertility due to several factors including anovulation. This has been attributed to a disturbed gonadotropic axis as in polycystic ovary syndrome (PCOS), but there is no precise evaluation. Our aim was to evaluate the gonadotropic axis and LH pulsatility patterns and to determine factor(s) that could account for the potential abnormality of LH pulsatility.DesignCase/control study.MethodsSixteen CAH women (11 with the salt-wasting form and five with the simple virilizing form), aged from 18 to 40 years, and 16 age-matched women, with regular menstrual cycles (28±3 days), were included. LH pulse patterns over 6 h were determined in patients and controls.ResultsNo differences were observed between patients and controls in terms of mean LH levels, LH pulse amplitude, or LH frequency. In CAH patients, LH pulsatility patterns were heterogeneous, leading us to perform a clustering analysis of LH data, resulting in a two-cluster partition. Patients in cluster 1 had similar LH pulsatility patterns to the controls. Patients in cluster 2 had: lower LH pulse amplitude and frequency and presented menstrual cycle disturbances more frequently; higher 17-OH progesterone, testosterone, progesterone, and androstenedione levels; and lower FSH levels.ConclusionsLH pulsatility may be normal in CAH women well controlled by hormonal treatment. Undertreatment is responsible for hypogonadotropic hypogonadism, with low LH pulse levels and frequency, but not PCOS. Suppression of progesterone and androgen concentrations during the follicular phase of the menstrual cycle should be a major objective in these patients.

scholarly journals Optimized strategy for in vivo Cas9-activation in Drosophila

2017 ◽  
Vol 114 (35) ◽  
pp. 9409-9414 ◽  
Author(s):  
Ben Ewen-Campen ◽  
Donghui Yang-Zhou ◽  
Vitória R. Fernandes ◽  
Delfina P. González ◽  
Lu-Ping Liu ◽  
...  
Keyword(s):  
Large Scale ◽  
Target Gene ◽  
In Vivo Studies ◽  
Transcriptional Activators ◽  
Loss Of Function ◽  
Guide Rnas ◽  
Genome Wide ◽  
A Genome ◽  
Endogenous Loci

While several large-scale resources are available for in vivo loss-of-function studies in Drosophila, an analogous resource for overexpressing genes from their endogenous loci does not exist. We describe a strategy for generating such a resource using Cas9 transcriptional activators (CRISPRa). First, we compare a panel of CRISPRa approaches and demonstrate that, for in vivo studies, dCas9-VPR is the most optimal activator. Next, we demonstrate that this approach is scalable and has a high success rate, as >75% of the lines tested activate their target gene. We show that CRISPRa leads to physiologically relevant levels of target gene expression capable of generating strong gain-of-function (GOF) phenotypes in multiple tissues and thus serves as a useful platform for genetic screening. Based on the success of this CRISRPa approach, we are generating a genome-wide collection of flies expressing single-guide RNAs (sgRNAs) for CRISPRa. We also present a collection of more than 30 Gal4 > UAS:dCas9-VPR lines to aid in using these sgRNA lines for GOF studies in vivo.

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